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Department of Nutritional Sciences, University of California, Berkeley, CA 94720 and * Department of Animal/Range Sciences, North Dakota State University, Fargo, ND 58105
2To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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-glutamyltranspeptidase and ornithine decarboxylase gene expressions
than did tissue from the control group during early lactation of both
cycles. Mammary tissue from the compensatory nutrition group also had
fewer apoptotic cells than tissue from the control group during late
lactation of the first lactation cycle. These results suggest that the
compensatory nutrition regimen imposed during the peripubertal
developmental phase stimulated mammary growth and enhanced lactation
performance by affecting the expression of genes that regulate the cell
cycle.
KEY WORDS: • compensatory growth • lactation • cell proliferation • apoptosis • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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,
Park and Jacobson 1993
). In growing animals, either
overfeeding or severely restricting total nutrient intake inhibits
normal development of the mammary gland, especially the degree of
proliferation of parenchymal cells. Proper nutritional status at the
time of puberty and during pregnancy is critical to maximize mammary
cell proliferation and to enhance performance during the first and
subsequent lactations (Wilde et al. 1986
).
In an effort to develop a better understanding of mammary development,
differentiation and subsequent life-long lactation performance, our
laboratory has studied a stair-step compensatory nutrition regimen
for growing rats (Kim et al. 1998
, Park et al. 1994 and 1988
), gilts (Crenshaw et al. 1989a and 1989b
), and dairy and beef heifers (Park et al. 1989 and 1998
). This nutrition regimen is a combination of both energy
restriction and realimentation that allows minimal development of
mammary tissues during an energy restriction phase, whereas a
compensatory growth phase immediately after energy restriction
stimulates rapid and fuller development of the mammary gland. Our
studies (reviewed in Park 1998
) as well as those of
others (Barash et al. 1994
, Choi et al. 1997 and 1998
) have shown that a well-controlled compensatory
nutrition regimen imposed during hormone-sensitive growth stages
before first parturition can significantly affect mammary development
and lactation performance.
The cellular mechanisms responsible for the effects of nutritionally induced compensatory growth on development, differentiation and regression of the mammary gland are not well studied, despite the potentially beneficial role of a controlled nutrition regimen in improving lactation. A better understanding of how cell number is controlled may lead to strategies for prolonging lactation, not only by increasing peak yield, but also by reducing net loss of mammary cells. In a previous study using rats, we focused on the influence of a compensatory nutrition regimen on mammary development, ornithine decarboxylase gene expression and lactation potential during the first lactation cycle. This study was conducted with the following two goals: 1) to determine whether the enhancement of lactation potential by our stair-step compensatory nutrition regimen persists throughout the first and second lactation cycles and 2) to determine the extent to which a compensatory nutrition regimen permanently modulates cell proliferation, differentiation, and apoptosis and expression of genes in mammary tissues of female rats.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Female Sprague-Dawley rats (n = 122, Harlan Sprague Dawley, Indianapolis, IN), 4 wk of age, were housed individually in metal wire mesh–bottomed cages and isolated in a controlled environment of 25°C and 50% relative humidity with a 12-h light:dark cycle and free access to water. All experimental methods and procedures were approved by the University Animal Care and Use Committee.
During a 1-wk acclimation period, all rats had free access to the
control diet (AIN-93, Reeves et al. 1993
). At 5 wk of
age, rats were randomly assigned to either control or stair-step
compensatory nutrition regimen groups. The control group was offered
free access to the control diet throughout the trial period. The
compensatory nutrition group was subjected to an alternating
2–2-3–3-wk schedule beginning with an energy-restricted diet
(40% energy restriction) for 2 wk followed by a control diet (free
access to control diet) for 2 wk. This step was then repeated at 3-wk
intervals. The treatment diet during the energy restriction period was
formulated to provide an intake of protein, vitamins and minerals
similar to that of the control group; only the energy content was less
(Table 1
). During the energy restriction phase, treatment rats were fed the
energy-restricted diet at 60% of the average ad libitum intake of
the control group, i.e., if the control group consumed 10 g/d, the
amount of feed offered to the restricted group was 6 g/d. Feed intake
was recorded twice each week and average daily intake was estimated.
All rats were weighed weekly until parturition and every 3 d
during the lactation period.
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). Cell proliferation by immunohistochemistry.
Randomly selected early lactating rats (d 2) (8 rats for the first lactation; 5 rats for the second lactation) from each dietary treatment were injected intraperitoneally with bromodeoxyuridine (BrdU,3 a thymidine analog; 5 mg/kg body weight; Aldrich, Milwaukee, WI). At 1 h after the BrdU injection, the rats were killed using CO2. A small portion of mammary tissue from the fifth left mammary gland was collected, fixed in Carnoy's fixative for 4 h, transferred to 70% ethanol, embedded in paraffin, sectioned at 5 µm and mounted onto glass slides. Tissue sections were stained for BrdU by using a specific monoclonal antibody and indirect immunoperoxidase detection via the avidin:biotinylated peroxidase complex (ABC) method. The deparaffinized sections were treated with 2 mol/L HCI for 30 min to denature nuclear chromatin and subsequently treated with blocking buffer [PBS (0.01 mol/L sodium phosphate and 0.14 mol/L NaCI, pH 7.3) containing 1% v/v normal horse serum (Vector Laboratories, Burlingame, CA)] to block nonspecific binding with the antibody. Sections were incubated for 90 min at room temperature with mouse anti-BrdU monoclonal antibody (Boehringer Mannheim, Indianapolis, IN; 2 mg/L in blocking buffer). Control mammary tissue sections were incubated with mouse immunoglobulin G serum and ABC complex for 45 min each, and peroxidase substrate SG (Vector Laboratories) was used in the color development. Tissue sections were counterstained with Nuclear Fast Red to visualize labeled nuclei.
BrdU staining was observed with a Nikon Microphot-FX upright microscope (Melville, NY). A photomicrograph (400X) was taken of each of five randomly chosen fields (300–500 nuclei per field) per slide per animal. Labeled and unlabeled nuclei on the total area of each photomicrograph were counted. The labeling index was calculated as the percentage of BrdU-labeled nuclei relative to the total number of nuclei.
Apoptosis in situ by end-labeling.
Mammary tissues were collected from the fifth left mammary gland of
seven (first lactation) to five (second lactation) rats per treatment
during late lactation (d 19), fixed in 4% formalin fixative for
24 h, embedded in paraffin, sectioned at 5 µm and
mounted onto glass slides. The terminal deoxynucleotidyl transferase
(TdT)-mediated deoxyuridine triphosphate (dUTP)-biotin nick
end-labeling (TUNEL) method was performed according to the protocol
described (Gavrieli et al. 1992
). The deparaffinized
sections were digested with 20 mg/L proteinase K at room temperature
for 15 min and dipped in methanol containing 3% hydrogen peroxidase at
room temperature for 15 min to inhibit endogenous peroxidase activity.
The sections were soaked in buffer (30 mmol/L Tris-HCI, pH 7.2, 140
mmol/L sodium cacodylate, 1 mmol/L cobalt chloride and 1% gelatin; In
Situ Apoptosis Detection Kit, Oncor, Gaithersburg, MD) containing TdT
(0.3 units/µL) and 1 mmol/L biotinylated dUTP at
37°C for 1 h in a humidified chamber, followed by soaking in a
buffer (0.3 mol/L sodium chloride and 0.03 mol/L sodium citrate) for 10
min to stop the reaction. After being rinsed in 0.01 mol/L PBS, pH 7.2,
the sections were treated with ABC complex (Vectastain Elite ABC kit,
Vector Laboratories) and incubated with the peroxidase substrate, SG
(Vector Laboratories), for 5–10 min at room temperature. The nuclei
were counterstained with Nuclear Fast Red. Rat intestine was used as
the positive and negative control; the TdT was replaced with distilled
water for the negative control.
Staining was observed with a Nikon Microphot-FX upright microscope. A photomicrograph (400X) was taken of each of five randomly chosen fields per slide per rat. Labeled and unlabeled nuclei on the total area of the photomicrograph were counted. The labeling index was defined as the percentage of positive nuclei relative to the total number of nuclei counted.
RNA extraction and dot and Northern blot analyses.
Total RNA was extracted from fresh mammary tissue of early (d 2) and
late (d 19) lactating rats by the method of Chomczynski and Sacchi (1987)
. Briefly, the mammary tissues were pulverized in
liquid nitrogen and homogenized in lysis buffer containing 4 mmol/L
guanidine thiocyanate, 25 mmol/L sodium citrate, 0.5% sarcosyl, 0.1
mmol/L 2-mercaptoethanol. The homogenate was extracted with phenol and
chloroform. Total RNA in the aqueous phase was precipitated with
isopropanol and sodium acetate, pH 4.2, and dissolved in diethyl
pyrocarbonate–treated distilled deionized H2O. For dot
blotting, total RNA from individual mammary tissues was serially
diluted by twofold and dotted directly onto nylon membranes. For
Northern blotting, poly(A)+ RNA was isolated from pooled
total RNA using oligo dT cellulose columns (5 Prime 3 Prime, Boulder,
CO). Poly(A)+ RNA (6 µg per lane) was
fractionated by electrophoresis on agarose gels (12 g/L) containing 2.2
mmol/L formaldehyde and transferred to nylon membranes.
The membranes were baked for 1 h in a vacuum oven at 80°C and
then hybridized with cDNA probes: rat -glutamyltranspeptidase
(donated by Dr. M. Haas, University of Wisconsin, Madison) and rat
ornithine decarboxylase (donated by Dr. P. J. Blackshear, Duke
University, Durham, NC). The denatured cDNA probes (specific activity,
2.5 x 107 dpm) were labeled with [32P]
dATP by random priming method (Multiprime DNA Labeling Systems,
Amersham Life Science, Arlington Height, IL). Prehybridization and
hybridization solutions consisted of 50% formamide, 5X sodium
chloride:sodium dihydrogen phosphate:ethylenediaminetetraacetic acid
disodium salt (SSPE), 5X Denhardt's solution, 0.5% SDS, and 100 mg/L
denatured salmon sperm DNA. The membranes were prehybridized for 3 h at 42°C. Hybridization was performed for 18 h at 42°C. The
membranes were washed twice at room temperature in a solution
containing 5X SSPE and 0.5% SDS, followed by washing twice at 37°C
in a solution containing 1X SSPE and 0.5% SDS. The membranes were
exposed to X-ray film (Kodak Rochester, NY) with an intensifying
screen at -70°C. The relative intensities of the autoradiograms were
quantitated by laser densitometry (Personal Densitometer SI System,
Molecular Dynamics, Sunnyvale, CA).
Statistical analysis.
Data were analyzed with the SAS statistical package (SAS/STAT Version 6.11, SAS Institute, Cary, NC). Comparison of means was by Student's unpaired t test. Results are presented as means ± SEM. Differences were considered significant at P < 0.05.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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The daily energy allowance of the growing rat is a major determinant of
the subsequent ability to express her inherited capacity for milk
production (Park and Jacobson 1993
). Overall lactation
performance was improved in the compensatory nutrition regimen group
across two lactation cycles (Table 2). The daily pup gain determined on d 12 was greater in the
compensatory nutrition regimen group than in the control group during
the first (P < 0.05) and the second (P
< 0.05) lactations. The estimated milk yield was also higher in
the compensatory nutrition group than in the control group during both
lactation cycles; dams in the compensatory nutrition regimen groups
produced 8.5% (first lactation, P = 0.084) and 12.8%
(second lactation, P < 0.05) more milk than did dams
in the control group. These results are supported by our previous study
(Kim et al. 1998
) in which dams reared on the
compensatory nutrition regimen produced 17% more milk than did dams
fed the control diet during the first lactation cycle. It is important
to note that the improved lactation performance during the first
lactation stage was continued into the second lactation cycle.
Therefore, we can postulate that a controlled compensatory nutrition
regimen imposed during the hormone (e.g., insulin, glucagon, growth and
thyroid hormones) sensitive growing period may have a permanent
influence on mammary cell development and differentiation.
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). Cell proliferation was 1.3- and 0.5-fold greater in mammary
tissues (d 2) from the compensatory group during the first
(P < 0.05) and second (P = 0.088)
lactation cycles, respectively, compared with those from the control
group. Even though most secretory cell proliferation occurs by the end
of pregnancy, additional mammary gland growth takes place during the
early stages of lactation. A continuation of this growth occurs until
the peak of lactation; little cellular proliferation takes place
thereafter (Daniel and Silverstein 1987
). During
lactation, cell proliferation is due solely to alveolar epithelial cell
proliferation; myoepithelial cells fail to divide after achieving
functional maturity at the end of gestation (Traurig 1967
). The proliferation of mammary ducts peaks by early
gestation and remains low throughout lactation in rats (Joshi et al. 1986
). Therefore, the cell proliferation occurring during
early lactation probably reflects mainly epithelial cell proliferation.
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. This
consistent increase in cell proliferation in mammary tissues from the
compensatory nutrition group throughout two lactations may explain the
overall improvement in lactation performance.
Ornithine decarboxylase (ODC) is a key regulating enzyme in the
biosynthesis of polyamines in mammalian cells and appears to play an
important role in the control of a variety of biological processes,
including cellular metabolism, differentiation, proliferation and
membrane function (Russell and McVicker 1972
).
Nutritional status is an important factor in the regulation of ODC
synthesis and cell proliferation. Elevated activities of ODC are
normally present in rapidly growing tissues. This is true of the
mammary gland during the phase of rapid growth and differentiation
around parturition (Russell and McVicker 1972
). The
expression of ODC in mammary tissues from the compensatory nutrition
regimen group was 2.2- and 1.1-fold higher than that from the control
group tissue for first and second lactations, respectively (Fig. 1
A and
B). The increase in ODC gene expression observed in both lactation
cycles may be an indication of the metabolic shift necessary for the
increased cell proliferation and for the increased milk protein
synthesis during the early phase of lactogenesis.
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-glutamyltranspeptidase (GGT) has been found in most secretory
tissues (Meister 1973
), and it may play an important
role in the regulation of the entry of amino acids into the cell. Along
with the ODC gene, our compensatory nutrition regimen also modulated
the gene expression of GGT in mammary tissues. In general, the mammary
tissues from both early and late stages of lactation from the
compensatory nutrition regimen group had higher levels of GGT
transcripts compared with the control group for two lactation cycles
(Fig. 2
A and
B). This increase in the expression of GGT is directly related to the
increased protein metabolism associated with the rate of cell
proliferation as well as with the greater metabolic demand for amino
acids for the synthesis and secretion of milk protein during the onset
of lactation.
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). After the peak of lactation, cell loss is largely
responsible for the decline in milk yield, but activity per cell is
maintained (Park and Jacobson 1993
). Therefore, the
number of secretory cells is one of the basic elements that limits milk
production. For successful milk production through succeeding
lactations, an animal should possess more mammary secretory cells.
Mammary tissue from late lactation (d 19) from the compensatory
nutrition group exhibited less cell death (35 and 27%) during the
first (P < 0.05) and second (P = 0.087) lactation cycles, respectively (Table 3)
. It is possible that
the decreased apoptosis in the compensatory group was a physiologic
consequence of greatly increased endocrine and metabolic activities
(Shetty 1990
). In a study employing a single
stair-step nutrition model, we found little decrease in the number
of apoptotic mammary cells in the compensatory nutrition group
(Kim et al. 1998
). Therefore, we believe that a
multistep compensatory nutrition regimen may be more effective in the
suppression of apoptotic cell death in mammary tissues.
In conclusion, the compensatory nutrition regimen improved lactation
performance and persistency by modulation of cell proliferation,
differentiation and apoptosis for two lactation cycles. The permanent
improvement in overall mammary growth and lactation potential observed
from this study as well as from earlier works (Kim et al. 1998
, Park et al. 1988, 1989, 1994, and 1998
)
have led us to hypothesize that compensatory nutrition-directed
mammary hyperplasia and hypertrophy together with elevated metabolic
activities are permanently maintained. Through modulation of endocrine
status, energy restriction redirects energy flow to
energy-conserving activities, mainly maintenance and repair
functions, by down-regulating genes involved in cell proliferation.
A realimentation phase after energy restriction is synchronous with one
or more critical hormonal stages of development (puberty through late
gestation); this phase induces compensatory allometric growth. The
synergistic interaction of nutritionally induced compensatory growth
with developmentally related allometric growth activates endocrine
status which, in turn, may cause a cascade of up-regulation of
various genes affecting cell proliferation. Increased cellular
proliferation causes hyperplasia and hypertrophy for maximum
parenchymal tissue growth in the mammary gland. Further, a physiologic
consequence of the compensatory mammary hyperplasia may be the
suppression of apoptotic cell death, thereby improving persistency of
lactation. The elevated metabolic signals induced through compensatory
growth enhance galactopoiesis (i.e., maintenance and/or enhancement of
milk secretion) throughout the first and subsequent lactations.
After the peak of lactation, cell loss is largely responsible for the decline in milk yield. The declining rate of milk secretion that prevails for much of the lactation cycle in dairy animals has long been a source of dismay to the producer and a challenge to the research scientist. Manipulation of mammary function to prevent or reduce this decline, thereby increasing the persistency of lactation, would be a major advancement in improving production efficiency.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: ABC, avidin:biotinylated
peroxidase complex; BrdU, bromodeoxyuridine; dUTP, deoxyuridine
triphosphate; GGT, -glutamyltranspeptidase; ODC, ornithine
decarboxylase; SSPE, sodium chloride:sodium dihydrogen
phosphate:ethylenediaminetetraacetic acid disodium salt; TdT, terminal
deoxynucleotidyl transferase; TUNEL, TdT-mediated dUTP-biotin nick
end-labeling.
Manuscript received December 3, 1998. Initial review completed December 30, 1998. Revision accepted February 15, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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1. Barash H., Bar-Meir Y., Bruckental I. Effects of low-energy diet followed by a compensatory diet on growth, puberty and milk production in dairy heifers. Livest. Prod. Sci. 1994;39:263-268
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4. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 1987;162:156-159[Medline]
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