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Structure and biochemistry of normal and mutant (hubhub ) mouse mammary tissue during gestation and lactation
Camp. Biochem. Physiol.
Vol. 112A. Nos. 314, pp. 527-536, 1995 Copyright 0 1995 Elsevier Science Inc. Printed in Great Britain. All rights reserved 0300-%29/95 $9.50+ .OO
Pergamon 0300-9629(95)02022-C
Structure and biochemistry of normal and mutant (hub/hub) mouse mammary tissue during gestation and lactation Brenda Alston-Mills,* Donna Brauns Department
Eugene J. Eisen, Sandra Anderson
of Animal Science, North Carolina State University,
and
Raleigh, NC 27695, U.S.A.
Mammary gland structure and function in mutant hub/hub (hyper-unconjugated hiliruhinemia) mice were compared to controls (+/hub). Pups from normal and mutant mice have decreased weight gain when suckled by mutant dams. Samples examined by light microscopy showed no apparent differences when excised at mid- (day 9-11) and late- (day 17-18) gestation, and early (day 3-4) lactation. There was some evidence that early involution may be occurring in the hub mice at midlactation (day 10-11). Total DNA was greater (33%) when sampled at midgestation in the normal mice but lower (20%) at late gestation when compared to the hub mice. Total protein concentration was higher (132%) during late gestation in hub mice but was lower (34%) by early lactation when compared to normal mice. Epidermal growth factor (EGF) in normal mice was 79% and 71% higher in early and midlactation, respectively, than in mutant mice. There were no differences in serum concentrations of progesterone between strains at any stage. From these results, there is a suggestion of premature production of proteins, possibly followed by early involution in hub mice. Milk yield was less in mutant mice than in normal mice. Low concentrations of EGF during lactation in mutant mice may partially account for the decreased growth observed in mutant and normal pups suckling mutant dams. Key words: Progesterone;
Mammary gland; DNA; Protein.
Comp. Biochem.
Lactation;
Gestation;
hub/hub;
Mutation;
Mice;
EGF;
Physiol. 112A, 527-536, 1995.
Introduction An autosomal single gene recessive mutation named hub, because of the condition of hyperunconjugated bilirubinemia, was described in an ICR strain (Saxton et al., 1985). All homozygotes (hub/hub) are phenotypically characterized by neonatal jaundice resulting from a bilirubin-UDP-glucuronosyl transferase deficiency (Burkhart ef al., 1995). Homozygous
Correspondence
lo: Brenda
Alston-Mills,
Deut. of Animal
Science, North Carolina State University, Raleigh, NC 27695, U.S.A. Received 7 February 1995; revised 21 June 1995; accepted 23 June 1995.
males are sterile. Homozygous females have normal conception rates, but their pups have a reduced birth weight and lower neonatal survival compared with normal ( +/?) siblings (Saxton ef al., 1985). Postnatal growth is retarded in both normal (+/?) and mutant (hub/ hub) pups nursed by mutant mothers (Saxton et al., 1985). Thus, inadequate mammary gland development and/or dysfunction is indicated. The interplay of hormones from the pituitary and the ovary are essential in the regulation of mammary gland structural development and functional differentiation. These hormones also influence the production and/
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or secretion of growth factors. Epidermal growth factor (EGF) is a polypeptide initially purified from the salivary glands of male mice and is resistant to trypsin, chymotrypsin, and pepsin degradation. Injections of EGF into newborn mice cause early incisor eruption and eyelid opening (Cohen, 1962). EGF plays a major role in the proliferation of mammary epithelium in pregnant mice. In culture of mouse and human mammary epithelial cells, it has been shown that EGF is ineffective alone and, therefore, other factors such as insulin or IGF-1 are needed (Taketani and Oka, 1983a; Imagawa er al., 1986). Using whole organ culture, prolactin and EGF act synergistically to promote lobular-alveolar development (Plaut, 1993). With the use of slow-release plastic implants, in viva studies have demonstrated that end bud growth can be initiated in ovariectomized animals (Coleman et al., 1988). Further, in hormonally intact animals, lobular-alveolar growth can be initiated in vivo (Vonderhaar, 1987) with slow-release implants containing EGF. As lactogenesis occurs, corticosteroids and prolactin provide for milk protein synthesis and a concomitant increase in plasma EGF mRNA in viva (Fenton and Sheffield, 1991). The role of EGF during lactogenesis is less clear. Early studies showed that EGF inhibited functional differentiation of mouse mammary epithelial cells in culture (Taketani and Oka, 1983b). When EGF in the submaxillary gland was measured during various reproductive stages, it was determined that there is an increase after the onset of lactation; that increase is maintained through lactation and after lactation (Kurachi and Oka, 1985). The in virvo studies of Sankaran and Topper (1987) showed that EGF is inhibitory to casein synthesis, particularly in the presence of supraphysiological concentrations of insulin. A plausible explanation is that EGF has bifunctional activity. The addition of EGF to insulin + aldosterone will increase the mRNA of p- and a-caseins in mammary glands of pregnant mice. However, in the presence of prolactin, EGF is inhibitory to casein gene expression and a-lactalbumin synthesis in tissues from pregnant and virgin rats and mice (Vonderhaar and Nakhasi, 1986). This finding does not preclude the presence of untranslated casein mRNA. During gestation, estrogens stimulate terminal end bud development; estrogen and progesterone, in the presence of anterior pituitary hormones, are responsible for ductal cell proliferation. Lobular-alveolar differentiation is attributed to progesterone (see Tucker, 1985; Forsyth, 1983 for reviews). A high progesterone level, as observed during gestation, also
ef a/.
suppresses milk synthesis. It is postulated that the withdrawal of progesterone is the stimulus for the onset of lactogenesis to occur. In rats (21-day gestation), steroidogenesis decreases over the last 9 days of gestation. Luteal 20ahydroxysteroid dehydrogenase, responsible for the reduction of progesterone to 20 (Ydihydroprogesterone, is induced approximately 36 hr prior to parturition. This phase is thought to complete the process of luteal progesterone reduction. The objective of this initial study was to compare the morphology and differentiation as measured by total DNA and protein of hub/ hub mouse mammary glands to those of control (+/hub) mice during gestation and lactation. Tissue EGF will be measured because of its effect on mammary cell proliferation and possible influence on milk protein production. Because abnormal progesterone levels can effect both structural development and/or subsequent lactogenesis, a second objective was to assess concentrations of serum progesterone during the same time period.
Materials and Methods Animals All mutants and normal control siblings were obtained from the original ICR strain described by Saxton et al. (1985). The mice were maintained at the Mouse Genetics Laboratory at North Carolina State University with food and water available ad fibitum. Females were fed Purina Mouse Chow 5015 (Purina Mills, Richmond, IN) during gestation and lactation. The animal laboratory was maintained at a temperature of 22”C, 55% relative humidity, and a 12-hr light/l2-hr dark cycle beginning with light at 0700. Weigh-suckle-weigh
study
The purpose of this study was to confirm the reduced lactational performance of hub/ hub females. Mutant (hub/hub) and normal (+/hub) females were mated to normal ( + / hub) males. One day after birth, the phenotypes of mutant pups were identified by their jaundiced appearance. Previous studies have shown that there are no differences in body weights of males and females through 12 days of age, so that no correction for sex of the pups was needed (Eisen, unpublished data). Eight mutant or 8 normal pups were assigned randomly to be nursed by either a mutant or normal dam in a factorial arrangement. At 6 and again at 12 days of age, dams and litters were weighed, separated for 3 hr, reweighed, rejoined to nurse for 1 hr and weighed again. The litter weight gain and dam weight loss dur-
Mammary gland development ing the 1-hr suckling period were used as measures of lactational performance.
Mammary
gland tissue and blood samples
For the purpose of obtaining mammary gland tissue and blood samples, mutant and control mice were produced by crossing hub/ hub females to +/hub males. This experiment had two replicates. Mice and litters were weighed immediately before euthanization. Adult females were anesthetized using sodium pentobarbital and blood was collected by cardiac puncture. Abdominal and inguinal mammary glands were removed by sterile procedure at midgestation (MG; 9-11 days), late gestation (LG; 17-18 days), early lactation (EL; 3-4 days) and midlactation (ML; lo-11 days). Samples were taken for histology and immediately fixed in Carnoy’s solution. Tissue for total DNA and protein and EGF measurements was rinsed in cold physiological saline and blotted on filter paper, then weighed and stored at - 10°C for later analyses. Serum was collected for quantitation of progesterone. Histology Samples of mammary gland tissue for histology were placed in Carnoy’s fixative overnight at 4°C followed by dehydration in a series of ethanols. Samples were then stained with Harris’ Modified hematoxylin and eosin B. For DNA analysis, approximately 100-150 mg of mammary tissue was added to 1 x TNE buffer (10 x = 100 mM Tris base, 10 mM EDTA, 20 M NaCl) and homogenized for 30 set using a Polytron homogenizer at setting 4 (Brinkmann Instruments, Westbury, NY) and placed on ice. The assay was done using a TKO 102 Standards Kit and read on a Hoeffer TKO 100 Mini-fluorometer following the procedure suggested by the manufacturer (Hoeffer Scientific, San Francisco, CA). Protein Approximately 100 mg of tissue were homogenized in 2.5 ml 0.2% NaCl (ice cold). To precipitate the protein, 1.25 ml of 10% TCA was added to the homogenate, vigorously shaken, and placed on ice for 20 min. Samples were centrifuged at 1800 rpm for 15 min at 4°C. The supernatant was discarded and the pellet resuspended in 0.5 ml of 0.2% NaCl and mixed. Total protein was measured using the bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL). EGF For measurement of EGF in mammary tissue, Radioimmunoassay Kit (BT-530) was
in mutant (hub/hub) mice
529
used following the instructions of the manufacturer (Biomedical Technologies, Inc. Stoughton, MA). Progesterone Serum progesterone was measured by a solid-phase radioimmunoassay with the antibody immobilized to the wall of polypropylene tubes. The RIA kit and procedures were supplied by Diagnostic Products Corporation (Los Angeles, CA). Statistical analysis The weigh-suckle-weigh experiment was analyzed by analysis of variance procedures (Steel and Torrie, 1980). The model included the effects of genotype of nurse dam, genotype of litter, interaction of genotype of nurse dam by genotype of litter, and error. The data from the mammary gland study were based on a statistical model that included the effects of replication, dam genotype, stage of mammary gland development, interaction of genotype x stage of mammary gland development, and error. DNA analysis also included the weight of the dam as a covariate. Least-squares means were compared by preplanned LSD tests.
Results Weigh-suckle-weigh
study
On day 6 of lactation, litter weights of +/? females were greater than those of hub/hub females (33.4 + 1.0 g, n = 20 vs 29.2 -t 0.9 g, n = 8; P < O.Ol), but there were no differences (P > 0.10) in dam weights (38.6 2 0.7 g vs 37.2 2 0.7 g), or litter weight gains (0.20 t 0.22 g vs 0.40 t 0.21 g) or dam weight loss (-0.90 + 0.53 g vs -0.80 5 0.50 g) in the I-hr suckling period. Genotype of the litter and dam genotype by litter genotype interaction were not significant for any traits on day 6 of lactation. Least-squares means are presented in Table 1 for data at day 12 of lactation. Litter and dam weights, and litter weight gain and dam weight loss in the 1-hr suckling period were significantly greater for normal than for mutant females. No significant effects were present for litter genotype and dam genotype x litter genotype interaction. The partial correlation between litter weight gain and dam weight loss on day 12 of lactation was -0.73 (P < O.Ol), which indicates that these two criteria of the estimate of lactational performance are highly correlated. Litter Characteristics Regarding mice used for tissue and blood collection, no significant differences were ob-
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Table 1. Least-squares
Trait Litter wt. Dam wt. Litter wt. gain Dam wt. loss Sample size
means f SE (g) for litter and dam traits and analysis of variance (ANOVA) probabilities at day 12 of lactation
C-C” 58.3 ” 1.8 42.3 2 1.1 1.13 k 0.19 -2.1 ” 0.26 12
Mean + SE (litter-dam) ___~___.~ C-Ma M-C” 48.7 ” 40.7 % 0.47 2 - 1.49 f 21
1.4 0.8 .14 0.20
55.5 42.1 0.81 - 1.86
2 + k -c 8
ANOVA urobabilitv ___~ Dam genotype Interaction
Litter genotype
M-M”
2.3 1.4 0.23 0.32
47.2 k 38.3 + 0.34 ? - 1.00 t 7
2.4 1.5 0.25 0.34
NS* NS NS NS
0.01 0.05 0.01 0.01
NS NS NS NS
“First letter is genotype of the litter and second letter is genotype of the dam; C = normal controls ( + /?); and M = mutant (hub/hub). *NS = not significant (P > 0.10).
served for genotype of litter and dam genotype for litter size and weight or dam weights (Table 2). Histology
When comparing the glandular structure of control mice to mutant mice at midgestation, no differences were observed (Fig. la and lb). Generally, just prior to and after parturition, numerous milk colloids can be observed in the lumina. After the newborn pups begin to suckle, the secretory activity of the epithelial cells is stimulated so that any given lobularalveolar structure is filling and emptying. At the light microscopic level, there were no obvious differences between the mutant mice and the control mice from late gestation to early lactation (Fig. 2a-2d). By midlactation, it appeared that more stromal cells, mainly adipose, were replacing secretory cells in the mutant mice compared to the control mice (Fig, 3a and 3b). In some of the tissue sections from the mutant mice, there was also evidence of pycnotic nuclei characteristic of early involution. No quantitative histomorphometry was done.
Table 2. Litter characteristics
Total glandular DNA
Least squares means of mammary gland DNA are presented in Fig. 4. Analysis of variance results indicated that overall genotype was not significant (P > 0. lo), but stage of mammary gland development, genotype by stage of mammary gland development interaction, and body weight of dam were significant (P < 0.01). The significant interaction is indicative of changes in ranking of the genotypes, depending on the stage of mammary gland development. From our results, at midgestation (day 911), the DNA in the control mice (n = 7) was 33% higher (P < 0.05) than in the mutant mice (n = 10). By late gestation (day 17-l@, there was a significant increase (87%; P < 0.01) in total DNA in the mutant mice (n = 18) but not in the control mice (n = 15). An interesting observation was that the glands from the control mice contained less DNA than the mutant at this time. By early lactation (hub II = 12; control n = lo), and late lactation (hub n = 10; control n = 12), DNA values were the same for both genotypes.
(means 2 SE) of hub/hub
and control (-+/hub)dams
Gestation +lhub
(hub/hub)
Trait
MG (n = 10)
LG (n = 18)
MG (n = 7)
LG (n = 15)
29.6 k .9 12.0 r+_.7 0.8 2 .7
35.6 + .7 11.2 + .5 16.5 k .5
31.9 “_ 1.1 11.8 k .8 1.6 k .8
37.6 4 .8 13.5 ? .6 18.7 k .6
Dam wt. (g) Litter size Litter + uterine wt. (a)
Trait Dam wt. (g) *Litter size *Litter wt.
Day 1 10.3 * .6 17.4 2 .6
*Litters were standardized
to 8; characteristics
37.4 2 .9 -
40.0 t .9 -
12.1 2 .7 20.3 5 .7
prior to separation for fostering.
39.7 i .9 -
42.2 2 .9 -
Mammary gland development
in mutant (hub/hub) mice
Fig. 1. Sample of mammary tissue taken at midgestation showing ductal tissue interspersed among stromal tissue. No differences are observed between the control (a) and mutant (b) genotypes, x 200.
Protein Genotype effects for protein in the mammary gland were not significant (P > 0. lo), but stage of mammary gland development and genotype by stage of mammary gland development interaction were significant (P < 0.01). Least squares means are plotted in Fig. 5. No genotypic differences in mammary gland protein were present at midgestation, but by late gestation hub/hub females had protein values that were 132% higher than in the controls (P < 0.01). However, by early lactation, the protein concentrations in the control mice were significantly higher (P < 0.01) than in the mutant mice. Both strains showed a significant (P < 0.01) increase between late gestation and early lactation. In the mutant genotype, there was a further 42% increase in protein concentration from early to midlactation (P < O.Ol), whereas the normal genotype did not increase further. There was no difference between genotypes by midlactation (P > 0.10). EGF Significant differences (P c 0.01) in EGF were observed for genotype, stage of mammary gland development, and genotype by stage of development effects. No genetic differences in EGF were observed during midand late gestation (Fig. 6). Although both mu-
tant and normal females showed significant (P < 0.01) increases in EGF from late gestation to early lactation and from early lactation to midlactation, EGF in normal females increased at a greater rate (P < 0.01). As a result, by midlactation the control females were 71% higher in EGF content than the mutant females. Progesterone Analysis of variance results indicated no significant (P < 0.01) genetic or genetic by stage of development interaction effects on progesterone level. Significant stage of development effects were found (P < 0.01). Mean comparisons showed that progesterone level at midgestation was higher (P < 0.01) than at each of the subsequent 3 periods, none of which were significantly different from each other (Fig. 7).
Discussion This study compared structural and biochemical characteristics of mammary glands in mutant (hub/hub) and control mice. During the first half of gestation, interlobar ducts and terminal end bud development are evident. By day 12, intralobular ducts and alveolar buds are formed. However, alveoli are visually devoid of secretory material. These
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B. Alston-Mills et al.
Fig. 2. Sample of mammary tissue taken at late gestation (top) and early lactation (bottom). Inclusion of fat in epithelial cells and secretory material in lumina are obvious in both control (a and c) and mutant (b and d) genotypes x 200.
Mammary gland development
in mutant (hub/hub) mice
533
Fig. 3. Sample of mammary tissue from midlactation. Distension of ducts and lumina obvious for both control (a) and mutant (b). Note, however, that in the mutant, adipose cells are beginning to replace glandular tissue as indicated by the arrow. x 200.
characteristics were observed in both genotypes. Additionally, lipid droplets are stored in cytoplasm comprising approximately 20% of the volume of the cell. As gestation proceeds, the alveoli show progressive enlargement and, by late gestation (day 18), the alveoh are seen with secretory granules apparent
'1
n
hub/hub
H
+/hub
at i
n
hub/hub
q
+/hub
d
al 3
;
40
rn ._ c)
‘E i5l w
in the lumina. Cytoplasm of the epithelial cells is enlarged and engorged by fat droplets. From our observations, both hub and control mice are structurally similar. It is generally accepted that measuring total DNA is an index of cell number. The ratio of DNA content to the number of nuclei is
2
m
30
5 E
E 20
a
e ._ Q
1
zi
5
10
ti 0 0
Stage
of
Development
MG Stage
Fig. 4. Least-square means f SE of mammary gland DNA adjusted for weight dam. MC = midgestation; LG = late eestation: EL = earlv lactation: ML = late lactation. aGMeans ‘with no letters in common differ at P < 0.05.
LG of
EL
ML
Development
Fig. 5. Least-square means 2 SE of mammary gland protein concentrations. MG = midgestation; LG = late gestation; EL = early lactation- ML = late lactation. abCMeans with no letters in common differ at P < 0.05.
B. Alston-Mills et al.
534
100
1
w
hub/hub
q
+/hub d
Md Stage
LG
EL
of
Development
ML
Fig. 6. Least-square means k SE of EGF concentrations in the mammary gland. MG = midgestation; LG = late gestation; EL = early lactation; ML = late lactation. abCMeans with no letters in common differ at P < 0.05.
relatively constant from day 7 of gestation to day 12 of lactation (See Munford, 1964, for review). In mice, DNA peaks that are indicative of mammary cell proliferation are bimodal, with one peak occurring at approximately day 4 of gestation and the other at about day 12 of gestation. At about day 2 of lactation, there is mitotic activity stimulated by suckling, and this continues until about day 7 (Traurig, 1967). From these results, an increase in DNA at early lactation was expected and observed in the control group, but the significant increase appeared prematurely in the mutant group at late gestation. However, it is important to understand that the type of cell, parenchymal or stromal, cannot be detern
MG
LG
Stage
of
hub/hub
EL
ML
Development
Fig. 7. Least-square means 2 SE of serum progesterone concentrations. MG = midgestation; LG = late gestation; EL = early lactation: ML = midlactation. “bMeans with no letters in common differ at P < 0.05.
mined. Maximal growth is attained by approximately day lo-11 of lactation, which corresponds to peak lactation (Munford, 1964). In ICR mice, derivatives of which were used in this study, it was shown that the number of nuclei per alveolus peaks at about day 10 with the proportion of glandular tissue maximum at about day 12 of lactation (Hanrahan and Eisen, 1970). During involution, epithelial cells show large vacuoles containing fat droplets and casein micelles. As weaning progresses, cell depletion occurs. By midlactation, there was some suggestion of adipose cells replacing secretory cells prior to normal involution at postweaning. Ultrastructural analysis is underway to elucidate these changes. It is known that the mRNA for proteins such as the caseins appear during early gestation. In mice, the appearance of casein occurs about 2 days prior to parturition. As observed in this study, by day 18, luminal secretory proteins were evident histologically in both mutant and control mice. The significantly higher protein and DNA values found in the mutant mice compared to control mice at late gestation suggested premature development of the gland. These increases are not a function of EGF because EGF values are the same at late gestation. By early lactation, the protein concentrates in the control mice were significantly higher than the mutant mice. This also suggested that milk production occurred prematurely. The similar protein concentration at midlactation was an interesting finding. In light of that, it is important to note that we can make no distinction among types of proteins (e.g., milk proteins, synthetic, or housekeeping proteins) at this time. Studies are actively underway to identify these proteins. Little information is available on the concentration or source of EGF in the mammary gland at various stages of reproduction. According to Kurachi and Oka (1985) there are increased salivary and plasma concentrations during pregnancy and lactation. However, the mammary gland, as part of its exocrine function, may make its own EGF or sequester the polypeptide. Values for tissue EGF in midand late-gestation were the same in both genotypes. A major difference observed was the highly significant difference in EGF concentrations between the control and mutant mice, especially during midlactation, which corresponds to the time when milk production is at its height. EGF may have a role in controlling casein message translation or protein turnover (Vonderhaar and Nakhasi, 1986), which could account for the decreased protein production in the mutants at early lactation. However,
Mammary gland development
this argument does not hold true when considering protein vs. EGF concentrations at midlactation. EGF is found in the milk and there is evidence that EGF can promote growth in gastrointestinal tracts of newborn animals, including pigs and rats (Widdowson et al., 1976; Berseth et al., 1983). Concentrations of EGF found in colostrum and milk should be a reflection of glandular content as well as plasma content, although values in both colostrum and milk are greater than values in the plasma (Beardmore and Richards, 1983). If that is, in fact, the case, it could partially account for the improved growth of normal and mutant pups suckling normal, rather than mutant, dams. This observation is in addition to the fact that milk yield in mutant dams is less than that of normal mice. Despite the fact that, for this study, the assay of progesterone for late gestation was limited to day 18 gestation, there was no way of determining exactly when parturition would occur. At this stage, a few hours can influence differences observed among animals. For all the sampling times, blood was taken within minutes of each other for both strains but found to be highly variable in their concentrations. In mice, serum values of progesterone for day 1 of gestation are < 10 ng/ml, increasing to 40-50 ng/ml from day 3-4, decreasing to 18-35 ng/ml at midgestation and, again, increasing steadily on day 15-16 to maximal values of 80- 115 ng/ml. Just before parturition at about day 19, values fall to approximately 7 ng/ml (Cowie et al., 1980). The earliest sample in this study was taken on days 9-10 of gestation, at which time the concentrations should be within the 18-35 ng/ml range. By late gestation, values should again be declining with lowest values observed as lactation occurs. These data fall within the expected levels (Fig. 7) and have no relevant significant influence on parameters of this study. Traditionally, abnormal growth and development of a system are manipulated experimentally. The occurrence of a natural mutant is an interesting model to examine structural and functional differentiation of the mammary gland to understand factors affecting regulation. On first impression, it appeared that the mutant dams had premature increases in DNA and protein, as noted during late gestation. These observations suggested precocious development and lactogenesis so that, by the time of parturition, overall milk production was on the decline. This was corroborated by decreased milk production in hub mice in the weigh-suckle-weigh experiment. Further, the presence of adipose cells was observed by midlactation. The subsequent increase of pro-
in mutant (hub/hub) mice
535
tein in the glands of the mutant mice may have been induced by the suckling stimulus which, in turn, stimulates prolactin. The persistance of suckling through midlactation possibly allowed protein production to “catch-up” to the control protein values. In conclusion, from the milk yield study, mutant dams can nurse control pups with enough milk to have weight gain, though not as effectively as the control dams. During lactation, mutant dams have lower EGF concentrations when compared to normal dams. Definite evidence for premature lactation and involution are not available from this study, but active investigation to identify the proteins and ultrastructural changes is underway . Acknowledgements-The research reported in this publication was funded by the North Carolina Agricultural Research Service (NCARS), Raleigh, NC 27695-7643, U.S.A. The use of trade names in this publication does not imply endorsement by the NCARS, nor criticism of similar ones not mentioned.
References Beardmore J. M. and Richards R. C. (1983) Concentrations of epidermal growth factor in mouse milk throughout lactation. J. Endocr. 96, 287-292. Berseth C. L., Lichtenberger L. M., Morriss F. H. (1983) Comparison of gastrointestinal growth promoting effects of rat colostrum and mature milk in newborn rats in vivo. Am. .I. Chin. Nutr. 31, 52-60. Burkhart J. G., Armstrong F. B. and Eisen E. J. (1995) A unique bilirubin-UDP-glucuronosyltransferase deficiency related to neonatal jaundice in mice. Biochem. Genetics (in press). Cohen S. (1962) Isolation of a mouse submaxillary gland protein accelerating incisor eruption and eyelid opening in the newborn animal. J. Biol. Chem. 237, 1555-1562. Coleman S., Silberstein G. B. and Daniel C. W. (1988) Ductal morphogenesis in the mouse mammary gland: Evidence supporting a role of epidermal growth factor. Dev. Biol. 127, 304-315. Cowie A. T., Forsyth I. A. and Hart I. C. (1980) Growth and development of the mammary gland. In Hormonal Control of Lactation (Edited by Gross F.. Labhart A., Mann T. and Zander J.), pp. 58-145. Springer-Verlag, New York. Fenton S. E. and Sheffield L. G. (1991) Lactogenic hormones increase epidermal growth factor messenger RNA content of mouse mammary glands. Biochem. Biophys. Res. Commun. 181, 1063-1069. Forsyth I. A. (1983) The Endocrinology of Lactation. In Biochemistry of Lactation. Edited by Mepham, T. B. pp. 309-349. Elsevier Science Publishers, New York. Hanrahan J. B. and Eisen E. J. (1970) A lactation curve for mice. Lab Animal Care 20, 101-104. Imagawa W., Spencer E. M., Larson L. and Nandi S. (1986) Somatomedin-C substitutes for insulin for the growth of mammary epithelial cells from normal virgin mice in serum-free collagen gel culture. Endocrinology 119, 2695-2699.
Kurachi H. and Oka T. (1985) Changes in epidermal growth factor concentrations of submandibular gland, plasma, and urine of normal and sialoadenoectomized female mice during various reproductive stages. J. Endoer. 106, 197-202.
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Munford R. E. (1964) A review of anatomical and biochemical change in the mammary gland with particular reference to quantitative methods of assessing mammary development. Dairy Sci. Abst. 26, 293-304. Plaut K. (1993) Role of epidermal growth factor and transforming growth factors in mammary development and lactation. J. Dairy Sci. 16, 1526-1538. Sankaran L. and Topper Y. J. (1987) Is EGF a physiological inhibitor of mouse mammary casein synthesis? Unphysiological responses to pharmacological levels Biochem. Biophys. Res. Comm. of hormones. 146, 121-125. Saxton A. M., Eisen E. J., Johnson B. H. and Burkhart J. G. 1985. New mutation causing_”iaundice in mice. J. Heredity 76, 441-446.
Steel R. G. D. and Torrie J. H. (1980) Principles and Procedures of Sratistics, 2nd ed., McGraw-Hill Book Co., New York. Taketani Y. and Oka T. (1983a) Biological action of epidermal growth factor and its functional receptors in normal mammary epithelial cells. Proc. natl Acad. Sci. U.S.A.
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Taketani Y. and Oka T. (1983b) Epidermal growth factor stimulates cell proliferation and inhibits functional differentiation of mouse mammary epithelial cells in culture. Endocrinology 113, 811-877. Traurig H. H. (1967) Cell proliferation in the mammary gland during late pregnancy and lactation. Anat. Rec. 157,489-504. Tucker H. A. (1985) Endocrine and neural control of the mammary gland in lactation. (Edited by Larson, B. L.) pp. 39-79. Iowa State University Press, Ames. Vonderhaar B. K. (1987) Local effects of EGF, a-TGF and EGF-like growth factors on labulo-alveolar development in the-mouse mammary gland in vivo. J. Cell Phvsiol. 132. 581-584.
Vonderhaar B: K. and Nakhasi H. L. (1986) Bifunctional activity of epidermal growth factor on a- and K-casein gene expression in rodent mammary gland in vitro. Endocrinology 119, 1178-l 184. Widdowson E. M., Colombo V. E. and Artavanis C. A. (1976) Changes in the organs of pigs in response to feeding for the first 24 hours after birth. Il. The digestive tract. Biol. Neonate 28, 272-281.
Vol. 112A. Nos. 314, pp. 527-536, 1995 Copyright 0 1995 Elsevier Science Inc. Printed in Great Britain. All rights reserved 0300-%29/95 $9.50+ .OO
Pergamon 0300-9629(95)02022-C
Structure and biochemistry of normal and mutant (hub/hub) mouse mammary tissue during gestation and lactation Brenda Alston-Mills,* Donna Brauns Department
Eugene J. Eisen, Sandra Anderson
of Animal Science, North Carolina State University,
and
Raleigh, NC 27695, U.S.A.
Mammary gland structure and function in mutant hub/hub (hyper-unconjugated hiliruhinemia) mice were compared to controls (+/hub). Pups from normal and mutant mice have decreased weight gain when suckled by mutant dams. Samples examined by light microscopy showed no apparent differences when excised at mid- (day 9-11) and late- (day 17-18) gestation, and early (day 3-4) lactation. There was some evidence that early involution may be occurring in the hub mice at midlactation (day 10-11). Total DNA was greater (33%) when sampled at midgestation in the normal mice but lower (20%) at late gestation when compared to the hub mice. Total protein concentration was higher (132%) during late gestation in hub mice but was lower (34%) by early lactation when compared to normal mice. Epidermal growth factor (EGF) in normal mice was 79% and 71% higher in early and midlactation, respectively, than in mutant mice. There were no differences in serum concentrations of progesterone between strains at any stage. From these results, there is a suggestion of premature production of proteins, possibly followed by early involution in hub mice. Milk yield was less in mutant mice than in normal mice. Low concentrations of EGF during lactation in mutant mice may partially account for the decreased growth observed in mutant and normal pups suckling mutant dams. Key words: Progesterone;
Mammary gland; DNA; Protein.
Comp. Biochem.
Lactation;
Gestation;
hub/hub;
Mutation;
Mice;
EGF;
Physiol. 112A, 527-536, 1995.
Introduction An autosomal single gene recessive mutation named hub, because of the condition of hyperunconjugated bilirubinemia, was described in an ICR strain (Saxton et al., 1985). All homozygotes (hub/hub) are phenotypically characterized by neonatal jaundice resulting from a bilirubin-UDP-glucuronosyl transferase deficiency (Burkhart ef al., 1995). Homozygous
Correspondence
lo: Brenda
Alston-Mills,
Deut. of Animal
Science, North Carolina State University, Raleigh, NC 27695, U.S.A. Received 7 February 1995; revised 21 June 1995; accepted 23 June 1995.
males are sterile. Homozygous females have normal conception rates, but their pups have a reduced birth weight and lower neonatal survival compared with normal ( +/?) siblings (Saxton ef al., 1985). Postnatal growth is retarded in both normal (+/?) and mutant (hub/ hub) pups nursed by mutant mothers (Saxton et al., 1985). Thus, inadequate mammary gland development and/or dysfunction is indicated. The interplay of hormones from the pituitary and the ovary are essential in the regulation of mammary gland structural development and functional differentiation. These hormones also influence the production and/
527
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or secretion of growth factors. Epidermal growth factor (EGF) is a polypeptide initially purified from the salivary glands of male mice and is resistant to trypsin, chymotrypsin, and pepsin degradation. Injections of EGF into newborn mice cause early incisor eruption and eyelid opening (Cohen, 1962). EGF plays a major role in the proliferation of mammary epithelium in pregnant mice. In culture of mouse and human mammary epithelial cells, it has been shown that EGF is ineffective alone and, therefore, other factors such as insulin or IGF-1 are needed (Taketani and Oka, 1983a; Imagawa er al., 1986). Using whole organ culture, prolactin and EGF act synergistically to promote lobular-alveolar development (Plaut, 1993). With the use of slow-release plastic implants, in viva studies have demonstrated that end bud growth can be initiated in ovariectomized animals (Coleman et al., 1988). Further, in hormonally intact animals, lobular-alveolar growth can be initiated in vivo (Vonderhaar, 1987) with slow-release implants containing EGF. As lactogenesis occurs, corticosteroids and prolactin provide for milk protein synthesis and a concomitant increase in plasma EGF mRNA in viva (Fenton and Sheffield, 1991). The role of EGF during lactogenesis is less clear. Early studies showed that EGF inhibited functional differentiation of mouse mammary epithelial cells in culture (Taketani and Oka, 1983b). When EGF in the submaxillary gland was measured during various reproductive stages, it was determined that there is an increase after the onset of lactation; that increase is maintained through lactation and after lactation (Kurachi and Oka, 1985). The in virvo studies of Sankaran and Topper (1987) showed that EGF is inhibitory to casein synthesis, particularly in the presence of supraphysiological concentrations of insulin. A plausible explanation is that EGF has bifunctional activity. The addition of EGF to insulin + aldosterone will increase the mRNA of p- and a-caseins in mammary glands of pregnant mice. However, in the presence of prolactin, EGF is inhibitory to casein gene expression and a-lactalbumin synthesis in tissues from pregnant and virgin rats and mice (Vonderhaar and Nakhasi, 1986). This finding does not preclude the presence of untranslated casein mRNA. During gestation, estrogens stimulate terminal end bud development; estrogen and progesterone, in the presence of anterior pituitary hormones, are responsible for ductal cell proliferation. Lobular-alveolar differentiation is attributed to progesterone (see Tucker, 1985; Forsyth, 1983 for reviews). A high progesterone level, as observed during gestation, also
ef a/.
suppresses milk synthesis. It is postulated that the withdrawal of progesterone is the stimulus for the onset of lactogenesis to occur. In rats (21-day gestation), steroidogenesis decreases over the last 9 days of gestation. Luteal 20ahydroxysteroid dehydrogenase, responsible for the reduction of progesterone to 20 (Ydihydroprogesterone, is induced approximately 36 hr prior to parturition. This phase is thought to complete the process of luteal progesterone reduction. The objective of this initial study was to compare the morphology and differentiation as measured by total DNA and protein of hub/ hub mouse mammary glands to those of control (+/hub) mice during gestation and lactation. Tissue EGF will be measured because of its effect on mammary cell proliferation and possible influence on milk protein production. Because abnormal progesterone levels can effect both structural development and/or subsequent lactogenesis, a second objective was to assess concentrations of serum progesterone during the same time period.
Materials and Methods Animals All mutants and normal control siblings were obtained from the original ICR strain described by Saxton et al. (1985). The mice were maintained at the Mouse Genetics Laboratory at North Carolina State University with food and water available ad fibitum. Females were fed Purina Mouse Chow 5015 (Purina Mills, Richmond, IN) during gestation and lactation. The animal laboratory was maintained at a temperature of 22”C, 55% relative humidity, and a 12-hr light/l2-hr dark cycle beginning with light at 0700. Weigh-suckle-weigh
study
The purpose of this study was to confirm the reduced lactational performance of hub/ hub females. Mutant (hub/hub) and normal (+/hub) females were mated to normal ( + / hub) males. One day after birth, the phenotypes of mutant pups were identified by their jaundiced appearance. Previous studies have shown that there are no differences in body weights of males and females through 12 days of age, so that no correction for sex of the pups was needed (Eisen, unpublished data). Eight mutant or 8 normal pups were assigned randomly to be nursed by either a mutant or normal dam in a factorial arrangement. At 6 and again at 12 days of age, dams and litters were weighed, separated for 3 hr, reweighed, rejoined to nurse for 1 hr and weighed again. The litter weight gain and dam weight loss dur-
Mammary gland development ing the 1-hr suckling period were used as measures of lactational performance.
Mammary
gland tissue and blood samples
For the purpose of obtaining mammary gland tissue and blood samples, mutant and control mice were produced by crossing hub/ hub females to +/hub males. This experiment had two replicates. Mice and litters were weighed immediately before euthanization. Adult females were anesthetized using sodium pentobarbital and blood was collected by cardiac puncture. Abdominal and inguinal mammary glands were removed by sterile procedure at midgestation (MG; 9-11 days), late gestation (LG; 17-18 days), early lactation (EL; 3-4 days) and midlactation (ML; lo-11 days). Samples were taken for histology and immediately fixed in Carnoy’s solution. Tissue for total DNA and protein and EGF measurements was rinsed in cold physiological saline and blotted on filter paper, then weighed and stored at - 10°C for later analyses. Serum was collected for quantitation of progesterone. Histology Samples of mammary gland tissue for histology were placed in Carnoy’s fixative overnight at 4°C followed by dehydration in a series of ethanols. Samples were then stained with Harris’ Modified hematoxylin and eosin B. For DNA analysis, approximately 100-150 mg of mammary tissue was added to 1 x TNE buffer (10 x = 100 mM Tris base, 10 mM EDTA, 20 M NaCl) and homogenized for 30 set using a Polytron homogenizer at setting 4 (Brinkmann Instruments, Westbury, NY) and placed on ice. The assay was done using a TKO 102 Standards Kit and read on a Hoeffer TKO 100 Mini-fluorometer following the procedure suggested by the manufacturer (Hoeffer Scientific, San Francisco, CA). Protein Approximately 100 mg of tissue were homogenized in 2.5 ml 0.2% NaCl (ice cold). To precipitate the protein, 1.25 ml of 10% TCA was added to the homogenate, vigorously shaken, and placed on ice for 20 min. Samples were centrifuged at 1800 rpm for 15 min at 4°C. The supernatant was discarded and the pellet resuspended in 0.5 ml of 0.2% NaCl and mixed. Total protein was measured using the bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL). EGF For measurement of EGF in mammary tissue, Radioimmunoassay Kit (BT-530) was
in mutant (hub/hub) mice
529
used following the instructions of the manufacturer (Biomedical Technologies, Inc. Stoughton, MA). Progesterone Serum progesterone was measured by a solid-phase radioimmunoassay with the antibody immobilized to the wall of polypropylene tubes. The RIA kit and procedures were supplied by Diagnostic Products Corporation (Los Angeles, CA). Statistical analysis The weigh-suckle-weigh experiment was analyzed by analysis of variance procedures (Steel and Torrie, 1980). The model included the effects of genotype of nurse dam, genotype of litter, interaction of genotype of nurse dam by genotype of litter, and error. The data from the mammary gland study were based on a statistical model that included the effects of replication, dam genotype, stage of mammary gland development, interaction of genotype x stage of mammary gland development, and error. DNA analysis also included the weight of the dam as a covariate. Least-squares means were compared by preplanned LSD tests.
Results Weigh-suckle-weigh
study
On day 6 of lactation, litter weights of +/? females were greater than those of hub/hub females (33.4 + 1.0 g, n = 20 vs 29.2 -t 0.9 g, n = 8; P < O.Ol), but there were no differences (P > 0.10) in dam weights (38.6 2 0.7 g vs 37.2 2 0.7 g), or litter weight gains (0.20 t 0.22 g vs 0.40 t 0.21 g) or dam weight loss (-0.90 + 0.53 g vs -0.80 5 0.50 g) in the I-hr suckling period. Genotype of the litter and dam genotype by litter genotype interaction were not significant for any traits on day 6 of lactation. Least-squares means are presented in Table 1 for data at day 12 of lactation. Litter and dam weights, and litter weight gain and dam weight loss in the 1-hr suckling period were significantly greater for normal than for mutant females. No significant effects were present for litter genotype and dam genotype x litter genotype interaction. The partial correlation between litter weight gain and dam weight loss on day 12 of lactation was -0.73 (P < O.Ol), which indicates that these two criteria of the estimate of lactational performance are highly correlated. Litter Characteristics Regarding mice used for tissue and blood collection, no significant differences were ob-
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Table 1. Least-squares
Trait Litter wt. Dam wt. Litter wt. gain Dam wt. loss Sample size
means f SE (g) for litter and dam traits and analysis of variance (ANOVA) probabilities at day 12 of lactation
C-C” 58.3 ” 1.8 42.3 2 1.1 1.13 k 0.19 -2.1 ” 0.26 12
Mean + SE (litter-dam) ___~___.~ C-Ma M-C” 48.7 ” 40.7 % 0.47 2 - 1.49 f 21
1.4 0.8 .14 0.20
55.5 42.1 0.81 - 1.86
2 + k -c 8
ANOVA urobabilitv ___~ Dam genotype Interaction
Litter genotype
M-M”
2.3 1.4 0.23 0.32
47.2 k 38.3 + 0.34 ? - 1.00 t 7
2.4 1.5 0.25 0.34
NS* NS NS NS
0.01 0.05 0.01 0.01
NS NS NS NS
“First letter is genotype of the litter and second letter is genotype of the dam; C = normal controls ( + /?); and M = mutant (hub/hub). *NS = not significant (P > 0.10).
served for genotype of litter and dam genotype for litter size and weight or dam weights (Table 2). Histology
When comparing the glandular structure of control mice to mutant mice at midgestation, no differences were observed (Fig. la and lb). Generally, just prior to and after parturition, numerous milk colloids can be observed in the lumina. After the newborn pups begin to suckle, the secretory activity of the epithelial cells is stimulated so that any given lobularalveolar structure is filling and emptying. At the light microscopic level, there were no obvious differences between the mutant mice and the control mice from late gestation to early lactation (Fig. 2a-2d). By midlactation, it appeared that more stromal cells, mainly adipose, were replacing secretory cells in the mutant mice compared to the control mice (Fig, 3a and 3b). In some of the tissue sections from the mutant mice, there was also evidence of pycnotic nuclei characteristic of early involution. No quantitative histomorphometry was done.
Table 2. Litter characteristics
Total glandular DNA
Least squares means of mammary gland DNA are presented in Fig. 4. Analysis of variance results indicated that overall genotype was not significant (P > 0. lo), but stage of mammary gland development, genotype by stage of mammary gland development interaction, and body weight of dam were significant (P < 0.01). The significant interaction is indicative of changes in ranking of the genotypes, depending on the stage of mammary gland development. From our results, at midgestation (day 911), the DNA in the control mice (n = 7) was 33% higher (P < 0.05) than in the mutant mice (n = 10). By late gestation (day 17-l@, there was a significant increase (87%; P < 0.01) in total DNA in the mutant mice (n = 18) but not in the control mice (n = 15). An interesting observation was that the glands from the control mice contained less DNA than the mutant at this time. By early lactation (hub II = 12; control n = lo), and late lactation (hub n = 10; control n = 12), DNA values were the same for both genotypes.
(means 2 SE) of hub/hub
and control (-+/hub)dams
Gestation +lhub
(hub/hub)
Trait
MG (n = 10)
LG (n = 18)
MG (n = 7)
LG (n = 15)
29.6 k .9 12.0 r+_.7 0.8 2 .7
35.6 + .7 11.2 + .5 16.5 k .5
31.9 “_ 1.1 11.8 k .8 1.6 k .8
37.6 4 .8 13.5 ? .6 18.7 k .6
Dam wt. (g) Litter size Litter + uterine wt. (a)
Trait Dam wt. (g) *Litter size *Litter wt.
Day 1 10.3 * .6 17.4 2 .6
*Litters were standardized
to 8; characteristics
37.4 2 .9 -
40.0 t .9 -
12.1 2 .7 20.3 5 .7
prior to separation for fostering.
39.7 i .9 -
42.2 2 .9 -
Mammary gland development
in mutant (hub/hub) mice
Fig. 1. Sample of mammary tissue taken at midgestation showing ductal tissue interspersed among stromal tissue. No differences are observed between the control (a) and mutant (b) genotypes, x 200.
Protein Genotype effects for protein in the mammary gland were not significant (P > 0. lo), but stage of mammary gland development and genotype by stage of mammary gland development interaction were significant (P < 0.01). Least squares means are plotted in Fig. 5. No genotypic differences in mammary gland protein were present at midgestation, but by late gestation hub/hub females had protein values that were 132% higher than in the controls (P < 0.01). However, by early lactation, the protein concentrations in the control mice were significantly higher (P < 0.01) than in the mutant mice. Both strains showed a significant (P < 0.01) increase between late gestation and early lactation. In the mutant genotype, there was a further 42% increase in protein concentration from early to midlactation (P < O.Ol), whereas the normal genotype did not increase further. There was no difference between genotypes by midlactation (P > 0.10). EGF Significant differences (P c 0.01) in EGF were observed for genotype, stage of mammary gland development, and genotype by stage of development effects. No genetic differences in EGF were observed during midand late gestation (Fig. 6). Although both mu-
tant and normal females showed significant (P < 0.01) increases in EGF from late gestation to early lactation and from early lactation to midlactation, EGF in normal females increased at a greater rate (P < 0.01). As a result, by midlactation the control females were 71% higher in EGF content than the mutant females. Progesterone Analysis of variance results indicated no significant (P < 0.01) genetic or genetic by stage of development interaction effects on progesterone level. Significant stage of development effects were found (P < 0.01). Mean comparisons showed that progesterone level at midgestation was higher (P < 0.01) than at each of the subsequent 3 periods, none of which were significantly different from each other (Fig. 7).
Discussion This study compared structural and biochemical characteristics of mammary glands in mutant (hub/hub) and control mice. During the first half of gestation, interlobar ducts and terminal end bud development are evident. By day 12, intralobular ducts and alveolar buds are formed. However, alveoli are visually devoid of secretory material. These
532
B. Alston-Mills et al.
Fig. 2. Sample of mammary tissue taken at late gestation (top) and early lactation (bottom). Inclusion of fat in epithelial cells and secretory material in lumina are obvious in both control (a and c) and mutant (b and d) genotypes x 200.
Mammary gland development
in mutant (hub/hub) mice
533
Fig. 3. Sample of mammary tissue from midlactation. Distension of ducts and lumina obvious for both control (a) and mutant (b). Note, however, that in the mutant, adipose cells are beginning to replace glandular tissue as indicated by the arrow. x 200.
characteristics were observed in both genotypes. Additionally, lipid droplets are stored in cytoplasm comprising approximately 20% of the volume of the cell. As gestation proceeds, the alveoli show progressive enlargement and, by late gestation (day 18), the alveoh are seen with secretory granules apparent
'1
n
hub/hub
H
+/hub
at i
n
hub/hub
q
+/hub
d
al 3
;
40
rn ._ c)
‘E i5l w
in the lumina. Cytoplasm of the epithelial cells is enlarged and engorged by fat droplets. From our observations, both hub and control mice are structurally similar. It is generally accepted that measuring total DNA is an index of cell number. The ratio of DNA content to the number of nuclei is
2
m
30
5 E
E 20
a
e ._ Q
1
zi
5
10
ti 0 0
Stage
of
Development
MG Stage
Fig. 4. Least-square means f SE of mammary gland DNA adjusted for weight dam. MC = midgestation; LG = late eestation: EL = earlv lactation: ML = late lactation. aGMeans ‘with no letters in common differ at P < 0.05.
LG of
EL
ML
Development
Fig. 5. Least-square means 2 SE of mammary gland protein concentrations. MG = midgestation; LG = late gestation; EL = early lactation- ML = late lactation. abCMeans with no letters in common differ at P < 0.05.
B. Alston-Mills et al.
534
100
1
w
hub/hub
q
+/hub d
Md Stage
LG
EL
of
Development
ML
Fig. 6. Least-square means k SE of EGF concentrations in the mammary gland. MG = midgestation; LG = late gestation; EL = early lactation; ML = late lactation. abCMeans with no letters in common differ at P < 0.05.
relatively constant from day 7 of gestation to day 12 of lactation (See Munford, 1964, for review). In mice, DNA peaks that are indicative of mammary cell proliferation are bimodal, with one peak occurring at approximately day 4 of gestation and the other at about day 12 of gestation. At about day 2 of lactation, there is mitotic activity stimulated by suckling, and this continues until about day 7 (Traurig, 1967). From these results, an increase in DNA at early lactation was expected and observed in the control group, but the significant increase appeared prematurely in the mutant group at late gestation. However, it is important to understand that the type of cell, parenchymal or stromal, cannot be detern
MG
LG
Stage
of
hub/hub
EL
ML
Development
Fig. 7. Least-square means 2 SE of serum progesterone concentrations. MG = midgestation; LG = late gestation; EL = early lactation: ML = midlactation. “bMeans with no letters in common differ at P < 0.05.
mined. Maximal growth is attained by approximately day lo-11 of lactation, which corresponds to peak lactation (Munford, 1964). In ICR mice, derivatives of which were used in this study, it was shown that the number of nuclei per alveolus peaks at about day 10 with the proportion of glandular tissue maximum at about day 12 of lactation (Hanrahan and Eisen, 1970). During involution, epithelial cells show large vacuoles containing fat droplets and casein micelles. As weaning progresses, cell depletion occurs. By midlactation, there was some suggestion of adipose cells replacing secretory cells prior to normal involution at postweaning. Ultrastructural analysis is underway to elucidate these changes. It is known that the mRNA for proteins such as the caseins appear during early gestation. In mice, the appearance of casein occurs about 2 days prior to parturition. As observed in this study, by day 18, luminal secretory proteins were evident histologically in both mutant and control mice. The significantly higher protein and DNA values found in the mutant mice compared to control mice at late gestation suggested premature development of the gland. These increases are not a function of EGF because EGF values are the same at late gestation. By early lactation, the protein concentrates in the control mice were significantly higher than the mutant mice. This also suggested that milk production occurred prematurely. The similar protein concentration at midlactation was an interesting finding. In light of that, it is important to note that we can make no distinction among types of proteins (e.g., milk proteins, synthetic, or housekeeping proteins) at this time. Studies are actively underway to identify these proteins. Little information is available on the concentration or source of EGF in the mammary gland at various stages of reproduction. According to Kurachi and Oka (1985) there are increased salivary and plasma concentrations during pregnancy and lactation. However, the mammary gland, as part of its exocrine function, may make its own EGF or sequester the polypeptide. Values for tissue EGF in midand late-gestation were the same in both genotypes. A major difference observed was the highly significant difference in EGF concentrations between the control and mutant mice, especially during midlactation, which corresponds to the time when milk production is at its height. EGF may have a role in controlling casein message translation or protein turnover (Vonderhaar and Nakhasi, 1986), which could account for the decreased protein production in the mutants at early lactation. However,
Mammary gland development
this argument does not hold true when considering protein vs. EGF concentrations at midlactation. EGF is found in the milk and there is evidence that EGF can promote growth in gastrointestinal tracts of newborn animals, including pigs and rats (Widdowson et al., 1976; Berseth et al., 1983). Concentrations of EGF found in colostrum and milk should be a reflection of glandular content as well as plasma content, although values in both colostrum and milk are greater than values in the plasma (Beardmore and Richards, 1983). If that is, in fact, the case, it could partially account for the improved growth of normal and mutant pups suckling normal, rather than mutant, dams. This observation is in addition to the fact that milk yield in mutant dams is less than that of normal mice. Despite the fact that, for this study, the assay of progesterone for late gestation was limited to day 18 gestation, there was no way of determining exactly when parturition would occur. At this stage, a few hours can influence differences observed among animals. For all the sampling times, blood was taken within minutes of each other for both strains but found to be highly variable in their concentrations. In mice, serum values of progesterone for day 1 of gestation are < 10 ng/ml, increasing to 40-50 ng/ml from day 3-4, decreasing to 18-35 ng/ml at midgestation and, again, increasing steadily on day 15-16 to maximal values of 80- 115 ng/ml. Just before parturition at about day 19, values fall to approximately 7 ng/ml (Cowie et al., 1980). The earliest sample in this study was taken on days 9-10 of gestation, at which time the concentrations should be within the 18-35 ng/ml range. By late gestation, values should again be declining with lowest values observed as lactation occurs. These data fall within the expected levels (Fig. 7) and have no relevant significant influence on parameters of this study. Traditionally, abnormal growth and development of a system are manipulated experimentally. The occurrence of a natural mutant is an interesting model to examine structural and functional differentiation of the mammary gland to understand factors affecting regulation. On first impression, it appeared that the mutant dams had premature increases in DNA and protein, as noted during late gestation. These observations suggested precocious development and lactogenesis so that, by the time of parturition, overall milk production was on the decline. This was corroborated by decreased milk production in hub mice in the weigh-suckle-weigh experiment. Further, the presence of adipose cells was observed by midlactation. The subsequent increase of pro-
in mutant (hub/hub) mice
535
tein in the glands of the mutant mice may have been induced by the suckling stimulus which, in turn, stimulates prolactin. The persistance of suckling through midlactation possibly allowed protein production to “catch-up” to the control protein values. In conclusion, from the milk yield study, mutant dams can nurse control pups with enough milk to have weight gain, though not as effectively as the control dams. During lactation, mutant dams have lower EGF concentrations when compared to normal dams. Definite evidence for premature lactation and involution are not available from this study, but active investigation to identify the proteins and ultrastructural changes is underway . Acknowledgements-The research reported in this publication was funded by the North Carolina Agricultural Research Service (NCARS), Raleigh, NC 27695-7643, U.S.A. The use of trade names in this publication does not imply endorsement by the NCARS, nor criticism of similar ones not mentioned.
References Beardmore J. M. and Richards R. C. (1983) Concentrations of epidermal growth factor in mouse milk throughout lactation. J. Endocr. 96, 287-292. Berseth C. L., Lichtenberger L. M., Morriss F. H. (1983) Comparison of gastrointestinal growth promoting effects of rat colostrum and mature milk in newborn rats in vivo. Am. .I. Chin. Nutr. 31, 52-60. Burkhart J. G., Armstrong F. B. and Eisen E. J. (1995) A unique bilirubin-UDP-glucuronosyltransferase deficiency related to neonatal jaundice in mice. Biochem. Genetics (in press). Cohen S. (1962) Isolation of a mouse submaxillary gland protein accelerating incisor eruption and eyelid opening in the newborn animal. J. Biol. Chem. 237, 1555-1562. Coleman S., Silberstein G. B. and Daniel C. W. (1988) Ductal morphogenesis in the mouse mammary gland: Evidence supporting a role of epidermal growth factor. Dev. Biol. 127, 304-315. Cowie A. T., Forsyth I. A. and Hart I. C. (1980) Growth and development of the mammary gland. In Hormonal Control of Lactation (Edited by Gross F.. Labhart A., Mann T. and Zander J.), pp. 58-145. Springer-Verlag, New York. Fenton S. E. and Sheffield L. G. (1991) Lactogenic hormones increase epidermal growth factor messenger RNA content of mouse mammary glands. Biochem. Biophys. Res. Commun. 181, 1063-1069. Forsyth I. A. (1983) The Endocrinology of Lactation. In Biochemistry of Lactation. Edited by Mepham, T. B. pp. 309-349. Elsevier Science Publishers, New York. Hanrahan J. B. and Eisen E. J. (1970) A lactation curve for mice. Lab Animal Care 20, 101-104. Imagawa W., Spencer E. M., Larson L. and Nandi S. (1986) Somatomedin-C substitutes for insulin for the growth of mammary epithelial cells from normal virgin mice in serum-free collagen gel culture. Endocrinology 119, 2695-2699.
Kurachi H. and Oka T. (1985) Changes in epidermal growth factor concentrations of submandibular gland, plasma, and urine of normal and sialoadenoectomized female mice during various reproductive stages. J. Endoer. 106, 197-202.
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Munford R. E. (1964) A review of anatomical and biochemical change in the mammary gland with particular reference to quantitative methods of assessing mammary development. Dairy Sci. Abst. 26, 293-304. Plaut K. (1993) Role of epidermal growth factor and transforming growth factors in mammary development and lactation. J. Dairy Sci. 16, 1526-1538. Sankaran L. and Topper Y. J. (1987) Is EGF a physiological inhibitor of mouse mammary casein synthesis? Unphysiological responses to pharmacological levels Biochem. Biophys. Res. Comm. of hormones. 146, 121-125. Saxton A. M., Eisen E. J., Johnson B. H. and Burkhart J. G. 1985. New mutation causing_”iaundice in mice. J. Heredity 76, 441-446.
Steel R. G. D. and Torrie J. H. (1980) Principles and Procedures of Sratistics, 2nd ed., McGraw-Hill Book Co., New York. Taketani Y. and Oka T. (1983a) Biological action of epidermal growth factor and its functional receptors in normal mammary epithelial cells. Proc. natl Acad. Sci. U.S.A.
80, 2647-2650.
Taketani Y. and Oka T. (1983b) Epidermal growth factor stimulates cell proliferation and inhibits functional differentiation of mouse mammary epithelial cells in culture. Endocrinology 113, 811-877. Traurig H. H. (1967) Cell proliferation in the mammary gland during late pregnancy and lactation. Anat. Rec. 157,489-504. Tucker H. A. (1985) Endocrine and neural control of the mammary gland in lactation. (Edited by Larson, B. L.) pp. 39-79. Iowa State University Press, Ames. Vonderhaar B. K. (1987) Local effects of EGF, a-TGF and EGF-like growth factors on labulo-alveolar development in the-mouse mammary gland in vivo. J. Cell Phvsiol. 132. 581-584.
Vonderhaar B: K. and Nakhasi H. L. (1986) Bifunctional activity of epidermal growth factor on a- and K-casein gene expression in rodent mammary gland in vitro. Endocrinology 119, 1178-l 184. Widdowson E. M., Colombo V. E. and Artavanis C. A. (1976) Changes in the organs of pigs in response to feeding for the first 24 hours after birth. Il. The digestive tract. Biol. Neonate 28, 272-281.