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This is to certify that the

thesis entitled

MECHANISMS REGULATING PARENCHYMAL DEVELOPMENT
IN THE BOVINE MAMMARY GLAND

presented by

Laurie Ellen Davis

has been accepted towards fulfillment
of the requirements for

M. S . degree in Animal Science

Major professor

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6/01 c:/CIRC/DateDue.p65-p. 15

MECHANISMS REGULATING PARENCHYMAL DEVELOPMENT IN THE
BOVINE MAMMARY GLAND

By

Laurie Ellen Davis

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE
Department of Animal Science

2002

ABSTRACT

MECHANISMS REGULATING PARENCHYMAL DEVELOPMENT IN THE
BOVINE MAMMARY GLAND

By

Laurie Ellen Davis

The objective was to examine the proliferative response of mammary epithelial
cells to extracts of parenchymal tissue from different regions of the mammary glands of
dairy heifers. Mammary extracts were prepared from parenchyma collected from
proximal and distal regions within the glands of prepubertal heifers (n=3, BW = 213 i 16
kg). “Proximal” was defined as the 1/3 region closest to the teat and “distal” was defined
as the 1/3 region farthest from the teat. MAC-T bovine mammary epithelial cells were
cultured on collagen gels in basal medium with or without 1, 3 or 5% mammary extracts,
using 3 wells per treatment in 3 separate assays. After 40 h incubation with treatments,
total cellular DNA per well was measured. Mammary extracts of tissue from proximal
regions stimulated 11% more cell proliferation than extracts of tissue from distal regions

=0.02). Addition of mammary extracts stimulated a level of cell proliferation that was
108% greater than basal medium. Concentrations of IGF-I and leptin in extracts did not
differ between regions (P=O. l 8; P=O.37, respectively). Distal regions tended to have
greater abundance of IGFBP-3 than proximal regions (P=0.06). Abundance of IGFBP-2,
a 28-kD and a 24-kD BP did not differ by region (P>O.1). In conclusion, extracts from
proximal regions are more mitogenic than extracts from distal regions of the developing

mammary parenchyma in prepubertal dairy heifers.

I dedicate this thesis to my family, who have given me the
love, support and courage to undergo any challenge...
Flora and Jim Williams, my mother and step-father;
Julie and Brad Smith, my sister and brother-in-law;

Crystal, Johnney, Mark, Sara and Beth Grove, my farm family.

iii

ACKNOWLEDGEMENTS

I would first like to thank my major professor, Dr. Miriam Weber Nielsen, for the
opportunity to come to Michigan State and for the care and guidance she has given me
over many years. Your encouragement throughout the years, both in Virginia and
Michigan, has allowed me to focus on reaching my goals and making plans for the future.
Also, many thanks to my guidance committee, Dr. Mike VandeHaar, Dr. Cathy Ernst and
Dr. Don Jump, for your many thought provoking questions and ideas to make this
research project more productive and complete.

Special thanks to the lab group of 2270, especially to Luis Silva for your
mentoring in the culture hood and sharing your knowledge of cell culture. Also, to Jim
Liesman for all of your time and patience in helping me learn the in’s and out’s of ligand
blots, RIAs and SAS!

Last, and certainly not least to my groups of insanity relief! A special thanks to
Dr. Joe Domecq for your friendship, guidance and for allowing me to enter ‘your’ dairy
judging world! To my office mates, Marci Charest, Christel Moons and Dave Mello,
thanks for all of the talks in 1261 and for your life-long fi'iendships. Also, thanks to
Melissa and B. J. Macfarlane and Ashley Peterson for your friendship especially during

that wonderful thesis writing phase!

iv

TABLE OF CONTENTS

LIST OF TABLES ............................................................................................................ vii
LIST OF FIGURES ......................................................................................................... viii
LIST OF ABBREVIATIONS ............................................................................................. x
INTRODUCTION .............................................................................................................. 1
LITERATURE REVIEW ................................................................................................... 4
Physiology of Mammary Development ......................................................................... 4
Proximal and Distal Regions of Mammary Parenchyma ............................................... 6
Quantitating Mammary Gland Development ................................................................. 7
Mammary Extracts ......................................................................................................... 9
MAC-T: Mammary Alveolar Cell Large T Antigen .................................................... 10
Endocrine Regulation of Mammary Growth ................................................................ 12
IGF-I / Somatotropin Axis ........................................................................................ 12

IGF Ligands, Receptor Structure and Localization .................................................. 16
Insulin-Like Growth Factor Binding Proteins (IGFBP) ........................................... 18
IGFBP-3 ................................................................................................................ 21
IGFBP-2 ................................................................................................................ 23

Other Growth Factors and Hormones ....................................................................... 24
Transforming Grth F actor-B (TGF-B) .............................................................. 25

Leptin .................................................................................................................... 25
Fibroblast Grth Factor (F GF) ........................................................................... 27
Keratinocyte Growth Factor (KGF) ...................................................................... 27
Estrogen ................................................................................................................ 28
Summary ...................................................................................................................... 29
MATERIALS AND METHODS ...................................................................................... 30
Preparation of Mammary Extracts ............................................................................... 3O
MAC-T Cell Culture Experiments ............................................................................... 31

[3 H] Thymidine Incorporation Assay ........................................................................... 32
Total DNA Assay ......................................................................................................... 33
Total Protein Assay ...................................................................................................... 33
IGF-I Radioimmunoassay (RIA) .................................................................................. 34
Western Ligand Blot .................................................................................................... 37
Leptin Radioimmunoassay .......................................................................................... 39
Statistical Analysis ....................................................................................................... 40
RESULTS ......................................................................................................................... 41
Cell Culture Experiments ............................................................................................. 41
Protein Content ............................................................................................................. 53

Abundance of IGFBP in Mammary Extracts ............................................................... 53

IGF-I Concentrations in Mammary Extracts ................................................................ 57
Leptin Concentrations in Mammary Extracts .............................................................. 62
DISCUSSION ................................................................................................................... 64
SUMMARY AND CONCLUSIONS ............................................................................... 73
BIBLIOGRAPHY ............................................................................................................. 74
REFERENCES ................................................................................................................. 75
APPENDIX ....................................................................................................................... 87
Introduction .................................................................................................................. 87
Primary Bovine Mammary Epithelial Cell (PMEC) Isolation ..................................... 88
PMEC Observations and Controlling Fibroblast Contamination ................................. 92
Irnmunohistochemistry ................................................................................................. 94
PMEC Culture Experiments ....................................................................................... 100
Preparation of Rat Tail Collagen ................................................................................ 105
MAC-T Cell Culture Experiments ............................................................................. 106
Mitogenic Activity of Mammary Extracts from Prepubertal and Postpubertal Heifers
.................................................................................................................................... 111

vi

LIST OF TABLES

Table 1. The effect of region of the mammary parenchyma on the abundance of IGF-
binding proteins (IGFBP) in mammary extracts ....................................................... 56

Table 2. Summary table of overall results ........................................................................ 63

Table 3. Differences between primary bovine mammary epithelial cell isolation
procedures from five different heifers. ..................................................................... 91

vii

LIST OF FIGURES

Figure 1. Overview of the GH / IGF-I axis ...................................................................... 15
Figure 2. The role of IGFBPs in the IGF-I system .......................................................... 20

Figure 3. Effect of increasing doses of IGF-I on [3 H] thymidine incorporation into DNA
of MAC-T cells ......................................................................................................... 42

Figure 4. Effect of increasing doses of IGF-I on total DNA of MAC-T cells after 18 h..43
Figure 5. Effect of increasing doses of IGF-I on total DNA of MAC-T cells after 40 h..44

Figure 6. Effect of mammary extracts on [3 H] thymidine incorporation into DNA of

MAC-T cells ............................................................................................................. 47
Figure 7. Effect of mammary extracts on total DNA of MAC-T cells after 18 h ............ 48
Figure 8. Effect of mammary extracts on total DNA of MAC-T cells after 40 h ............ 49

Figure 9. Effect of mammary extracts fiom proximal and distal regions of the mammary
gland on total DNA of MAC-T cells after 40 h ........................................................ 51

Figure 10. Effect of mammary extracts from proximal and distal regions of the
mammary gland on total DNA of MAC-T cells afier 18 and 40 h ........................... 52

Figure 11. Regulation of IGFBP abundance in mammary tissue extracts prepared from
proximal and distal regions of the mammary parenchyma ....................................... 55

Figure 12. Fractionation of BSA and 125I-IGF-I by size exclusion gel chromatography on
Sephadex G-50 gel column. ...................................................................................... 58

Figure 13. IGFBP abundance in mammary tissue extracts from 2 different fractions
collected from a Sephadex G-50 gel column ............................................................ 61

Figure 14. Illustration of primary bovine mammary epithelial cells stained using
immunohistochemistry: Vectastain ABC kit and monoclonal antibody against
Cytokeratin Peptide 18 .............................................................................................. 97

Figure 15. Illustration of primary bovine mammary epithelial cells stained using

irnmunohistochemistry: Vectastain ABC kit and monoclonal antibody against
Vimentin ................................................................................................................... 98

viii

Figure 16. Illustration of primary bovine mammary epithelial cells stained using
immunohistochemistry: Vectastain ABC kit and monoclonal antibody against
Cytokeratin Peptide 17 .............................................................................................. 99

Figure 17. Effect of increasing doses of IGFBP-3 on [3 H] thymidine incorporation into
DNA of primary bovine mammary epithelial cells ................................................. 103

Figure 18. Inhibition of DNA synthesis of primary bovine mammary epithelial cells in
medium containing IGF-I alone or with IGFBP-3. ................................................. 104

Figure 19. Effect of increasing doses of IGFBP-3 on [3 H] thymidine incorporation into
DNA of MAC-T cells ............................................................................................. 108

Figure 20. Inhibition of DNA synthesis of MAC-T cells in medium containing IGF-I
alone or with IGFBP-3 ............................................................................................ 109

Figure 21. Effect of increasing doses of TGF-B ..... 1 on [3 H] thymidine incorporation into
DNA of MAC-T cells ............................................................................................. 110

Figure 22. Effect of mammary extracts from prepubertal and postpubertal heifers on total
DNA of MAC-T cells after 40 h. ............................................................................ 113

ix

LIST OF ABBREVIATIONS

ALS = Acid labile subunit

BM = Basal medium

BCA = Bicinchroninic acid

BSA = Bovine serum albumin

bST = Bovine somatotrophin

CFP = Cleared fat pad

CV = Coefficient of variation

CT = Computed tomography

CPM = Counts per minute

DMSO = Dimethyl sulfoxide

DPM = Disintegrations per minute

EGF = Epidermal growth factor

EDTA = Ethylenediaminetetraacetic acid
FBS = Fetal bovine serum

FCS = Fetal calf serum

FGF = Fibroblast growth factor

GLM = General linear model

GH = Growth hormone

GH—R = Growth hormone receptor
GHRF = Growth hormone releasing factor

GHRH = Growth hormone releasing hormone

HBSS = Hanks’ buffered salt solution

HU = Houndsfield units

IGF = Insulin-like growth factor

IGFBP = Insulin-like growth factor binding protein
LAP = Latency activated protein

LS means = Least square means

MAC-T = Mammary alveolar cell large T-antigen
MFP = Mammary fat pad

PCA = Perchloric acid

PBS = Phosphate buffered saline

PMEC = Primary mammary epithelial cells

RIA = Radioimmuno assay

RT-PCR = Reverse transcription polymerase chain reaction
rpm = revolutions per minute

SV-40 = Simian virus 40

SDS = Sodium dodecyl sulfate

SRIF = Somatotrophin releasing inhibitory factor
SEM = Standard error of the mean

TGF = Transforming growth factor

TCA = Trichloroacetic acid

TEMED = Tetramethylethylenediamine

TBS = Tris buffered saline

xi

INTRODUCTION

Interactions between hormones and growth factors regulate mammary growth in
prepubertal dairy heifers. Many reviews have focused on the importance of
understanding mammary development in heifers and how this foundation for subsequent
development and future milk yield can be affected by factors during early life (Akers,
1990; Knight, 2000; Tucker, 1981). A clearer understanding of the physiology and
development of the mammary gland could lead to techniques that enhance mammary
growth, thereby increasing milk yield. This could result in an increase in profitability for
the dairy producer.

Since the 1950’s, many scientists have studied factors affecting the number and
dividing rate of mammary epithelial cells. A high correlation exists between milk yield
and measurement of total mammary DNA (Sinha and Tucker, 1966). Milk yield is
positively correlated with the number of mammary epithelial cells present in the gland
and the activity of these secretory cells (Broster and Broster, 1984).

The mitogenic activity in the mammary gland is partially the result of interactions
between grth factors and their binding proteins. These interactions can be synergistic
and / or antagonistic in vivo and in vitro (Forsyth et al., 1998; Oka et al., 1991).
Understanding mammary development and growth is the goal of numerous labs focused
on many different species: murine (Beauloye et al., 1999; Robinson et al., 1996;
Silberstein et al., 1990; Soriano et al., 1998), ovine (Hovey et al., 1998b; McFadden et
al., 1990), caprine (Prosser et al., 1990; Prosser et al., 1994), porcine (Kraetzl et al.,
1998), and human (Blat et al., 1994; Chen et al., 1994; Reiss and Barcellos-Hoif, 1997),

in addition to the bovine (Akers, 1990).

Few studies have evaluated the relationship between mitogenic activity and
location of epithelial cells within the mammary gland (review by Akers, 1990; Ellis et al.,
2000; McFadden et al., 1990). Because these studies demonstrated that epithelial cells
from the distal border of the growing parenchyma are more rapidly proliferating, it is
logical that a “gradient” of mitogenic activity may exist within the mammary
parenchyma. However, these studies have only focused on the mitogenic capacity of the
epithelial cells from different regions within the mammary gland. Therefore, evaluation
of the mitogenic activity of parenchyma tissue, which not only includes epithelial cells
but also fibroblasts and adipocytes, should give more insight into the paracrine
mechanism of mammary development. Furthermore, the mitogenic activity of
parenchymal tissue isolated from different regions may differ due to the synthesis of
factors by the mammary fat pad (Hovey et al., 1998a).

Insulin-like growth factor-I (IGF-I) has been shown in many studies to be a major
mitogen in the mammary gland (Collier et al., 1993; Prosser et al., 1994; Shamay et al.,
1988). IGF-I is produced in the stromal portion of the gland (Hauser et al., 1990) and
IGF-1 receptors were characterized on microsomes from bovine mammary tissue (Hadsell
et al., 1990). Hence, a paracrine role for IGF-I action on the epithelium has been
proposed (Hauser et al., 1990). Furthermore, to fully understand the mechanism of IGF-I
growth stimulation in mammary development, the role of the IGF binding proteins must
also be taken into consideration.

A novel protein, leptin, was recently shown to reduce the mitogenic activity of
IGF-I when both were applied to bovine mammary epithelial cells in culture (Silva et al.,

1999). Leptin is produced by adipocytes and MAC-T cells, a bovine mammary epithelial

cell line (Houseknecht et al., 1998; Smith and Sheffield, 2002). Receptors for leptin have
been identified on bovine mammary epithelial cells (Silva et al., 2001).

Therefore, the objectives of this study focused on the mitogenic activity in
extracts of tissue isolated from different parenchymal regions within the prepubertal
heifer mammary gland. In addition, this project further characterized the mammary
extracts by comparing the concentrations of IGF-I and leptin, and the abundance of

IGFBP in extracts prepared from two different regions within the parenchymal tissue.

OBJECTIVES:

1. To compare the mitogenic activity in extracts of mammary tissue collected from
proximal and distal regions of the parenchyma.

2. To compare the concentrations of IGF-I and leptin within mammary extracts.

3. To compare the abundance of IGFBPs in mammary extracts.

LITERATURE REVIEW

Physiology of Mammary Development:

 

During development of the embryo and after mammary gland differentiation, a
mammary line or ridge is formed from the thickening of epithelial cells (Anderson,
1978). This ridge thickens to a hillock that differentiates into buds that form pairs of
glands. The bovine has two pairs of glands that form in the inguinal area. The cells in
this region differentiate into what will become the mammary parenchyma. Mammary
buds determine the number of glands and teats an organism will have, corresponding to
four in the bovine. These sprouts will later give rise to the gland cistern and major ducts
of the mammary gland. The mesenchyme (fat pad) grows outwardly creating pressure
that forms the shape of the teat. The fat pad provides the space for future development,
as the parenchymal tissue grows into the fat pad in later stages during prepubertal
development.

During prepubertal development, the mammary glands grow at a faster rate than
the rest of the body (Sinha and Tucker, 1969). During this period of development, the
allometric phase, there is rapid ductal growth into the fat pad in the rodent model.
However, no alveoli are formed during this phase (Hovey, 1996). Puberty and the
maturation of the ovaries signal the end of the allometric phase. At the end of the
allometric period, weight of total mammary tissue is 2 to 3 kg, with 0.5 to 1 kg of this
being parenchymal tissue. This parenchyma consists of 10-20% epithelium, 40-50%

connective tissue and 30-40% adipose tissue (Sejrsen et al., 1982).

During the allometric phase, a high rate of gain in heifers leads to earlier ages at
puberty but has negative effects on mammary development (Sejrsen et al., 1982). In this
study, ad libitum feeding compared to restricted feeding (60%) during the prepubertal
period reduced the wet weight of mammary secretory tissue by 23% and DNA content by
32%. Increases in growth rate from 400 to 850 g/d decreased the average age at first
estrus from 16.6 to 8.4 months (Sejrsen, 1994). Thus, the onset of puberty is more
closely related to nutrition and body development than actual chronological age
(Daughaday and Rotwein, 1989; Schillo et al., 1992).

From approximately the third estrus until pregnancy, the growth of the mammary
gland occurs at the same rate as body growth, which is referred to as isometric growth
(Sinha and Tucker, 1969). During this time, high rates of gain do not negatively affect
development of the gland (Sejrsen et a1, 1982; Sejrsen et al., 2000).

This change from the allometric to the isometric phase is largely influenced by
shifts in concentration of circulating hormones. As puberty approaches, luteinizing
hormone (LH) pulse frequency increases, stimulating development of large dominant
follicles and leading to an increase in estrogen production and secretion. After puberty,
secretion of progesterone is also thought to contribute to the shift from the allometric to
the isometric grth phase (Knight and Peaker, 1982).

Mammary secretory cell numbers increase rapidly during pregnancy until the
onset of lactation (Tucker, 1981). These cells become firlly differentiated between
parturition and peak lactation. There is a gradual loss of mammary cells during the

course of a lactation (Capuco et al., 2001 ). After peak lactation, the rate of cell atrophy

becomes greater than cell division and milk yield begins to decrease (Knight, 2000;
Knight and Wilde, 1987).

Much research on mammary development focuses on either the bovine or murine
model. However, large differences exist between murine and bovine mammary anatomy
and development. Ducts are more widely dispersed with a rounded profile in the rodent
(Akers, 1990). Ductal elongation involves terminal endbuds that elongate and grow to
the distal borders of the murine fat pad (Hovey, 1996). The ruminant has a more
complex tubular structure. The periphery of the duct in the bovine gland is surrounded
by connective tissue. Therefore, those interested in the bovine should consider these

differences even though there is a larger cost associated with using the bovine model.

Proximal and Distal Regions of Mammy Parenchma:

During the allometric phase of development, the ductal portion of the gland grows
rapidly into the fat pad. Recent work has been reported on the activity of cells isolated
from different locations within the developing gland. “Distal” epithelial cells penetrate
into the mammary fat pad and are those epithelial cells farthest from the teat cistern. The
“proximal” cells are those that are closest to the teat cistern. Proximal epithelial cells are
involved in local formation of ductal branches and later become part of the gland cistern
and a branched ductal structure. Differences exist in the mitogenic capacity of epithelial
cells from different regions. Ellis et a1. (2000) defined these two regions as “peripheral”
(referred to as “distal” in this thesis project) and “medial” (i.e., “proximal”). Primary
epithelial cells were isolated from the distal and proximal sections of the bovine gland

(Ellis et al., 2000). Distal cells, compared to proximal cells, were two to three times

more sensitive tothe mitogenic actions of IGF-I or serum in cell culture. Ellis and co-
workers attributed this difference to an increase in IGF-I receptor number in distal versus
proximal cell regions. The same group (Akers, 1990; McFadden et al., 1990) observed
tritiated thymidine labeled cells in cross sections of ovine mammary ducts and found
more labeling on the distal groups of ducts compared with proximal portions, indicating a
greater rate of cell division in the peripheral region.

Composition of parenchyma varies with location of tissue within the mammary
gland. Sejrsen and co-workers (1982) used tissue slices stained for light microscopy for
histological determination of individual cell types. Results showed that the area occupied
by epithelial cells decreased as distance from the base of the gland (“proximal” region)
increased. The percentage of fat cells in the parenchyma increased as distance from the
base of the gland increased.

The fat pad is an important source of mitogenic activity within the mammary
gland of mice (Hovey et al., 1998a). However, little research has been published on the
role of the fat pad in ruminants. The distal cells are in closer vicinity to the fat pad than
the proximal regions. Bovine mammary epithelial cells were growth inhibited when co-
cultured with fat pad explants (McFadden and Cockrell, 1993). This inhibitory role may

relate to the production of leptin within adipocytes (Houseknecht et al., 1998).

Quantitating Mammary Gland Development:
Many techniques have been used to measure mammary development in the
bovine, including palpation scores, teat length (Lammers et al., 1999) and mammary

biopsies. During the 1950’s (Davidson and Leslie, 1950), quantifying DNA through

either determination of the phosphorus present or measuring intensity of light absorption
due to purine and pyrimidine components became a popular way of measuring
proliferation. Although inexpensive, these techniques do not allow direct assessment of
epithelial: fat cell ratios and are not good estimates of total weight of the mammary
parenchyma.

Size of the gland and the degree of development can be assessed through
slaughter of the animal and collection of the udder. Proportion and weight of
parenchyma and stroma can be measured in slices of the gland, along with differences in
structure. Mammary gland growth is assessed by measurement of total DNA of
mammary tissue. Irnmunohistochernistry staining of cells or histology staining of cross
sections can determine organization of organelles, types of tissue and percentage of each
cell type within the mammary gland. Growth factor receptor mRN A expression can be
assessed by in situ hybridization. In addition, mammary tissue can be digested with
enzymes to isolate a primary epithelial cell population. Measurement of DNA synthesis
or accumulation of DNA in these primary cells can evaluate total epithelial cell number
after in vitro treatment and the sensitivity of these cells to different mitogens.
Homogenizing mammary tissue and preparation of extracts can be used to analyze the
mitogenic activity of the tissue for mammary epithelial cell growth in vitro. This
technique will be discussed further in the “Mammary Extracts” section.

Another technique is the use of computed tomography (CT) scanning which is
measured in Houndsfield Units (HU). The amount of parenchyma and extraparenchymal
tissue (fat) was estimated using this technique (Sejrsen et al., 1986; Sorensen et al., 1987)

on thawed sections of bovine mammary glands. Tissue above 20 HU is considered

parenchymal tissue and tissue with values of 20 HU or below is labeled extra-
parenchymal tissue. In these studies, the CT scanning technique gave results similar to
the dissection technique. Ayadi and co-workers (2000) used a non-invasive,
ultrasonographic procedure to estimate milk cistern volume to study the evolution of milk
stored in the gland at different milking intervals. Ultrasound and CT scanning should be
further investigated for possible measurement of mammary development by assessing the
portion of gland that is fat pad versus parenchyma. These techniques may allow
measurement of gland development in an animal over time without the need for

slaughter.

Mammag Extracts:

Waksman and colleagues (1991) conducted the first experiment using bovine
mammary tissue extracts at a 3:1 saline to tissue ratio. Aqueous extracts of mammary
tissue stimulated proliferation of undifferentiated bovine mammary epithelial cells in
vitro in a dose-dependent manner. The mitogenic activity of extracts was synergistic
with the addition of fetal calf serum (FCS), IGF-I or IGF-I plus epidermal growth factor
(EGF). The addition of EGF to extracts did not stimulate epithelial cell proliferation
significantly compared to extracts alone. Mean protein content of the extracts was 18
mg/ml and extracts were used at a 10% concentration in media in cell proliferation
assays.

Weber and colleagues (1999; 2000b) used mammary extracts in two different
studies. Addition of mammary extracts (5% concentration in medium) to bovine primary

mammary epithelial cells in culture stimulated DNA synthesis ([3 H] thymidine

incorporation) 545 % compared to basal medium treatment. Addition of IGFBP-3 to
mammary extracts inhibited extract-induced proliferation of primary cells. Further,
addition of IGF-I antibodies to mammary extracts inhibited up to one half of the
mitogenic activity of the mammary extracts (Weber et al., 1999). Mammary extracts
were also prepared from mammary tissue of prepubertal heifers fed at a high or low
feeding level and treated with growth hormone or placebo. A 5 % mammary extract
treatment stimulated greater [3 H] thymidine incorporation into primary epithelial cells
than fetal bovine serum (FBS, 10% of medium) or 100 pg IGF-I treatments. Extracts
from heifers fed at a low feeding level stimulated significantly greater [3 H] thymidine
incorporation than mammary extracts of heifers fed at a high level (Weber et al., 2000b).
Mammary tissue extracts from heifers treated with growth hormone (GH) compared with
placebo were higher in concentrations of IGF-I, higher in abundance of IGFBP-3, and
lower in abundance of IGFBP-2 (Weber et al., 2000a). This mammary extract procedure
could also be used to compare the mitogenic activity of tissue from heifers of the
prepubertal and postpubertal stages or to compare tissue collected from different regions

of the mammary parenchyma.

MAC-T: Mammary Alveola; Cell nge T Antiien:

The experiments in this study focused on evaluating the mitogenic activity in
mammary parenchyma and how it influences proliferation of mammary epithelial cells.
Use of mammary explants allows study of the interactions between different cell types

and mitogens produced by each. However, this technique does not allow the

10

characterization of the response of an isolated cell type, in particular the epithelial cell, to
mitogens in cell culture.

The MAC-T (Mammary Alveolar Cell large T antigen) cell line was chosen for
use in this project. The MAC-T cell line has been used in numerous studies as a model
for growth and morphological development of bovine mammary epithelial cells. Huynh
and colleagues established this cell line using mammary epithelial cells from a lactating
cow and transfecting these cells with the Simian Virus 40 (SV-40) large T antigen
(Huynh et al., 1991). This cell line is immortalized, not transformed, and maintains many
similar characteristics as primary cells in vitro and mammary tissue in vivo. This cell
line has been well defined and has growth characteristics similar to mammary epithelial
cells grown on both plastic and collagen gels. Zhao and co-workers (1992) showed that
MAC-T cells are responsive to IGF-I as measured by both [3 H] thymidine incorporation
and total DNA assay, and have high affinity IGF-I receptors. Also, Zhao used Scatchard
analysis of an IGF-I binding assay to determine that IGF-I binds to MAC-T cell surface
receptors (Zhao et al., 1992). MAC-T cells are able to synthesize lactose, lipid, and or
and B casein (Huynh et al., 1991). Also, MAC-T cells proliferate in response to insulin,
IGF-l (including Des (1-3) and Long R3 forms), IGF-II, and bovine serum albumin
(BSA). This group further characterized this cell line, showing that MAC-T cells lack a
mitogenic response to human and mouse EGF, bovine growth hormone (GH), bovine
prolactin, estrogen, progesterone, and display a small response to transforming growth
factor-0t (TGF-or) (Woodward et al., 1994, 1996). MAC-T cells, like primary bovine
mammary epithelial cells, synthesize IGFBP-2,-3,-4 and -6 (Cohick, 1998). Thus, MAC-

T cells are considered a useful model for in vitro studies of mammary cell proliferation.

11

Endocrine Regulation of Mamma_ry Growth:

 

Older experiments have clearly demonstrated that growth hormone (GH) is
required for normal mammary development (F orsyth, 1989; review by Sejrsen, 1994).
Exogenous administration of GH increases milk production in cows (Peel and Bauman,
1987) and stimulates peri-pubertal mammary growth in sheep (McFadden et al., 1990)
and dairy heifers (Sandles and Peel, 1987; Sejrsen et al., 1986). However, local infusion
of GH into the mammary gland does not increase milk production in sheep (McDowell
and Hart, 1984) indicating that GH does not act directly on mammary epithelial cells to
stimulate production. Moreover, addition of GH does not stimulate cell proliferation in
cultures of mammary epithelial cells (Sejrsen, 1994). Specific binding of GH to
receptors in mammary tissue has not been detected by the use of GH-receptor (GH-R)
binding studies (Akers, 1985; Pump et al., 1993). However, Glirnm and colleagues
(1990) used molecular hybridization analysis to detect GH-R mRN A (4.4 kb and 9.2 kb
transcripts) in lactating bovine mammary tissue. The presence of GH-R mRNA suggests
that the cells possess the GH-R. The receptor gene may be transcribed but it is not
known whether the mRNA is translated. Further research is needed to understand these

discrepancies.

GH / IGF-I Axis:

Much evidence suggests the effect of GH on mammary gland development is
mediated indirectly through the insulin-like growth factor (IGF) system (Akers, 1985;
Kleinberg, 1997; Sejrsen, 1994). The idea that IGF-I was important to mammary

development and lactation originated from studies using exogenous somatotropin (GH) as

12

 

a treatment to increase milk production. Vander Kooi and co-workers (1995) observed
increases in serum IGF-I concentrations, hepatic IGF-I mRNA abundance, and serum
IGFBP-3 abundance after intravenous infusing lactating cows with either GH or GH
Releasing Factor (GHRF). A subcutaneous injection of GH in lactating dairy cows and
goats resulted in increases in milk yield (Bauman and Vernon, 1993; Faulkner, 1999). A
greater increase of IGF-I in milk than plasma was noted and Faulkner suggested that after
treatment with GH, changes in IGF-I concentrations within the mammary gland occur
prior to the general circulation. IGF-I treatment stimulates the growth of primary bovine
(Shamay et al., 1988) and ovine (Winder et al., 1989) mammary epithelial cells and a
bovine mammary epithelial cell line (Zhao et al., 1992). This stimulation is caused by
IGF-I binding to membrane receptors on secretory cells and is detected as an increase in
DNA synthesis in myoepithelial cells, ductal and alveolar epithelial cells (Baumrucker
and Stemberger, 1989; McGrath et al., 1991).

IGF-I also acts locally in vivo. Close arterial infusion of IGF-I or IGF-II into the
external pudic artery of the goat mammary gland increased milk yield by increasing
blood flow to the gland and by increasing the persistency of lactation (Prosser et al.,
1990; Prosser et al., 1994). Infusion of IGF-I into the mammary gland of non-lactating
beef cows (Collier et al., 1993) and dairy heifers (Silva et al., 2002) increased mammary
growth. However, regulation of IGF-I synthesis within the mammary gland remains
undefined. Walden and colleagues (1998) hypothesized that GH stimulates IGF-I
production in the stromal compartments of the gland. Then, IGF-I acts on the ductal
portion of the gland and stimulates formation of terminal end buds in mice. IGF-I (4') null

mice had significantly less mammary development than same age wild-type controls

13

(Ruan & Kleinberg, 1999). Administration of IGF-I, but not GH or estrogen, had a
stimulatory effect on mammary development in these null mice. Thus, even when GH is
present, mammary development is reduced unless IGF-I is present.

In 1983, Sejrsen and others suggested that the negative influence of excess
feeding on mammary growth in prepubertal heifers might be associated with a decrease
in circulating GH concentrations (Sejrsen et al., 1983). Unlike GH, circulating IGF-I is
greater in animals fed for high rates of gain compared to low rates of gain (Breier et al.,
1986). Mammary growth and milk production are positively correlated with
concentrations of GH (Bauman and Vernon, 1993; Sejrsen et al., 1983). During early
lactation, plasma IGF-I concentrations are low and concentrations are inversely related to
milk production. Rouge and others (1988) suggested that the relationship during early
lactation could represent an uncoupling of the GH / IGF axis due to negative energy
balance. These differences may also be due to the effect of IGF binding proteins
(IGFBP) and altered synthesis of IGFBP in response to different feeding patterns,
treatments, etc. More studies are now being done to investigate the effects of leptin
(Silva et al., 1999), compensatory growth (Ford and Park, 2001) and additional growth
factors (Woodward et al., 1995) and how these factors might influence growth of the
mammary gland.

Another explanation for these differences between GH (also known as
somatotropin, ST) and IGF-1 concentrations could be a negative feedback mechanism.
GH-releasing hormone (GHRH) is produced in the hypothalamus and regulates the
amount of GH synthesized and secreted from the pituitary (review by Thissen et al.,

1994) (see Figure 1). GH secretion is controlled by both the stimulatory influence of

14

GHRH and the inhibitory effect of somatostatin (SRIF, somatotrophin releasing
inhibitory factor) (see Figure l). The IGF-I peptide is primarily synthesized and secreted
by the liver when GH binds to hepatic GH receptors (Gluckman et al., 1987). When
pituitary cells were treated with GHRH, and with or without IGF-I (10 mM), IGF-I
inhibited GHRH-elicited GH secreted by 67%. High levels of circulating IGF-I may
provide a negative feedback mechanism by either decreasing the amount of GHRH
secreted or increasing the amount of SRIF secreted and thus decreasing the amount of

GH synthesized and secreted from the pituitary (Ceda et al., 1987).

u:**”"'

”TEPTHAbfflps BLOODSTREAM
GHRH SRIF

+\u [-

Hrunrhv -
6” 'Gfi UDDER

1!»

IGF-l

 

\/

 

--IGF4
”T‘IGFJRECEPTUR

LIVER

Figure 1. Overview of the GH / IGF-I axis. GH secretion from the anterior pituitary is
regulated by the stimulatory influence of GH-releasing hormone (GHRH) and the
inhibitory effect of somatostatin (SRIF), both produced in the hypothalamus. When GH
binds to hepatic GH receptors, the IGF-I peptide is synthesized and secreted by the liver.
High concentrations of circulating IGF-I may provide a negative feedback mechanism by
working through SRIF and GHRH to decrease GH production from the pituitary. IGF-I
found in the mammary gland is locally produced and / or travels to the gland via
circulation. IGF-I receptors are found on mammary epithelial cells; however, this cell
type does not produce IGF-I. IGF-I is produced in the stromal portion of the gland.

15

IGF Ligands, Receptor Structure and Loom:

IGF-I receptors are found on mammary epithelial cells as well as on pre-
adipocytes, myoblasts, chondrocytes, mesenchymal cells, fibroblasts, endothelial cells,
and hepatocytes (McCusker, 1998). Type I and 11 receptors have been found in the
mammary glands of cows (DeHoff et al., 1988). Bovine mammary epithelial cells
express IGF system receptors (IGF-I, -II, and insulin), do not produce IGF-I (Hadsell et
al., 1990), and do secrete IGF-II (Baumrucker et al., 1993). IGF-I is produced in the
stromal portion of the gland; therefore, a paracrine role for IGF-I action for the
epithelium has been suggested (Hauser et al., 1990). However, data from Hodgkinson
and co-workers (1991) suggests that blood is the major source of IGF-I in the mammary
epithelium.

IGF-I has a molecular weight of 7.6-kD with approximately 70 amino acids
(Wong et al., 1989). This ligand is composed of four domains on a single peptide chain
(Thissen et al., 1994). The IGF-I molecule shares identical amino acid sequence in the
bovine, human and porcine, and differs in the ovine by one amino acid and murine by
five amino acids (Wong et al., 1989). The liver is the primary source of circulating IGF-I
in the mouse (Sjogren et al., 1999) and other animals (Daughaday and Rotwein, 1989).
Many tissues produce the IGF-I peptide including heart, lung, skeletal muscle and the
gonads (D’Ercole et al., 1984). Weber and co-workers (1999) measured IGF-I
concentrations in mammary extracts and serum of heifers, finding averages of 34 and 107
ng/ml, respectively. The abundance of IGF-I in many different organs has led to the

suggestion that IGF-I may act in an endocrine, autocrine, and/or paracrine manner.

16

The type I IGF receptor is a heterotetramer and has high homology with the
insulin receptor. The receptor consists of two extracellular a subunits that contain the
ligand-binding domain where signal transduction is initiated by the ligand binding to the
domain’s cysteine-rich regions (review by Thissen et al., 1994). Each of these (1 subunits
is linked via disulfide bonds to a [3 subunit that houses both the transmembrane domain
and the intracellular tyrosine kinase catalytic domain. This tyrosine kinase activity is
located within the cytoplasm. Ligand binding causes autophosphorylation on tyrosine
residues and propagation of cytoplasmic signals. The Type I IGF-receptor binds IGF-I,
IGF-II and insulin. The Type I receptor has a 10-fold higher affinity for IGF-I than IGF-
II, and a 1000-fold lower affinity for insulin than IGF-I. The Kd for IGF-I is 1 nM. The
IGF-I and insulin receptors are both azfiz heterotetramers and have a high degree of
sequence homology. The insulin receptor binds insulin with highest affinity, IGF-I with
IOO-fold lower affinity, and IGF-II with an even lower affinity.

The Type II IGF receptor, also known as the mannose-6-phosphate receptor, is a
300-kD monomeric transmembrane glycoprotein (Thissen et al., 1994). This receptor
binds IGF-I with a 500-fold lower affinity than IGF-II and does not bind insulin.

Des (1-3) IGF-I and Long R3 IGF-I are two analogs of IGF-I that have a reduced
affinity for IGFBP. Des (1-3) IGF-I lacks the N-terminal tripeptide Gly-Pro-Glu and
shows similar or increased potency compared to normal IGF-I (Ross et al., 1989). The
Long R3 form of IGF-I has a substitution of an Arg for a Glu at position 3 and has a 13
amino acid extension peptide at the N terminus (Francis et al., 1992; Tomas et al., 1993).

The IGF-I ligand can be found in the circulation in three different forms. Little

free IGF-I is found in the circulation. Approximately 80% of all circulating IGF-I is

17

bound to IGFBP. Eighty percent of this bound IGF-I is part of a 140-kD ternary complex
[with the acid-labile subunit (ALS) and either IGFBP-3 or —5] and the other 20% is
bound to only binding proteins. These binary complexes are small enough to cross the
capillary endothelium, but the ternary complex is too large (Baxter, 1993). The half-life
of free IGF-I is extended from 10 min to 30-90 min when in binary complexes, and to

more than 12 hr when bound to the ternary complex (review by Boisclair et al., 2001).

Insulin-Lik:e Grth Factor Binding Proteins (IGFBP):

To fully understand the mechanism of IGF-I growth stimulation in mammary

 

development, the role of the IGF binding proteins must also be considered. The function
of a labile pool of IGF-I in the circulation is to provide an available source of IGF-I for
delivery to target tissues. Regulation of this labile pool is through the IGFBP. These
multifunctional proteins have the capacity to both inhibit and enhance IGF actions. The
IGFBP bind more than 80% of IGF in the circulation, either in binary or ternary
complexes. The binding proteins assist in transporting IGF-I to target organs and tissues
via the blood stream. Six high affinity BP (IGFBP 1-6) (Keifer et al., 1991) and more
recently, nine IGFBP-related proteins (IGFBP rP1-9) were characterized that bind to
IGFs with much lower affinity than the IGFBP (Hwa et al., 1999).

Bovine mammary epithelial cells from pregnant and lactating cows synthesize
IGFBP-2 (34-kD), IGFBP-3 (46 and 42-kD), IGFBP-4 (24—kD) and IGFBP-5 (30-kD)
(Gibson et al., 1999). All four of these binding proteins were detected in bovine blood,
milk and cell culture-conditioned media using western ligand blots. Also, Gibson and

others (1999) found that mRNA for IGFBP-3 is predominant in secretory epithelial cells

18

‘5'

 

l-‘i

versus other cell types. Mammary tissue extracts from prepubertal heifers contained
IGFBP-2 (32-kD), IGFBP-3 (40 to 43-kD), and IGFBP of 28-kD and 24-kD (putatively
IGFBP-1 and -4) are present in the heifer mammary gland (Weber eta1., 2000a).
Binding proteins function to extend the half-life of IGF-I in the circulation
(mainly IGFBP-3 associating with the ALS), transport IGF-I from vasculature to tissues
and localize IGF-I to specific target tissues and cells (Cohick, 1998). Binding proteins r
can have both stimulatory and inhibitory effects on the bioactivity of IGF-I (see Figure
2). Binding proteins may act by inhibiting the bioactivity of IGF-I through competition

with receptors, enhancing IGF-I activity by concentrating the growth factor on the cell

 

surface in receptor regions, prolonging the half-life of IGF-I in the circulation, interacting
with other grth factors, or by acting independently of IGF-I (Baumrucker and Erondu,

2000; Oh, 1998; review by Rajah et al., 1997).

19

 

\\ r/ t

 

In Circulation Putative IGFBP-3
Type! Receptor Receptor
Cell proliferation, 223:0" Oi 038k

differentation

Figure 2. The role of IGFBP in the IGF-I system. Approximately 80% of circulating
IGF-I is bound to IGFBP. IGFBP assist in transporting IGF-I to target tissues and in
prolonging the half-life of IGF-I. Free IGF-I is able to bind to the Type I receptor and is
involved in cell proliferation and cell differentiation. IGFBP can also inhibit IGF-I
actions by competing for binding with the receptor. In particular, IGFBP-3 is thought to
inhibit cell growth and may even cause apoptosis in certain cell types.

Both receptor numbers and circulating levels of IGF-I change over the lifetime of
the bovine. Expression of IGF-I is regulated by developmental factors. IGF-I
concentrations are low in the newborn calf and rise following birth (Skaar et al., 1994).
Plasma concentrations of IGF-I increased from 50 to 450 ng/ml from 1 to 45 weeks of
age. The postnatal rise in IGF-I is suggested to occur because of the appearance of
receptors in the liver and the onset of GH-dependent IGF-I release.

Concentrations of IGF-I and GH and IGFBP abundance continue to fluctuate
during adult life, depending on the physiological state of the cow. Growth hormone
concentrations increase during early lactation when milk yield is high and increasing.

Serum IGF-I concentrations and liver IGF-I mRN A abundance are low during early

20

lactation, increase as lactation persists and concentrations are high during the dry period
(Shanna et al., 1994; Vicini et al., 1991). IGF-I blood concentrations are inversely
related to milk production (Ronge et al., 1988; Vega et al., 1991). This is opposite of
IGFBP-2, for which concentrations are higher in early versus late lactation (Cohick,
1998). During pregnancy, protease activity either appears or significantly increases,
leading to degradation of IGFBP, especially IGFBP-3 (Hossenlopp et al., 1990).

During feed deprivation, concentrations of serum IGFBP-3 and IGF-1 are
generally decreased, while IGFBP-1 and IGFBP-2 are increased. These results are
similar to the profile during early lactation (see above). The GH / IGF-I axis is said to be
uncoupled during severe feed restriction and this restriction may even abolish the ability
of GH to increase IGF-I (review by Bauman, 1999). In summary, many of these studies
show a general positive relationship between IGF-I and IGFBP-3 and an inverse

relationship between IGF-I and IGFBP-2.

IGFBP-3:

Insulin-like growth factor binding protein-3 constitutes the majority of IGFBP in
the serum of heifers (McGrath et al., 1991) and in bovine mammary tissue extracts
(Weber et al., 20003). This binding protein has 6 to 24 times the circulating half-life of
IGFBP-2 or IGFBP-4 (McGuire et al., 1998). IGFBP-3 serum levels are normally
positively correlated with IGF-1. Because of the above information, much research has
focused on the relationship between IGFBP-3 and IGF-1.

IGFBP-3 is identified as a doublet of proteins approximately 38 and 42-kD. In

the circulation, IGFBP-3 forms a 140-kD ternary complex with the ALS and with either

21

IGF-I or IGF-II. In 1997, Erondu and colleagues cloned the bovine IGFBP-3 gene, a
single copy gene. This gene is approximately 10 kb and is divided into 5 exons with the
fifth containing a 3’ untranslated region. The gene contains a TATA box, a transcription
start site, a GC rich sequence element, and two overlapping AP-2 binding elements, and
does not have a CAAT box. Using transfection techniques, 80% of the basal promoter
activity was shown to be located in the first 130 bp of the 5’ end and this region
responded to IGF-I.

IGF-I is a potent regulator of IGFBP-3. Conditioned media from MAC-T cells
(Cohick and Turner, 1998) and primary epithelial cells GVIcGrath et al., 1991) treated
with IGF-I showed greater IGFBP-3 secretion into media compared to cells treated with
basal medium (BM). Exposure of cells to IGF-I increased the half-life of IGFBP-3. IGF-
I activates transcriptional and post-transcriptional mechanisms to stimulate IGFBP-3
synthesis in MAC-T cells (Cohick et al., 2000).

IGFBP-3 displays both a stimulatory and an inhibitory effect on proliferation of
mammary epithelial cells. The ability of IGF-I to stimulate DNA synthesis is increased
when MAC-T cells are transfected with IGFBP-3 (Cohick, 1998). However, in placental
fibroblasts, IGFBP-3 is shown to inhibit IGF-I stimulation of DNA synthesis in a dose-
dependent manner (Rodgers et al., 1996). When IGFBP-3 is added in culture, it is
probable that it binds to IGF-I and reduces the chance for IGF-I to bind to IGF-I receptors
on epithelial cells (Oh, 1998; Rajah et al., 1997). When IGF-I is allowed to bind to its
own receptor, it causes a stimulation of cell proliferation or differentiation (see Figure 2,
p. 19). If IGFBP-3 binds to its own putative receptor, it causes an inhibition of cell

proliferation and in some systems, such as cancer cells, cell death can occur (Oh, 1998).

22

 

However, IGF-I binding to IGFBP-3 prevents these effects. Therefore, Rajah and co-
workers (1997) imply that IGFBP-3 can mediate apoptosis by both IGF-I-independent
and IGF-I-dependent mechanisms.

Limited evidence exists to support an IGF-I-independent effect of IGFBP-3 in
bovine mammary tissue. When rhIGFBP-3 was added as a treatment to mammary
epithelial cells in vitro, mitogenic activity of serum and IGF-1 treatments was abolished. E”
Addition of 40 and 800 ng/ml of IGFBP-3 to basal medium inhibited DNA synthesis of
bovine primary mammary epithelial cells by approximately 25% and reduced mitogenic

activity in mammary tissue extracts by 35% (Weber et al., 1999). Mammary epithelial

 

cells do not secrete IGF-I (Hadsell et al., 1990), showing that this inhibitory effect was t"
independent of IGF-I. Rodgers and co-workers (1996) transfected an IGFBP-3 gene into

mouse embryo fibroblasts that lacked an IGF-I receptor, resulting in a decrease in the

growth rate of the transfected cells. Grill and Cohick (2000) transfected MAC-T cells

with IGFBP-3. Basal DNA synthesis was lower for the transfected cells but these cells

responded more strongly to addition of IGF-I than did mock cells. Overexpression of

IGFBP-3 inhibits epithelial cell proliferation (Cohen et al., 1993). These results suggest

that the inhibitory effect of IGFBP-3 is independent of the IGF-I receptor dependent

pathway.

IGFBP-2:
IGFBP-2 may have a greater function in transporting IGF-I than previously
thought. Its role becomes important when IGFBP-3 is insufficient to bind all of the free

IGF-I (Zapf, 1995). IGFBP-2 is the predominant form of IGFBP secreted by bovine

23

primary mammary epithelial cells cultured in serum-free media (McGrath et al., 1991).
IGFBP-2 is secreted in a 34-kD form and is dimeric at 72-kD (Bourner et al., 1992).
Serum IGFBP-2 concentrations are increased when ruminants enter a catabolic state such
as early lactation or fasting (Sharma et al., 1994). IGFBP-2 concentrations are higher
during early lactation compared to the dry period and a trend exists for concentrations to

be higher during early versus late lactation. Administration of bST results in lower

 

F-
concentrations of serum IGFBP-2, in contrast to serum and mRNA levels of IGF-I ll
(Sharma et al., 1994; Vicini et al., 1991). When IGF-1 was upregulated by generating
transgenic rabbits carrying fusion genes of synthetic DNA coding for human IGF-I, the
increase in local hIGF-I peptide in milk also upregulated IGFBP-2 but not any other i“-

binding proteins (Wolf et al., 1997). Few studies have been published showing the

effects of IGFBP-2 on bovine mammary epithelial cells. Thus, the role that IGFBP-2 has

in mammary development and the IGF-I system is unclear.

Other Growth Factors and Hormones:

Many growth factors and their receptors have been identified within the bovine
mammary gland. Many cell types are abundant in the mammary gland. Each of these
cell types secrete and have receptors for different types of growth factors. This pool of
growth factors, acting on epithelial cells in both paracrine and autocrine fashions,
regulates mammary development. Some are thought to act synergistically with IGF-I,

and others are thought to inhibit cell proliferation independently of IGF-I.

24

Transforming Growth Factor-B (TGF-B):

Transforming growth factor -B (TGF-B) mRNA has been identified in the
mammary gland (Maier et al., 1991; Plant, 1993). The TGF family can be sub-divided
into three groups: TGF-BS, activins and bone morphogenetic proteins (BMP). TGF-l3 is a
homodimer of two 12 tol 5-kD subunits linked by a single di-sulfide bond, or it is
released as a biologically inactive precursor containing a dimer of the N-terminal pro-
region / LAP (latency-activated) protein. There are three forms of TGF-B. The mature
forms have 9 conserved cysteine residues.

Physiologically, TGF-B1 has a biphasic response. That is, depending on the
concentrations used in cell culture, TGF -01 can be either inhibitory or stimulatory to cell
proliferation. When used as a treatment on primary bovine mammary cells (Purup et al.,
2000), lower concentrations of TGF-[31 (25, 50 pg/ml) stimulated proliferation of cells,
while higher concentrations (>100 pg/ml) inhibited DNA synthesis. Addition of TGF-B1
to cells inhibited the mitogenic response of IGF-I. Purup suggests that the bi-phasic
response in vitro of TGF-B] may have a concentration-dependent role in mammary
growth. TGF-[31 was implanted into the mammary gland of mice (Daniel etal., 1989). In
pregnant mice, TGF-B1 inhibited ductal elongation and inhibited DNA synthesis in the

epithelitun, but not in the stroma.

Leptin:

Leptin is a 16-kD protein secreted by white adipocytes and MAC-T cells
(Houseknecht et al., 1998; Smith and Sheffield, 2002). Leptin is involved with the

regulation of food intake, energy expenditure and energy balance via interaction with

25

neurotransmitters (Houseknecht et al., 1998). Secretion of leptin is highly correlated with
body fat mass and adipocyte size. Insulin and cortisol stimulate expression of leptin.
Leptin is found in the circulation bound to proteins that may modulate the function of this
hormone and its half-life. Leptin receptor mRN A expression has been shown in tissues
such as kidney, lung, liver, and muscle (Jensen et al., 1999).

Leptin is of interest to scientists studying mammary development because of its

F
suggested role in nutritional regulation, especially by dietary energy, of mammogenesis. 5
Recently leptin receptor expression and protein were characterized in bovine mammary :
and other tissues using reverse-transcription PCR (RT-PCR) and immunohistochemical
staining, respectively (Silva et al., 2001). Physiological concentrations of leptin (2 to 6 ‘—

 

ng/ml) reduced the mitogenic effect of IGF-I on MAC-T cells (Silva et al., 1999). Leptin
is suppressed during infusion of rhIGF-I in normal rats (Boni-Schnetzler et al., 1999).
This suppression was due to a reduction in circulating insulin leading to increased fat
mobilization. This study also showed that leptin was not suppressed by rhGH, suggesting
that it is unlikely that GH regulates leptin in the rat model. However, when added with
insulin or dexamethasone in vitro, GH attenuated the ability of insulin or dexamethasone
to stimulate leptin expression in adipose tissue explants taken from young cattle
(Houseknecht et al., 2000). This study also reported that young cattle treated with GH
showed increased leptin concentrations in adipose tissue. Leptin abundance was

positively correlated to adipose tissue IGF—I mRNA.

26

Fibroblast Growth Factor (FGF):

Fibroblast growth factor (F GF ) is a heparin binding protein and was purified from
prepartum bovine mammary gland secretion (Lametsch et al., 2000). The isolated cDNA
is 1170 bp and the theoretical mass of the protein is 22.5-kD. The F GF family of growth
factors is involved in tissue remodeling, angiogenesis and morphogenesis. This family
has a dual-receptor system consisting of four high affinity tyrosine kinase receptors and a
low affinity heparan sulfate proteoglycan (HSPG). FGF alone exhibited a dose-
dependent mitogenic activity when used as treatment on bovine primary mammary
undifferentiated cells (Sandowski et al., 1993). This mitogenic effect was synergistic
when IGF-I was added. Also, mammary stromal cells have been shown to be stimulated

by acidic FGF (aFGF), as well as by IGF-I (Hovey et al., 2001).

Kgatinogyte Growth Factor (KGF):

KGF, produced by the stroma, modulates stromal-epithelial interactions (Rubin et
al., 1989). Mammary epithelial cells respond differently to increasing doses of
keratinocyte growth factor (KGF). Media supplemented with 10 ng/ml KGF increased
DNA synthesis in MAC-T cells by 390% (Hovey et al., 2001). However, at higher
concentrations of KGF (50 to 100 ng/ml KGF), proliferative responses were not different
from those seen at 0 ng/ml KGF. Ovine stromal cells did not respond to KGF but did
proliferate in response to IGF-I and IGF-II. KGF mRNA is detected in bovine mammary
stromal cells but not in mammary epithelial cells (Hovey et al., 2001). In this same
study, the mammary fat pad (MFP) had significantly higher levels of KGF mRNA than

the epithelium-cleared fat pad.

27

I “C ‘ “Fifi.

 

The above results show the importance of the interactions between the stromal
and epithelial portions of the gland, and the paracrine actions of many growth factors

within the mammary gland.

Estrogen:

For many years, estrogen has been thought to stimulate mammary ductal growth
and progesterone to stimulate lobulo-alveolar development (Reece, 1956). In 1993,
Purup and co-workers ovariectomized prepubertal heifers at 2.5 months of age and
slaughtered at 9.5 months of age. Ovariectomized heifers had a greater percentage of
stromal tissue and a lower percentage of epithelial tissue mass. Treatment of intact
heifers with GH increased mammary tissue by 16%, but this treatment did not affect
ovariectomized heifers. Therefore, this study suggested that exogenous GH had no effect
in the absence of estrogen. In another ovariectomy study, ewe lambs given exogenous
estrogen had higher levels of IGF-I mRNA expression in the mammary fat pad (Hovey et
al., 1998b). Recent evidence has shown that parenchyma removed fiom heifers treated
with estrogen and GH had increased incorporation of [3 H] thymidine into DNA than
controls (Berry et al., 2001). This study also showed that estrogen treatments increased
IGF-I concentrations in parenchyma and the mammary fat pad tissues. These studies
confirm the importance of ovarian secretions in mammary development and growth.
However, estrogen is incapable of stimulating growth of mammary epithelium in vitro
(Oka et al., 1991) similar to progesterone, growth hormone, prolactin and thyroxine.

These hormones have been shown to be involved in the growth of the gland during

28

pregnancy by using in vivo ablation and hormone replacement experiments (N andi,

1958).

Summary:

Many growth factors and hormones regulate mammary development in heifers.
As the mammary parenchyma extends into the surrounding fat pad, regional differences
in its anatomy become evident. These regions later differentiate into distinct structures in
the mature gland. Locally-acting growth factors contribute to parenchymal tissue
development, although growth regulation is poorly understood. The objective of this
study was to determine if different regions of the developing mammary parenchyma
contained different amounts of mitogenic activity. The most potent mitogen known for
bovine mammary epithelial cells in vitro is IGF-I. Both concentrations of IGF-I and
leptin and a profile of the IGF-binding proteins were investigated to evaluate their role in

the overall mitogenic activity within each region.

29

MATERIALS AND METHODS

Preparation of Mammy Extracts:

Purchased Holstein heifers were weighed, stunned by captive bolt and slaughtered
by exsanguination at the Michigan State University Meats Laboratory. Heifers (n=3)
weighed 202, 205 and 232 kg at slaughter. Reproductive tracts were dissected to confirm
that heifers were prepubertal. Blood samples were collected from two of the three
heifers. After exsanguination, mammary glands were removed and transported to the
laboratory for dissection. The exterior surface of mammary glands was cleaned with
ethanol, and sterile blades were used to dissect the gland. Each glandular quarter was cut
open to expose the teat cistern, parenchyma and fat pad. The parenchyma was visually
separated into three sections: 'proximal', the third of the parenchyma closest to the teat
cistern; 'distal', the third of the parenchyma closest to the fat pad; and 'intermediate', the
parenchyma between the proximal and distal sections. Tissue was taken from each of
these three sections, placed in WhirlPak bags and fiozen at -80°C.

At a later date, 5 g of parenchymal tissue, each from proximal and distal regions,
was thawed and diced into tared beakers containing 12 ml of saline (0.9% sodium
chloride). Tissue was homogenized for 1 min using a Polytron (Switzerland, PT 10 20
350D). The tip of the Polytron was rinsed between samples with 3 ml of saline, which
was then combined with the homogenate to yield a total of 15 ml, with a 3:1 saline to
tissue ratio. Homogenate was placed into 50-ml tubes and shaken at 4°C for 90 min.
Homogenate was filtered through a double layer of gauze into 35-ml Beckman tubes

(Beckman Coulter, Fullerton, CA) and centrifuged (Sorvall Instruments, RC5C) at 10,000

30

 

x g and 4°C for 45 min. The supernatant was transferred to 10-ml Beckman tubes and
ultracentrifuged (Beckman, L-80) at 105,000 x g and 4°C for 60 min. Supernatant was
recovered, sterile filtered through a 0.22-micron filter and stored at -80°C until use in cell

culture experiments. Approximately 7 ml of mammary extract was recovered after these

centrifirgation steps.

MAC-T Cell Culture Experiments:

Cells used for all cell culture experiments were MAC-T cells (Huynh et al., 1991),
a transfected mammary epithelial cell line prepared from mammary tissue from a
lactating dairy cow. MAC-T cells of a single passage were grown up for use in
mammary extract experiments. Basal medium (Dulbecco’s Modified Eagle Medium,
DMEM/F12, Invitrogen) containing HEPES (Invitrogen, Carlsbad, CA, 7.4 g) and
sodium bicarbonate (cell culture grade, Sigma, St. Louis, MO, 2.478 g), was titrated to a
pH of 7.3 and sterile filtered. Basal medium (BM) also contained soybean trypsin
inhibitor (Type I-S, Sigma, 1 mg/L), transferrin (apo-bovine, Sigma, 5 mg/L), glutathione
(Sigma, 1 mg/L), insulin (bovine, 0.01 mg/L), sodium selenite (Sigma, 5 mg/L),
antibiotic/ antimycotic (Invitrogen, 10 ml/L), gentamicin(1nvitrogen, 2.5 ml/L), and
bovine serum albumin (BSA) (Invitrogen, 2.625 g/L). MAC-T cells were plated at 1 X
104 cells per well in a 24-well plate. Cells were plated on top of a collagen monolayer
(Type I, Becton Dickinson, Bedford, MA) in a solution containing BM supplemented
with 10% fetal bovine serum (FBS, Invitrogen). After 24 h in culture, medium was
aspirated, cells were washed with BM, and medium was replaced with 1 ml of BM.

Treatments were prepared and applied to the cells after 72 h in culture. All treatments

31

were done in triplicate allowing 8 different treatments per 24-well plate. Each plate
contained control treatments of BM and F BS at 10% concentration in BM. Treatments
included increasing amounts of IGF-I (0.25, 0.5, 1, 2, 4 and 8 nyml) and extracts of
mammary tissue fiom each of three heifers. For each of these three heifers, extracts from
proximal and distal regions were added at 1, 3 and 5% concentrations in BM.
Concentrations at 1, 3 and 5% were chosen after analysis of effects on cell proliferation
of a dose-response series of 1, 3, 5, 7 and 9% mammary extracts (see Results). Cell
proliferation was evaluated at 18 h after treatment addition by [3 H] thymidine
incorporation, at 18 h by total DNA assay and at 40 h by total DNA assay. An IGF-I
dose-response series was included in this experimental model as a test for normal cell
response to increasing doses of IGF-I. Basal medium was included as a control for the
basal level of cell proliferation and was included in each assay. The three assays were

each replicated on three different dates.

l3Hl Thymidine Incorporation Assay:

After 16 h of treatment, 1 uCi (2.2 x 10‘5 DPM) of [3H] thymidine (ICN
Pharmaceuticals, Irvine, CA) was added to each well in plates for analysis of [3H]
thymidine incorporation. After 2 h of incubation of cells with [3 H] thymidine, media was
aspirated and cells were exposed to a series of washes: Hanks’ Buffered Salt Solution
(HBSS, Sigma), 90% ethanol, 10% trichloroacetic acid (TCA, Sigma), and an additional
90% ethanol wash. Then wells were allowed to dry for 5 min. Lysing buffer (250 pl /
well, 0.5 M NaOH, 0.1% triton, Sigma) was added and plates were gently rocked for 30

min. A lOO-ul sample was taken from each well and placed into a scintillation vial

32

(Research Products International (rpi), Mount Prospect, IL). Scintillation cocktail (5 ml,
Safety-Solve, rpi) was placed into each vial and [3 H] was quantified on a beta counter
(TM Analytical Mark V 32 ARI). Calculated counts per minute (CPM) were adjusted to

disintegrations per minute (DPM) assuming 40% efficiency of the counter.

Total DNA Assay:

Eighteen hours after treatments were applied, media fi'om each well was aspirated
and cells were washed with 1 ml phosphate buffered saline (PBS, Invitrogen).
Ethylenediaminetetraacetic acid (EDTA, 1.4 ml / well, 10 nM solution; J .T. Baker,
Phillipsburg, NJ) was added and plates were incubated for 20 min. After this incubation
period, KH2P04 (100 pl / well, 1 M potassium phosphate; J .T. Baker) was added as a
neutralizer and pH was measured. Plates were then kept on ice and cells were sonicated
(Sonifier Converter, Model L, Branson Sonic Power, Danbury, CN). DNA standards (0,
0.624, 1.25, 2.5 and 5.0 ug/ml) and samples (160 pl) were pipetted into a black 96-
microwell plate (Dynex Microfluor, Chantilly, VA). Hoechst No. 33258 dye was mixed
with buffer (1 nM NaCl, 10 nM Tris) and added to each well (Labarca and Paigen, 1980).
The fluorescence of this dye was measured (CytoFluor, PerSeptive Biosystems,
Cambridge, England) as a quantification of total DNA per well. This procedure was

repeated on different plates 40 h after treatment addition.

Total Protein Assay:
The bicinchoninic acid (BCA) protein assay kit (BCA-200, Pierce, Rockford, IL)

was used to detect and quantitate total protein contained in the mammary extracts. A

33

dilution of 1:20 (mammary extractzdeionized H20) was used. Standards (BSA: 0, 250,
500, 750, 1000 pg / ml) and mammary extracts were pipetted (25 pl) onto a clear 96-
microwell plate. A working reagent (200 pl) was added that catalyzed a reduction of
Cu+2 to Cu+1 by protein in the sample. A reagent containing bicinchoninic acid was used
to colorimetrically detect the cuprous cation (CuH) product formed by the chelation of
two molecules of BCA with one cuprous ion (CuH). Plates were incubated for 30 min at
37°C and the purple-colored product was colorimetrically quantitated using a plate reader
(562 nm, SpectraMax 340, Molecular Devices, Sunnydale, CA) after cooling to room

temperature.

IGF-I Radioimmunoassay (RIA):

IGF-I was measured in mammary extracts and blood serum. Binding proteins
were separated from IGF-I by two different methods, acid-ethanol cryoprecipitation and
size exclusion gel column chromatography. Acid-ethanol cryoprecipitation was similar
to that described by Breier et a1. (1991). A HCl / ethanol mixture (87.5% ethanol, 12.5%
HCl (2 M), vol/vol) was added to each sample at a 4:1 ratio (1.2 ml HCl / ethanol at —
20°C and 300 pl of mammary extract). This mixture was vortexed, stored at -20°C for 1
h and then centrifuged at 4000 x g for 30 min at 4°C. A 750-pl aliquot of the supernatant
(containing IGF-I) was transferred to a 2-ml eppendorf tube and neutralized using Tris
base (300 pl at 0.855 M) at a 5:2 ratio. This mixture was incubated at -20°C for 1 h,
centrifuged at 4000 x g for 30 min at 4°C, and the supernatant (300 pl) was recovered.
The serum samples were reconstituted with 900 pl of deionized H20.

Extracted samples were assayed at 50 and 100 pl to check the parallelism of the

34

assay and of the extraction procedure. Samples were assayed in duplicates. Sample
volumes were equalized to 100 pl with a Tris based neutralizing buffer [53.5%
ethanol/HCL (87.5% ethanol, 12.5% 2 M HCl), 28.6% 0.855 M Tris, 17.9% deionized
H20]. Samples were reconstituted to 200 pl with a PBS-based neutralizing buffer (0.1 M
NaPO4, 0.01 M EDTA, 0.003 M Na Azide, 0.005% Tween 20; pH to 7.5).

Controls included 100-pl serum samples that were high (H), low (L) and very low
(LL) in IGF-I concentration. These serum samples were extracted to remove IGFBP
similar to the mammary extract samples. Extracted serum samples were analyzed at 100
and 200 pl per tube. These serum samples were used to standardize the assay. Non-
specific binding controls consisted of 200 pl of neutralizing buffer (1 part Tris
neutralizing buffer : 1 part PBS neutralizing buffer) mixed with 250 pl assay buffer (0.03
M NaH2PO4, 0.01 M EDTA, 0.02% Na Azide, 0.005% Tween 20, 0.02% protamine
sulfite; pH to 7.5). A standard curve was included in the assay, with IGF-I concentrations
of 0, 25, 50, 100, 200, 400, 800, 1600, 3200 and 6400 pg/ml. These standards were
assayed in duplicates.

The 125I-IGF-I isotope was diluted in assay buffer, so that 50 pl of the dilution
would contain approximately 20,000 CPM. A rabbit anti-human IGF-I antisera (Gro-
Pep, Adelaide, SA, Australia) was diluted with assay buffer 125,000 and 250 pl of
diluted antiserum was added to each sample for a final dilution of antiserum at 1:50,000.
This antibody was added to all tubes except the non-specific binding controls and total
count tubes. Samples were then vortexed and incubated overnight at 4°C.

Protein A (Staphylococcus aureus, Roche, Switzerland; Zymed, San Francisco,

CA) was added the next day at 0.001 g/tube and tubes were vortexed. After a 2 h

35

incubation at room temperature, 2 ml of assay buffer was added to each tube. Tubes were
centrifirged for 30 min at 3070 x g, liquid was decanted and radioactivity of the pellet
was quantified in a gamma counter (Cobra II Auto Gamma, Packard BioScience Co,
Dowers Grove, IL).

The second separation procedure, size exclusion gel column chromatography, was
similar to that used by Mohan and Baylink (1995). This method used a Sephadex G-50
(Pharmacia, Piscataway, NJ) gel column to separate proteins into different fractions
based on their molecular weight. The G-50 gel column has a fractionation range of 1.5 to
30-kD, indicating that any proteins greater than 30-kD are excluded from the gel and pass
through the gel in the liquid phase. Proteins of molecular weights within the
fractionation range penetrate the gel particles and elute from the column in order of
decreasing molecular size. Binding proteins predominant in the heifer mammary gland
range from 24 to 43-kD in size. Therefore, most of these binding proteins should be
excluded from the gel and elute first from the column.

Samples (200 pl) were acidified with 800 pl of 1.25 M acetic acid containing
0.125 M NaCl, incubated for 15 min and added to the column. The eluant solution
contained 1 M acetic acid and 0.1 M NaCl. The volume of each fraction was determined
after characterization of the column using samples of BSA (molecular weight = 60 kD;
and 125I-IGF-I (molecular weight = 7 kD; IGF-I volume). The amounts of each fraction
collected were as follows: ‘A’ (0-12 ml), ‘B’ (12-20 ml), ‘C’ (20-29 ml), ‘D’ (29-38 ml),
‘E’ (38-45 ml), and ‘F’ (45-54 ml).

A 1.8 ml sample from each collected fraction was dried (Heto Type VR-l Speed

Vacuum, Appropriate Technical Resources, Laurel, MD) to concentrate the sample and to

36

eliminate the acid. Samples were re-constituted in buffer (PBS neutralizing buffer) so
that original mammary extracts and serum samples were diluted by a factor of 20 and 40,
respectively. As a control for recovery of IGF-I from the column, 6 ng and 2 pg of IGF-I
was added to 1 ml of mammary extract and BSA, respectively. Samples with added IGF-
I were acidified and added to the column. In addition, recovery of IGF-I from the RIA
was tested by adding 7.5 ng of IGF-I to 1 ml of serum just prior to assaying the sample.
Mammary extracts were assayed at 50, 100 and 200 pl and serum samples were assayed
at 25, 50 and 100 p1 to confirm the parallelism of the assay. Volume of samples was
equalized with buffer (PBS neutralizing buffer) to 200 pl. The remainder of the assay
was similar to the RIA protocol as stated previously. Deviations from this assay included

the use of only the PBS neutralizing buffer and not the Tris-based neutralizing buffer.

Western Lgand Blot:

Western ligand blotting was used to analyze the relative abundance of IGFBP in
mammary extracts. This procedure was also used to confirm the absence of IGFBP in the
IGF-I fractions that were collected using the Sephadex G-50 gel column. A 12.5%
acrylamide running gel solution for four gels [24.9 ml monomer solution (30.8%
acrylamide, 2.7% bisacrylarnide), 15 ml 4X running gel buffer (1.5 M Tris-Cl, pH 8.8),
600 pl 10% SDS and 19.2 ml deionized H2O] was prepared and degassed. Tetramethyl-
ethylenediamine (TEMED, 20 pL, Eastman, Rochester, NY) and 10% ammonium
persulfate (300 pl, Sigma) solution was added to the degassed running gel solution.
Running gels were allowed to polymerize and the assembly was removed from the gel

apparatus and moved to the loading apparatus.

37

A 4% acrylamide stacking gel solution for four gels [2.64 ml monomer, 4.98 ml
4X stacking gel buffer (0.5 M Tris-Cl, pH 6.8), 198 pl 10% SDS and 12.18 ml deionized

H2O] was prepared and degassed. Immediately after TEMED (10 pl) and a 10%

ammonium persulfate solution (100 pl) was added to the stacking gel solution, the gel
was pipetted on top of the separating gel and a comb was inserted into each stacking gel.
The gel was then allowed to set up and the comb removed. Tank buffer (0.025 M Tris,
0.192 M glycine, 0.1% SDS, pH 8.3) was placed into each well and in the lower buffer
chamber.

The samples were prepared using equal parts of protein sample and 2X treatment
buffer (0.125 M Tris-Cl, 4% SDS, 20% glycerol, 0.02% bromophenol blue, pH 6.8). The
sample with treatment buffer was placed in a 70°C waterbath for 20 min to denature the
protein within the sample. The sample was cooled and loaded at a concentration of 200
pg protein per lane. A molecular ladder (BenchMark Prestained Protein Ladder,
Invitrogen) containing proteins from 10 to 190-kD was used as a marker for binding
protein sizes. Samples of IGFBP-2 (bovine, GroPep, Adelaide SA, Australia) and
IGFBP-3 (human recombinant, Upstate Biotechnology, Lake Placid, NY) were included
in the gels as positive controls for binding of [1251] IGF-I. The voltage remained constant
at 180 V throughout electrophoresis (75 min) and was stopped when the bromophenol
blue dye reached the bottom of the gel.

Gels and filter paper (BioRad, Hercules, CA) were placed into transfer buffer
(0.025 M Tris, 0.192 M glycine, 20% methanol, pH 8.3). Polyvinylidene difluoride
(PVDF, BioRad) membranes were cut to fit the size of the gels and pre-wetted in 100%

methanol before rinsing in transfer buffer. On the semi-dry transfer cell (BioRad

38

TransBlot SD), items were layered in the following order: filter paper, PVDF membrane,
gel and filter paper. Voltage was set constant at 25 volts for 160 min (BioRad PowerPac
200). Membranes were removed and placed in a Tris buffer [TBS with 1% BSA (RIA
grade, Sigma) and 0.1% Tween 20] for 1 h, incubated 12 h with 700,000 CPM [12511-1013-
1 per ml of buffer, washed 3X with TBS + 0.1% Tween 20 and 3X with TBS, and then
placed in a x-ray cassette (Autoradiography Cassette, Fisher Scientific, Pittsburgh, PA)
with two pieces of film (Kodak BioMax MS Film, Eastman). After approximately 24 and
48 h of exposure, each sheet of film was developed. Film was then scanned with a
densitometer (F luor-S Multilrnager, BioRad; Quantity One v4.1, BioRad) to quantify

differences in the density of the bands corresponding to various IGFBP.

Leptin Radioimmunoassay:

Serum and mammary extract samples were sent to Dr. Duane Keisler’s laboratory
at the University of Missouri. Leptin concentrations were determined as in Delavaud et
a1. (2000). A standard curve (0.08 to 4 ng) was included in the assay prepared from
recombinant ovine leptin. For samples, triplicate aliquots of 200 pl were assayed. Both
sample and standard tubes were incubated for 24 h at 4°C with 50 pl of a 1:1,500 dilution
of rabbit anti-ovine leptin antisera. After this incubation, tubes were incubated an
additional 20 hours after 20,000 CPM 125I-ovine leptin was added to each tube. Final
dilution of leptin antisera was 1:15,000. Bound and free leptin were separated by
addition of 100 pl of sheep anti-rabbit plasma and 2 ml of 4.4% polyethylene glycol,
followed by centrifugation. Radioactivity of the pellet was quantified with a gamma

counter (Cobra 11, Packard).

39

Statistical Analysis:

Statistical analysis of cell culture experiments was performed using the Mixed
procedure of SAS (SAS, Version 8.1, Cary, NC). The statistical model tested for the
main effect of parenchymal region and concentration. Random effects included heifer,
heifer*region interaction, and well (experimental date*heifer*treatment interaction).
Statistical differences were based on least square means (LS means) of
concentration*region. Linear and quadratic effects of mammary extract treatments were
analyzed across and within region. Coefficients of orthogonal polynomials for equally
spaced levels were used. Linear and quadratic effects of IGF-I treatments were analyzed
across and within treatments. Coefficients for non-equally spaced levels were determined
using Proc IML. For ligand blot analysis, contrast statements were used to test
differences in IGFBP density between parenchymal regions. Differences in protein
content were evaluated using the Tukey - multiple comparison test in Proc GLM.

Pearson correlations were calculated to determine the relationships between protein
content and mitogenic activity. Differences in IGF-I and leptin concentrations in
mammary extracts from different regions were analyzed using the Mixed procedure.
Statistical differences were based on LSmeans of region. Data are presented as LS means
i standard error of the means (SEM). Differences were considered significant at P s

0.05.

40

RESULTS

Cell Culture Experiments:

The main objective of the cell culture experiments was to evaluate the mitogenic
activity of mammary extracts of tissue collected from proximal and distal regions of the
mammary gland. The mitogenic capacity of mammary extracts for MAC-T cells plated
on collagen was analyzed using two different cell proliferation assays: [3 H] thymidine
incorporation assay and measurement of total DNA. An IGF—I dose response curve and
FBS were also included in assays to confirm that the MAC-T cells would respond to
known nritogens. Also, a dose response curve of mammary extracts was done in initial
experiments to determine which concentrations of extracts would be optimal for testing
the main objective.

MAC-T cells were treated with increasing doses of IGF-I (0.25, 0.5, 1, 2, 4 and 8
ng/ml) to test the cells’ growth response to a known mitogen. This dose range stimulated
cell proliferation in a dose-dependent manner (Figures 3, 4 and 5). Linear and quadratic
effects of IGF-I doses were significant in both total DNA assays (P<0.0001) and the
linear effect was significant in the [3 H] thymidine incorporation assay (P<0.0001). In all
three assays, IGF-I stimulated cell proliferation (P<0.01) at concentrations 2 0.5 ng/ml,
compared with BM. Addition of 8 ng IGF-I/ml stimulated cell proliferation 224%, 64%
and 78% in the [3 H] thymidine incorporation assay, total DNA assay at 18 h, and total
DNA assay at 40 h, respectively. A 10% concentration of FBS stimulated cell
proliferation 134%, 50% and 199% in the [3 H] thymidine incorporation assay, total DNA

assay at 18 h, and total DNA assay at 40 h, respectively.

41

15000

 

10000

 

DPM

5000

 

IGF-I (ng/ml)

Figure 3. Effect of increasing doses of IGF-I (0.25, 0.5, 1, 2, 4 and 8 ng/ml of media) on
[3 H] thymidine incorporation into DNA of MAC-T cells. Results are reported as DPM
(disintegrations per minute). Data are presented as LS means i SEM from three different
assays. Basal medium (BM) and 10% fetal bovine serum (FBS) were included as
controls. IGF-I treatments are different (P<0.001) from BM (0) at doses 2 0.5 ng/ml.

42

 

 

DNA (pg/well)

 

10% 0 0.25 0.5 l 2 4 8

IGF-I (ng/ml)

Figure 4. Effect of increasing doses of IGF-I (0.25, 0.5, 1, 2, 4 and 8 ng/ml of media) on
total DNA of MAC-T cells after 18 h. Results are reported as micrograms of DNA per
well. Data are presented as LS means i SEM from three different assays. Basal medium
(BM) and 10% fetal bovine serum (FBS) were included as controls. IGF-I treatments are
different (P<0.01) from BM (0) at doses 2 0.5 ng/ml.

43

 

 

 

 

 

DNA (pg/well)

 

 

10% 0 0.25 0.5 l 2 4 8

FBS
IGF-I (pg/ml)

Figure 5. Effect of increasing doses of IGF-I (0.25, 0.5, 1, 2, 4 and 8 ng/ml of media) on
total DNA of MAC-T cells after 40 h. Results are reported as micrograms of DNA per
well. Data are presented as LS means i SEM from three different assays. Basal medium
(BM) and 10% fetal bovine serum (FBS) were included as controls. IGF-I treatments are
different (P<0.001) from BM at doses 2 0.5 ng/ml.

Cells in all three assays were treated with increasing concentrations of mammary
extracts (1, 3, 5, 7 and 9% of BM) (Figures 6, 7 and 8). The purpose of this experiment
was to determine the concentrations of extracts that would be used in future experiments.
Equal amounts of mammary extracts processed from two different heifers and from both
proximal and distal regions were pooled for this experiment. Concentrations of l, 3 and
5% were chosen for use in later experiments. A dose of 1% mammary extract was
mitogenic in all three assays (P<0.001). Compared to BM, concentrations of 1, 3 and 5%
mammary extracts increased total DNA at 40 h by 38%, 122% and 159%, respectively.
The mitogenic activity of 5% extract after 40 h was not different from 7% (P=0.45) and
9% (P=0.12), showing a plateau effect (Figure 8). Therefore, 1% mammary extracts
gave a detectable mitogenic response and 5% mammary extracts showed a maximal
response after 40 h of treatment.

Subsequent assays focused on the treatment effects of mammary extracts at 3% of
BM because this concentration gave a response in the middle of the growth curve in the
total DNA assay at 40 h. Linear and quadratic effects of concentration (1, 3, 5, 7 and 9%)
were both significant (P<0.0001), thereby showing that there was a dose response and
also a plateau effect in this range of doses.

Both [3 H] thymidine incorporation and total DNA assays were performed for all
subsequent assays. However, the total DNA assay was used for further analysis because
the total DNA assay measures accumulation of DNA over 18 or 40 h, whereas the [3 H]
thymidine incorporation assay measures a snapshot of DNA synthesis during 2 h.
Because of this, the total DNA assay results are likely a more reliable indicator of cell

proliferation in response to treatment. The 18 h DNA assay was used to provide results

45

on DNA accumulation to complement the results of the [3 H] thymidine assay. MAC-T
cells responded in a dose-dependent manner to treatments of IGF-I but not to increasing
concentrations of mammary extracts in the [3 H] thymidine assay. A linear increase in
DNA synthesis (using a [3 H] thymidine assay) in response to increasing concentrations of
mammary extracts was noted using primary cells in a previous study (Weber et al., 1999),
however, it is not known why the MAC-T cells did not respond in a linear fashion. Thus,
conclusions of this study were based on the results of the 40 h DNA assay, to allow

measurement of treatment effects over a longer period of time.

46

 

 

Mammary Extract (% of media)

Figure 6. Effect of mammary extracts [1, 3, 5, 7 and 9% of basal medium (BM)] on [3H]
thymidine incorporation into DNA of MAC-T cells. Results are reported as DPM
(disintegrations per minute). Data are presented as LS means 1- SEM. BM (0) and 10%
fetal bovine serum (FBS) were included as controls. Marmnary extracts are different
(P<0.0001) from BM at concentrations of 2 1% of BM.

47

 

 

DNA (pg/well)

 

Mammary Extract (% of media)

Figure 7. Effect of mammary extracts [1, 3, 5, 7 and 9% of basal medium (BM)] on total
DNA of MAC-T cells after 18 h. Results are reported as micrograms of DNA per well.
Data are presented as LS means i SEM. BM (0) and 10% fetal bovine serum (FBS) were
included as controls. Mammary extracts are different (P<0.0001) from BM at
concentrations of 2 1% of BM.

48

 

 

 

 

DNA(pg/well)
o - N W -P- U1 0‘

 

Mammary Extract (% of media)

Figure 8. Effect of mammary extracts [1, 3, 5, 7 and 9% of basal medium (BM)] on total
DNA of MAC-T cells after 40 h. Results are reported as micrograms of DNA per well.
Data are presented as LS means i SEM. BM (0) and 10% fetal bovine serum (F BS) were
included as controls. Mammary extracts are different (P<0.001) from BM at
concentrations of z 1% of BM.

49

Results for extracts of tissue collected from three different heifers, each assayed
in three separate experiments, were pooled for statistical analysis. Mammary extracts at
1, 3 and 5% stimulated proliferation in a dose-dependent manner in the total DNA assay
at 40 h of treatment (Figure 9). Mammary extracts at 3% and 5% increased total DNA
108% and 122% respectively, compared to BM. Mammary extracts at 3% from proximal
regions increased total DNA more than those from distal regions (proximal = 3.74, distal
= 3.38 pg DNA/well; SEM = 0.11; P=0.02) (Figures 9, 10). In addition, 5% extracts
from proximal tissue tended (P=0.07) to be more mitogenic than those fiom distal
regions (Figure 9). In comparing the mitogenic response of mammary extracts to IGF-I,
mammary extracts at 3% stimulated cell proliferation 17% more than 8 ng of IGF-I/ml.

Significance existed for both linear (P=0.0001) and quadratic (P=0.0002) effects
at 40 h for both concentration of mammary extract and the interaction between
concentration and the parenchymal tissue region from which extracts were made. The
linear significance shows that there is a linear increase in mitogenic activity from 1, 3 and
5% mammary extracts. A quadratic effect indicates that the response is starting to
plateau, i.e. differences between 3 and 5% were smaller than between 1 and 3% extracts

(Figure 9).

50

 

 

 

 

 

 

E
i.
a; Proximal
3 I
< Distal
z I
Q

 

 

1 3 5

Mammary Extract (% of medium)

Figure 9. Effect of mammary extracts [1, 3 and 5% of basal medium (BM)] from
proximal and distal regions of the mammary gland on total DNA of MAC-T cells after 40
h. Results are reported as micrograms of DNA per well. Data are presented as LS means
i SEM from three different assays. Asterisks indicate treatment with extracts from
proximal regions increased DNA more than those from distal regions (at 3% of BM : *
P=0.02; at 5% of BM : T P=0.07).

51

 

 

DNA (pg/well)

 

Proximal Distal

Mammary Extract (3% of media)

Figure 10. Effect of mammary extracts [3% of basal medium (BM)] from proximal and
distal regions of the mammary gland on total DNA of MAC-T cells after 18 and 40 h.
Results are reported as micrograms of DNA per well. Data are presented as LS means i
SEM from three different assays. Asterisk indicates treatment with extracts from
proximal regions increased DNA more than those from distal regions (at 40 h: * P=0.02).

52

Coefficients of variation (CV) were calculated to test the variation within the cell
culture experiments. The average inter-assay variation was 5.2, 10.7 and 5.8% for [3 H]
thymidine incorporation assay, total DNA assay at 18 h and total DNA assay at 40 h,
respectively. Intra-assay variation was 14.3, 20.4 and 17.6% for [3 H] thymidine

incorporation assay, total DNA assay at 18 h and total DNA assay at 40 h, respectively.

Protein Content:

Protein content did not differ between mammary extracts (LS means = 12.6
mg/ml, SEM = 0.17; P=0.15). Basal medium contained bovine serum albumin (BSA) at
2.6 mg protein/ml of media. Addition of FBS to BM increased protein content an
additional 2.9 mg/ml of media. Addition of 1, 3 and 5% extracts to BM increased protein
content by 0.13 i 0.02, 0.38 :1: 0.04 and 0.63 i 0.07 mg/ml, respectively. Correlations,
using Pearson coefficients, were done between the protein concentration of extracts and
the mitogenic capacity of mammary extracts from different regions within each heifer.
This mitogenic activity was determined using data from total DNA assays at 40 h after
treatment addition, comparing extracts from different regions at 3% of media. Pearson
correlation coefficients did not show a strong correlation of extract protein concentration

with total DNA per well (r=0.05, P=0.55).

Abundance of IGFBP in Mammy Extracts:

Ligand blots were performed to determine the relative abundance of IGFBP in
each mammary extract (proximal and distal regions of three heifers). IGFBP-2 and

IGFBP-3 served as positive controls for [‘251]-IGF-I binding. The IGFBP-2 was clearly

53

visible on the autoradiograph (Figure 11); however, IGFBP-3 was not detected, possibly
because of degradation during storage. For statistical analysis, heifers were pooled so
that the main contrast compared extracts from proximal versus distal regions of the
mammary gland. A tendency existed for IGFBP-3 to be greater in distal than proximal
regions (P<0.06). Abundance of IGFBP-2, a 28-kD BP and a 24-kD BP did not differ by

region (P>O.1).

54

60-kD b

er 10’3
1 Q .1 . p' 4 43-kD
I Q o a» d 4 32-kD
1 a a a at 4 28-kD
' 4 24-kD

15-kD >

BP-3 444 444 452 452 510 510 BP-2
P D P D P D

Figure 11. Autoradiograph of a western ligand blot showing relative IGFBP abundance
in mammary tissue extracts prepared from proximal (‘P’) and distal (‘D’) regions of the
mammary parenchyma. ‘BP-3’ = IGFBP-3 standard, ‘BP-2’ = IGFBP-2 standard; ‘444’,
‘452’ and ‘510’ indicate three different heifers. Extracts (200 pg protein/lane) were
fi'actionated on a gel, transferred to a membrane and hybridized with [lzsfl-IGF-I. Mr =
relative molecular mass.

55

Table l. The effect of proximal (‘P’) and distal (‘D’) region of the mammary
parenchyma on the abundance of IGF-binding proteins (IGFBP) in mammary extracts.
Values are based on the densitometric units (Odu/mmz) recorded on the autoradiograph.
Data represent means for each region for three heifers.

 

IGFBP Mean P Mean D SE Probability

 

43-kD 54.9 70.6 5.2 0.06
32-kD 27.9 24.6 1.4 0.14
28-kD 28.0 24.3 1.8 0.17
24-kD 11.1 9.6 1.5 0.53

 

56

IGF-I Concentrations in Mammary Extracts:

Assays of samples that were separated by acid-ethanol cryoprecipitation did not
yield valid results due to problems with linearity of the assay over concentrations tested
and large coefficients of variation (>10%). However, RIAs using samples of fractions
from column chromatography yielded valid results. The RIA was linear when different
amounts of sample were assayed. Average inter-assay variation was 4.5 and 5.1%.
Average intra-assay variation was 9.4 and 8.0%.

The volumes of each fraction collected fi'om the column were as follows: ‘A’ (0-
12 ml), ‘B’ (12-20 ml), ‘C’ (20-29 ml), ‘D’ (29-38 ml), ‘E’ (38-45 ml), and ‘F’ (45-54
ml). The ‘B’ fraction contained the proteins that were greater than the upper limit and
those of greatest molecular weight within the fractionation range (1.5 to 30-kD). The ‘B’
fraction was identified by measuring the protein content of fractions collected after BSA
was applied to the Sephadex G-50 gel column (Figure 12). To capture the binding
proteins less than 30-kD, 2 to 3 ml additional were collected than the volume in the BSA
peak. The ‘C’ fraction contained the majority of 125I-IGF-I, as determined by
quantification using a gamma counter, and was used to characterize the fiaction that
would include the 7.6-kD protein, IGF-I. Two other peaks were noted during the
characterization of the column with the labeled ligand. The first peak (‘B’) likely

contained degraded 125I-IGF-I, while the third peak (‘E’) likely included free iodine.

57

l----A---||--B--ll--C--II--D--lI-E--||--F---l

 
  
 

 

   

 

200-. —~ 0.6 E
E
'7" 150! R 1’ 0'5 8
‘3 — 0.4 S
g 100~~ ~~o3 8

~~ 0.2
8 50+ 1 E
’0.1 [—

M "
0 4‘ $431.. 0.0 a
2 6 10 14 18 22 26 30 34 38 42 46 50 54 a.
FRACTION(ml)
—'-CPM—’—PROTEIN

Figure 12. Characterization of protein flow from Sephadex G-50 size exclusion gel
chromatography column using BSA and ”SI-IGF-I. The BSA contains proteins
(albumin) greater than the upper limit of the fractionation range (1.5-kD to 30-kD) and is
measured as “protein” in optical density (CD) as shown on the z-axis. The 125I-IGF-I
penetrates the gel particles, is eluted within the fractionation range, and was quantified
using a gamma counter where CPM = counts per minute on the y-axis. Each tick mark
represents a 4-ml fraction on the x-axis. Letters ‘A’ through ‘F’ represent the fractions
that were collected for all samples.

58

Concentrations of IGF-I in mammary extracts did not differ between proximal
and distal regions of the parenchyma (P=0.18). Mean IGF-I concentrations were 30.5
ng/ml for the proximal region and 27.9 ng/ml for the distal region (SEM = 2.2) of
mammary extract or 116.8 ng/ g of mammary parenchymal tissue. This calculation is
based on the 3:1 ratio of salineztissue used in preparation of mammary extracts. Serum
samples contained 231.9 i 17.3 ng/ml of serum.

Samples containing added IGF-I were used for validation of column
chromatography and the RIA. Thirty nanograrns of IGF-I were added to a mammary
extract sample before addition to the Sephadex column, and 108% (i 7) was recovered.
Also, 2 ng of IGF-I was added to a sample of 5% BSA and 113% (i 16) was recovered.
Recovery for 6 ng of IGF-I added to a serum sample just before the RIA was 97% (i 6).

The effectiveness of the IGFBP separation by column chromatography also was
evaluated by ligand blotting of fractions collected from the column. Fractions ‘A’, ‘D’
and ‘E’ did not contain IGF-I as quantitated in the RIA. The ‘B’ fraction containing
proteins greater than the upper limit and those of greatest molecular weight within the
fractionation range, should have contained the majority of the IGFBP. Assay of IGF-I
concentrations in the ‘B’ fraction by RIA did not yield linear results (i.e., increasing
amounts of sample decreased amount of bound [1251]-IGF-I in a manner that was not
parallel to the standard curve) over the range of amounts assayed. The ‘C’ fraction that
penetrated the gel particles contained IGF-I, similar to previous results in characterization
of the column using 125I-IGF-I, and yielded linear results in the RIA. Both ‘B’ and ‘C’
fractions were firrther characterized using ligand blots (Figure 13). The ‘B’ fraction for

both mammary extracts and serum samples contained four IGFBP, similar to results for

59

ligand blotting of mammary extracts. These included IGFBP-3, IGFBP-2 and lower
molecular weight IGFBP (28 and 24-kD). However, as expected, the subsequent ‘C’
fraction collected from the column for either mammary extracts or serum samples did not
contain detectable binding proteins. As positive controls for [1251]-IGF-I binding,
IGFBP-2 and IGFBP-3 were included on all blots. Also, a molecular weight ladder and a

non-fractionated serum sample were included on each gel.

60

60-kD
Mr x 10’3

443-kD

4 32-kD
4 28-kD

4 24-kD

lS—kD >

 

BP-Z BP-3 S 444 444 452 452 510 510
P D P D P D

60-kD >
Mr x 10‘1

4 43-kD

4 32-kD
4 28-kD

4 24-kD

lS-kD>

 

BP-Z BP-3 S 444 444 452 452 510 510
P D P D P D

Figure 13. Autoradiograph of a western ligand blot showing relative IGFBP abundance
in mammary tissue extracts from two different fractions collected from a Sephadex G-50
gel column. Panel A shows proteins from fraction ‘B’ and Panel B shows proteins from
fraction ‘C’. ‘L’ = molecular weight ladder; ‘BP-3’ = IGFBP-3 standard; ‘BP-2’ =
IGFBP-2 standard; ‘444’, ‘452’and ‘510’ indicate three different heifers; ‘P’ and ‘D’
refer to proximal and distal regions of the mammary parenchyma, respectively; ‘S’ =
serum. Mr = relative molecular weight. Extracts (200 pg protein/lane) were fractionated
on a gel, transferred to a membrane and hybridized with [1 5I]-IGF-I.

Leptin Concentrations in Mammy Extracts:

Leptin concentrations did not differ between proximal and distal regions of the
mammary parenchyma (P = 0.37). Mean leptin concentrations were 3.9 ng/ml for the
proximal region and 3.6 ng/ml for the distal region (SEM = 0.3) of mammary extract or
15.2 ng/ g of parenchymal tissue. Serum samples for two of the three heifers contained

3.6 (i 1.2) ng leptin/ml of serum.

62

Table 2. Summary table of overall results.

 

Proximal Distal % Change P value
MAC-T Assay (3%) Higher Lower 11.1 0.02
IGF-I Concentrations Higher Lower 10.9 0.18
IGFBP-3 Abundance Lower Higher 12.9 0.06
IGFBP-2 Abundance Higher Lower 11.3 0.14
28-kD IGFBP Abundance Higher Lower 1 1.5 0.17
24-kD IGFBP Abundance Higher Lower 11.6 0.53
Leptin Concentrations Higher Lower 10.8 0.37

 

63

DISCUSSION

A clearer understanding of prepubertal heifer marnmogenesis could lead to
techniques that enhance mammary growth, resulting in increased milk production and
profitability for the dairy producer. Future milk yield is positively correlated with the
number of mammary epithelial cells present and the activity of these cells (Broster and
Broster, 1984). Many stimulatory and inhibitory factors that modulate mammary
epithelial cell proliferation are produced within the mammary gland. Thus, greater
knowledge of the regulation of synthesis and action of these factors could enable
researchers to increase mammary cell proliferation. This study focuses on the mitogenic
activity within the mammary parenchyma and how this activity differs by region of the
heifer mammary gland.

To study the mitogenic activity of specific regions of the parenchyma within the
mammary gland, mammary extracts were prepared from tissue collected from proximal
and distal regions of the parenchyma. Mammary extracts of tissue from the proximal
region stimulated proliferation of MAC-T cells 11% more than extracts from distal
regions, as measured by total DNA content per culture well. Protein content of extracts
was not correlated to the amount of mitogenic activity in mammary extracts used as
treatments on MAC-T cells. The amount of increase in protein concentration in basal
medium with the addition of extracts was minimal. Thus, the greater mitogenic activity
in parenchyma fi'om proximal compared with distal regions is not explained by

differences in total protein content.

64

One of the factors known to stimulate mammary development is IGF-I. IGF-I
mRNA has been detected in stromal cells (Hauser et al., 1990). IGF-I is a potent mitogen
for the bovine MAC-T mammary epithelial cell line (Zhao et al., 1992, Woodward et al.,
1994) and for primary bovine (Shamay et al., 1988) and ovine (Winder et al., 1989)
mammary epithelial cells. MAC-T cells (Huynh et al., 1991) were chosen for this thesis
project because of the problems associated with harvesting a pure mammary epithelial
cell population fiom prepubertal heifers as described by Sharnay et a1. (1988) (see
Appendix). MAC-T cells proliferated in a dose-dependent manner in response to
increasing doses of IGF-I, similar to what has been shown previously (Zhao et al., 1992).
Mammary epithelial cells have receptors for both IGF-I and IGF-II (McCusker, 1998).
However, little is known about the mechanisms required for growth and differentiation of
tissue within the developing mammary gland.

The mammary extracts in this experiment ranged in IGF-I concentrations from
27.5 to 45.5 ng/ml of extract, which is within the range of those determined in a previous
experiment (Weber et al., 2000a). Concentrations of IGF-I did not differ within
mammary extracts from different parenchymal regions. These results are similar to
another study that focused on the mitogenic response of mammary epithelial cells to
mammary tissue extracts. Mammary extracts of tissue collected from heifers fed at a
high compared to a low rate of gain stimulated less [3 H] thymidine incorporation (Weber
et al., 2000b). Extracts of mammary tissue from these two groups of heifers did not
differ in concentrations of IGF-I (Weber et al., 2000a). In both our experiment and the
Weber study, IGF-I concentrations did not explain the differences in mitogenic activity of

the mammary extracts.

65

Separation and removal of IGFBP fi'om IGF-I is essential for samples to be
assayed accurately without interference by the IGFBP in the radioimmunoassay. Initially
an IGFBP extraction procedure, similar to the acid-ethanol cryoprecipitation used by
Breier et a1. (1991) was used. Results of this extraction procedure indicated that this was
not a valid extraction procedure for mammary extract samples. This is in agreement with
studies that have compared different separation procedures (Breier et al., 1991; Frey et
al., 1994; Gutierrez et al., 1997; Mohan and Baylink, 1995). Size exclusion gel
chromatography was noted to be an efficient method for removal of IGFBPs from IGF-I
in all four references. Thus, IGFBP were separated fiom IGF-I in the mammary extract
and serum samples by applying acidified samples to a Sephadex G-50 gel column. RIA
results showed that two different fiactions gave readings in the IGF-I assay. The ‘B’
fraction should have contained proteins that were greater than the upper limit and those of
greatest molecular weight within the fiactionation range (1.5 to 30-kD). This fraction
yielded IGFBP when tested on a ligand blot and results of increasing concentrations of
the ‘B’ fraction in a RIA showed non-linearity and high coefficients of variation. This
indicates that the IGFBP did interfere by cross-reacting with the antigen and competing
for binding to the antibody. The ‘C’ fraction contained IGF-I and no IGFBP were
detected by western ligand blotting. This separation procedure yielded a valid RIA of
samples, showing linearity over the concentrations tested with low inter- and intra-assay
variation. These results further confirm that if binding proteins are not removed
successfully, these proteins will interfere with an IGF-I RIA.

The majority of IGF-I in the circulation is bound to IGFBP (Baxter, 1993). Gene

expression of IGFBP by specific tissues may regulate their local effects on IGF-I, while

66

the availability of serum IGF-I may be regulated by alterations in serum IGFBP levels
(Lemozy et al., 1994). Binding protein -2, -3, and the 28-kD and 24-kD binding proteins
(putatively IGFBP-1 and -4) are present in the heifer mammary gland (Weber et al.,
2000a). In this study, ligand blots of mammary extracts displayed IGFBP with relative
molecular masses of 40 to 43 (IGFBP-3), 32 (IGFBP-2), 28-kD and 24-kD, as seen
previously (Weber et al., 2000a). Abundance of IGFBP-2, a 24-kD and a 28-kD IGFBP
within the mammary extracts did not differ with parenchymal tissue region. Abundance
of IGFBP-3 showed a strong tendency to be greater in distal compared to proximal
regions. Hence, parenchymal tissue region does not influence IGF-I concentrations in
mammary extracts but does influence local abundance of IGFBP-3 in the mammary
gland. These observations agree with the above suggestion by Lemozy and others (1994)
that IGFBP gene expression by the mammary gland regulates the local effects of IGF-I.
The IGFBP-3 is of major importance because it constitutes the majority of IGFBP
in serum of heifers (McGrath etal., 1991) and in bovine mammary tissue extracts (Weber
et al., 2000a). In addition, mRNA for IGFBP-3 is predominant in the secretory epithelial
cell compared to other tissue cell types within the mammary gland (Gibson et al., 1999).
IGFBP-3 may act independently of IGF-I in certain environments or under specific
conditions. Purup et a1. (2000) suggested that IGFBP-3 in mammary tissue inhibits IGF-I
activity while IGFBP-3 in serum potentiates IGF-I activity. When acting independently
of IGF-I, IGFBP-3 usually has an inhibitory effect on epithelial cell proliferation (Cohen
et al., 1993; Rodgers et al., 1996). Little evidence is available for the role of IGFBP-2 in
mammary development. IGFBP-l is thought to be an inhibitor of IGF-I action (Lewitt et

al., 1991). IGFBP-3 was more abundant in mammary extracts of tissue from the distal

67

region that inhibited total DNA by 11% compared to extracts of tissue from the proximal
region of the mammary parenchyma. This indicates that IGFBP-3 may have had an
inhibitory effect on MAC-T epithelial cell proliferation.

This study was the first to analyze the mitogenic activity of mammary extracts of
parenchymal tissue collected from different regions within the bovine mammary gland.
Two previous studies reported the activity of cells from different regions within the
developing gland. Explants of mammary tissue taken from prepubertal lambs were
incubated with [3 H] thymidine (Akers, 1990). The majority of labeled cells were located
at the periphery of groups of ducts. Microsomes from the distal parenchyma region of a
prepubertal heifer bound more radiolabeled IGF-I than microsomes fiom the proximal
parenchyma (Ellis et al., 2000). In addition, epithelial cells from the peripheral (distal)
region were more responsive to both IGF-I and TGF-B (Ellis et al., 2000). Results of
these two studies suggest that epithelial cells were proliferating faster on the distal
regions of the parenchyma. In the present study the mitogenic activity within the
parenchyma was evaluated, which includes the products of multiple cell types.
Parenchymal tissue consists of fibroblasts, epithelial and adipocyte cells, which produce
different mitogens and have receptors for different growth factors. Overall, mitogenic
activity is a measure of the difference between the effects of stimulatory factors minus
those that are inhibitory to cell proliferation.

Three major morphological differences between the distal and proximal regions
are 1) the vicinity of the region to the fat pad, 2) the abundance of different cell types
within the parenchyma of the two regions and 3) the regional structural differences that

become evident later in development. First, tissue in the distal region is closer in vicinity

68

to the fat pad than the proximal region. Therefore, the distal parenchyma is also closer to
possible stimulatory and inhibitory factors produced by adipocytes within the fat pad.
Second, the proportion of cell types differs between different regions within the
mammary gland (Sejrsen et al., 1982). Using tissue slices stained for light microscopy
for histological determination of individual cell types, this group determined that the
epithelial cells decreased from 11.9% to 9.7% (P<0.01) as distance fiom the base of the
gland or teat (i.e., proximal region) increased. The percentage of fat cells in the
parenchyma increased from 25.4% to 45.4% (P<0.001) as distance from the base of the
gland increased. Lastly, later in development, the parenchyma in the proximal region
likely differentiates into the primary ductal structures, whereas the distal region
differentiates into pre-alveolar structures (Ellis et al., 2000). Different factors that
influence gene expression must be present in the two regions to allow the parenchymal
tissue to differentiate into different structures.

Leptin, a factor shown to inhibit the stimulatory action of IGF-I, is produced by
adipocytes, mammary tissue, and MAC-T cells (Houseknecht et al., 1998; Smith and
Sheffield, 2002). Bovine mammary epithelial cells from prepubertal heifers display
receptors for leptin (Silva et al., 2001). Partly because of recent interest in leptin, much
research has focused on the function of the mammary fat pad in mammary development.
The fat pad in mice is crucial to normal growth and development of the mammary gland
(Hoshino & Martin, 1974). Mouse mammary epithelial cells (COMMA-lD cells) grew
when co-cultured with and without floating murine mammary fat pad explants (Hovey et
al., 1998a). Hovey et a1. suggested that the murine mammary fat pad is a direct source of

mitogenic activity and that it is involved in modulating the response of epithelial cells to

69

certain mitogens. However, McGrath’s (1983) data showed that primary mouse
mammary epithelial cell growth, measured by [3 H] thymidine labeling, was inhibited
when in close contact with stromal cells.

The role of the fat pad in bovine mammary development is not clear. Woodward
et al. (1993) suggested that adipocytes have different roles in the induction of mammary
epithelial cell proliferation in rodents compared with ruminants. For example, the cow
has a lack of close association of epithelial cells and mammary adipocytes compared with
the rodent. Because of morphological differences, the results from rodent studies should
not be assumed to be the same for the bovine (Akers, 1990). Few studies have been
published on the role of the bovine fat pad in mammary development. Interestingly, fat
pad explants from both beef and dairy heifers significantly inhibited primary bovine
mammary epithelial cell growth in vitro (McFadden and Cockrell, 1993).

The importance of interactions between the mammary fat pad, stroma and the
developing epithelium has been demonstrated. Two studies have compared the intact
mammary fat pad (MFP) to the epithelium cleared fat pad (CF P) of heifers (Berry et al.,
2001) and ewe lambs (Hovey et al., 1998b). Berry and co-workers cleared epithelium
from a single fat pad in heifers at 1 mo of age; at 18 mo, heifers were treated with
estrogen before slaughter. Estrogen treatment increased the amount of IGF-I protein in
MFP and parenchyma but to a lesser extent in CF P. Estrogen tended to increase
abundance of IGF-I mRNA in MFP, but not CFP, implying that the regulation of IGF-I
expression is modulated by adjacent epithelium. IGF-I mRNA was more abundant in
MF P than CF P in ewe lambs, further indicating that IGF-I mRNA expression was

regulated by epithelial-stromal interactions (Hovey et al., 1998b). Hovey suggested that

70

proliferating epithelium might exert a positive feedback on surrounding stroma, causing
an increase in mRNA expression of IGF-I. Sheffield noted the importance of species
differences in stromal requirements for mammary epithelial growth and that this area
requires further investigation (Sheffield, 1988). By transplanting mammary epithelium
into gland free fat pads of mice, Sheffield showed that the mouse mammary epithelia
were capable of organizing into ductal structures if grown with the appropriate stroma.
However, bovine mammary epithelia were unable to proliferate when injected into gland
free mammary fat pads of athymic nude mice (Sheffield and Welsh, 1987). Thus,
existing data fiom the bovine indicates substantial differences in function compared with
the fat pad in mice. More research is needed to explain differences seen in these studies.
The evidence from this thesis indicates that bovine parenchyma tissue in closer vicinity to
the fat pad has less mitogenic activity than tissue more distant from the fat pad.

One potential explanation for the difference in mitogenic activity is the role of
leptin in mammary development. Leptin is produced by adipocytes (Houseknecht et al.,
1998). Parenchymal tissue in the distal region, which is closer to the fat pad than the
proximal region, is closer in vicinity to an area of leptin production. In addition, Sejrsen
and co-workers (1982) found that the percentage of fat cells in the parenchyma increased
as the distance from the teat increased. Therefore, a higher percentage of fat cells would
be located in the distal region than the proximal region of the parenchymal tissue.
Leptin, when used as a treatment in cell culture, is shown to inhibit bovine mammary
epithelial cell proliferation (Silva et al., 1999). Therefore, it would seem logical that
mammary extracts prepared from the proximal region would have lower concentrations

of leptin and would stimulate more DNA synthesis than extracts from the distal region.

71

However, when mammary tissue was homogenized to make mammary extracts, it was
noted that a large quantity of fat and also connective tissue remained on top of the gauze
when the homogenate was poured through a double layer of gauze. Data from this
experiment did show greater mitogenic activity of extracts prepared from parenchyma in
the proximal region but region did not influence leptin concentrations in mammary
extracts. Evidence from this research supports the idea of leptin as a potential local
regulator of mammary development, as leptin concentrations were easily detectable in
mammary tissue. Linearity of the leptin RIA for serum samples has been tested but not
for the mammary extract samples. Therefore, a question remains as to whether these
leptin concentrations are accurate for the mammary extract samples.

The observation that IGF-I and leptin concentrations did not differ between
regions suggests that there must be another stimulator or inhibitor that influences
mammary development. Further research is needed in this area to determine if there are
other regulators of mammary development that contributed to observed differences in
mitogenic activity. The use of antibodies against both stimulatory and inhibitory growth
factors such as IGF-I, IGFBP, leptin, F GF , KGF, and TGF-[31, would be one technique
that could be used to quantitate the proliferative activity that each factor is contributing to

the overall mitogenic effect of these mammary extracts.

72

SUMMARY AND CONCLUSIONS

Mammary extracts of tissue from proximal regions of the parenchyma were 11 %
more mitogenic than extracts of tissue from the distal region. Two main differences
between these two regions, known prior to this study, included vicinity of the region to
the fat pad and the abundance of different cell types (epithelial and fat cells) within the
parenchyma of the tissue. In addition, as the parenchyma extends into the fat pad during
development, regional differences in its anatomy become apparent. This study further
characterized differences in region of the mammary parenchyma by measuring the
concentration of IGF-I and leptin and the abundance of IGFBP within mammary extracts
of tissue from these different regions. Concentrations of IGF-I were measured in
extracts because it is a known potent mitogen for mammary epithelial cells in vitro.
Furthermore, the role of the IGFBP must be considered to fully understand the
mechanism of IGF-I growth stimulation in mammary development. Concentrations of
leptin were analyzed because of its inhibitory effect in vitro on mammary epithelial cell
proliferation. The parenchymal tissue region did not influence IGF-I or leptin
concentrations in mammary extracts but did affect the local abundance of IGFBP-3 in the
mammary gland. These findings indicate that IGFBP-3 may have had an inhibitory role
in MAC-T epithelial cell proliferation. Because IGF-I and leptin concentrations did not
explain the difference in mitogenic capacity of the mammary extracts, differences in
IGFBP-3 or another local regulator likely contribute to regional differences in the

mammary gland during development.

73

BIBLIOGRAPHY

Laurie Ellen Davis was born in Waynesboro, Virginia on September 29, 1977.
She graduated from Wilson Memorial High School in Fishersville, Virginia in 1995.
Thereafter, she enrolled at Virginia Polytechnic Institute and State University with a
major in Dairy Science, a minor in Animal Science and earned a Bachelor of Science in
May 1999. In May 2002, she defended her thesis project and received a Master of
Science from Michigan State University in Animal Science in the summer of 2002. The
author is a member of the American Dairy Science Association and the American

Association for the Advancement of Science.

74

REFERENCES

Akers, R. M. 1985. Lactogenic hormones: binding sites, mammary growth, secretory cell
differentiation, and milk biosynthesis in ruminants. J. Dairy Sci. 68:501-519.

Akers, R. M. 1990. Lactation physiology: a ruminant animal perspective. Protoplasma
159:96-111.

Anderson, R. A. 1978. Embryonic and fetal development of the mammary apparatus.
Pages 3-40 in Lactation: A Comprehensive Treatise. Vol. IV. B. L. Larson, ed.
Academic Press, NY.

Ayadi, M., G. Caja, X. Such, and C. H. Knight. 2000. An ultrasonographic method for
estimating mammary cistern volume after different milking intervals in dairy cows. J.
Dairy Sci. 83(Suppl. l):166.

Bauman, D. E., and R. G. Vernon. 1993. Effects of exogenous bovine somatotrophin on
lactation. Ann. Rev. Nutr. 13:437-461.

Bauman, D. E. 1999. Bovine somatotropin and lactation: from basic science to
commercial application. Domest. Anim. Endocrinol. 17:101-116.

Baumrucker, C. R., W. M. Campana, C. A. Gibson, and D. E. Kerr. 1993. Insulin-like
growth factors (IGF) and IGF binding proteins in milk; sources and functions. Endocrine
Reg. 27:157-172.

Baumrucker, C. R. and N. E. Erondu. 2000. Insulin-like growth factor (IGF) system in
the bovine mammary gland and milk. J. Mam. Gland Biol. Neoplasia 5:53-64.

Baumrucker, C. R. and B. H. Stemberger. 1989. Insulin and insulin-like grth factor-I
stimulate DNA synthesis in bovine mammary tissue in vitro. J. Anim. Sci. 67:3503-3514.

Baxter, R. C. 1993. Circulating binding proteins for the insulin-like growth factors. TEM.
4:91-96.

Beauloye, V., S. M. Muaku, P. Lause, D. Portetelle, R. Renaville, A. R. Robert, J. M.
Ketelsledgers, and D. Maiter. 1999. Monoclonal antibodies to growth hormone (GH)
prolong liver GH binding and GH-induced IGF-I/IGFBP-3 synthesis. Am. Physiol. Soc.
E308-E315.

Berry, S. D., T. B. McFadden, R. E. Pearson, and R. M. Akers. 2001. A local increase in

the mammary IGF-I : IGFBP-3 ratio mediates the mammogenic effects of estrogen and
growth hormone. Domest. Anim. Endocrinol. 21:39-53.

75

Blat, C., J. Villaudy, and M. Binoux. 1994. In vivo proteolysis of serum insulin-like
growth factor (IGF) binding protein-3 results in increased availability of IGF to target
cells. J. Clin. Invest. 93:2286-2290.

Boisclair, Y. R., R. P. Rhoads, I. Ueki, J. Wang, and G. T. Ooi. 2001. Review: The acid-
labile subunit (ALS) of the 150 kDa IGF-binding protein complex: an important but
forgotten component of the circulating IGF system. J. Endocrinol. 170:63-70.

Boni-Schnetzler, M., C. Hauri, and J. Zapf. 1999. Leptin is suppressed during infusion of
recombinant human insulin-like growth factor I (rhIGF-I) in normal rats. Diabetologia
42: 160-166.

Boumer, M. J ., W. H. Busby Jr., N. R. Siege], G. C. Krivi, R. H. McCusker, and D. R.
Clemmons. 1992. Cloning and sequence determination of bovine insulin-like growth
factor binding protein-2 (IGFBP-2): comparison of its structural and functional properties
with IGFBP-1. J. Cell. Biochem. 48:215-226.

Breier, B. H., J. J. Bass, J. H. Butler, and P. D. Gluckman. 1986. The somatotrophic axis
in young steers: influence of nutritional status on pulsatile release of growth hormone and
circulating concentrations of insulin-like growth factor 1. J. Endocrinol. 111:209-215.

Breier, B. H., B. W. Gallaher, and P. D. Gluckrnan. 1991. Radioimmunoassay for insulin-
like growth factor —1: solutions to some potential problems and pitfalls. J. Endocrinol.
128:347-357.

Broster, W. H. and V. J. Broster. 1984. Reviews of the progress of Dairy Science: long
term effects of plane of nutrition on the performance of the dairy cow. J. Dairy Res.
51:149-196.

Capuco, A. V., D. L. Wood, R. Baldwin, K. Mcleod, and M. J. Paape. 2001. Mammary
cell number, proliferation, and apoptosis during a bovine lactation: relation to milk
production and effect of bST. J. Dairy Sci. 84:2177-2187.

Ceda, G. P. R. G. Davis, R. G. Rosenfeld, and A. R. Hoffman. 1987. The growth
hormone (GH)-releasing hormone (GHRH)-GH-somatomedin axis: evidence for rapid
inhibition of GHRH-elicited GH release by insulin-like growth factor I and II.
Endocrinology 120: 1658-1662.

Chen, J. C., Z. M. Shao, M. S. Sheikh, A. Hussain, D. LeRoith, C. T. Roberst, and J. A.
Fontana. 1994. Insulin-like growth factor-binding protein enhancement of insulin-like
growth factor-I (IGF-I) — mediated DNA synthesis and IGF-1 binding in a human breast
carcinoma cell line. J. Cell. Physiol. 158:69-78.

Cohen, R, G. Larnson, T. Okajima, and R. Rosenfeld. 1993. Transfection of the hmnan

insulin-like growth factor binding protein-3 gene into Balb/c fibroblasts inhibits cellular
growth. Mol. Endocrinol. 7:380-386.

76

Cohick, W. 1998. Role of the insulin-like growth factors and their binding proteins in
lactation. J. Dairy Sci. 81 :1769-1777.

Cohick, W. and J. Turner. 1998. Regulation of IGF binding protein synthesis by a bovine
mammary epithelial cell line. J. Endocrinol. 157:327-336.

Cohick, W. S., B. Wang, P. Verma, and Y. R. Boisclair. 2000. Insulin-like growth factor
I (IGF-I) and cyclic adenosine 3’,5’ — monophosphate regulate IGF-binding protein-3
gene expression by transcriptional and posttranscriptional mechanisms in mammary
epithelial cells. Endocrinology 141:4583-4591.

Collier, R. J ., M. F. McGrath, J. C. Byatt, and L. L. Zurfluh. 1993. Regulation of bovine
mammary growth by peptide hormones: involvement of receptors, growth factors and
binding proteins. Livest. Prod. Sci. 35:21-33.

Daniel, C. W., G. B. Silberstein, K. Van Horn, P. Strickland, and S. Robinson. 1989.
TGF-B 1-induced inhibition of mouse mammary ductal growth: developmental specificity
and characterization. Dev. Bio. 135220-30.

Daughaday, W. H. and P. Rotwein. 1989. Insulin-like growth factor I and II. Peptide
messenger ribonucleic acid and gene structures, serum, and tissue concentrations.
Endocrine Rev. 10:68-91

Davidson, J. N., and 1. Leslie. 1950. A new approach in the biochemistry of growth and
development. Nature 165 :49-53.

DeHoff, M. H., R. G. Elgin, R. J. Collier, and D. R. Clemmons. 1988. Both type I and II
insulin-like growth factor receptor binding increase during lactogenesis in bovine
mammary tissue. Endocrinology 122:2412-2417.

Delavaud, C., F. Bocquier, Y. Chilliard, D. H. Keisler, A. Gertler, and G. Kann. 2000.
Plasma leptin determination in ruminants: effect of nutritional status and body fatness on
plasma leptin concentration assessed by a specific RIA in sheep. J. Endocrinol. 165:519-
526.

D’Ercole, A. J ., A. D. Stiles, and L. E. Underwood. 1984. Tissue concentrations of
somatomedin C: Further evidence for multiple sites of synthesis and paracrine or
autocrine mechanisms of action. Proc. Natl. Acad. Sci. USA 81:935-939.

Ellis, 8., S. Purup, K. Sejrsen, and R. M. Akers. 2000. Growth and morphogenesis of
epithelial cell organoids from distal and medial mammary parenchyma of prepubertal
heifers. J. Dairy Sci. 83:952-961.

Erondu, N. E., B. Toland, M. Boes, B. Dake, D. R. Moser, and R. S. Bar. 1997. Bovine

insulin-like growth factor binding protein-3: organization of the chromosomal gene and
functional analysis of its promoter. Endocrinology 138:2856-2862.

77

Faulkner, A. 1999. Changes in plasma and milk concentrations of glucose and IGF-1 in
response to exogenous growth hormone in lactating goats. J. Dairy Res. 66:207-214.

Ford, J. A., and C. S. Park. 2001. Nutritionally directed compensatory growth enhances
heifer development and lactation potential. J. Dairy Sci. 84: 1669-1678.

Forsyth, I. A. 1989. Growth factors in mammary gland function. J. Reprod. Fertil.
85:759-770.

Forsyth, I. A., J. A. Taylor, and C. D. Moorby. 1998. DNA synthesis by ovine mammary
alveolar epithelial cells: effects of heparin, epidermal growth factor-related peptides and
interaction with stage of pregnancy. J. Endocrinol. 156:283-290.

Francis, G. L., M. Ross, F. J. Ballard, S. J. Milner, C. Senn, K. A. McNeil, J. C. Wallace,
R. King, and J. R. E. Wells. 1992. Novel recombinant fusion-protein analogues of
insulin-like growth factor (IGF)-I indicate the relative importance of IGF-binding protein
and receptor binding for enhanced biological potency. J. Mol. Endocrinol. 8:213-223.

Freshney, R. I. 1994. Culture of animal cells: a manual of basic technique. Vol III.
Wiley-Liss, NY.

Frey, R. S., M. R. Hathaway, and W. R. Dayton. 1994. Comparison of the effectiveness
of various procedures for reducing or eliminating insulin-like grth factor-binding
protein interference with insulin-like growth factor-I radioirnmunoassays on porcine
serum. J Endocrinol. 140:229-237.

Gibson, 0, M. Staley, and C. Baumrucker. 1999. Identification of IGF binding proteins
in bovine milk and the demonstration of IGFBP-3 synthesis and release by bovine
mammary epithelial cells. J. Anim. Sci. 77:1547-1557.

Glimm, D. R., V. E. Baracos, and J. J. Kennelly. 1990. Molecular evidence for the
presence of growth hormone receptors in the bovine mammary gland. J. Endocrinol.
126:R5-R8.

Gluckrnan, P. D., B. H. Breier, and S. R. Davis. 1987. Physiology of the somatotropic
axis with particular reference to the ruminant. J. Dairy Sci. 70:442-466.

Grill, C. J. and W. S. Cohick. 2000. Insulin-like growth factor binding protein-3 mediates
IGF-I action in a bovine mammary epithelial cell line independent of an IGF interaction.
J. Cell. Physiol. 183:273-283.

Gutierrez, C. G., B. K. Campbell, D. G. Armstrong, and R. Webb. 1997. Insulin-like
growth factor-I (IGF-I) production by bovine granulosa cells in vitro and peripheral IGF-I
measurement in cattle serum: an evaluation of IGF-binding protein extraction protocols.
J. Endocrinol. 153:231-240.

78

Hadsell, D. L., P. G. Campbell, and C. R. Baumrucker. 1990. Characterization of the
change in type I and II insulin-like growth factor receptors of bovine mammary tissue
during the pre- and postpartum periods. Endocrinology 126:637-643.

Hauser, S., M. McGrath, R. Collier, and G. Krivi. 1990. Cloning and in vivo expression
of bovine growth hormone receptor mRNA. Mol. Cell. Endocrinol. 72: 1 87-200.

Hodgkinson, S. C., G. S. G. Spencer, J. J. Bass, S. R. Davis, and P. D. Gluckman. 1991.
Distribution of circulating insulin-like growth factor-I (IGF-I) into tissues. Endocrinology
129:2085-2093.

Hoshino, K., and F. Martin. 1974. Parenchymal-stromal interactions during regeneration
of mammary isographs in mice. Anatomy Record. 178:379-406.

Hossenlopp, P., B. Segovia, C. Lassarre, M. Roghani, M. Bredon, and M. Binoux. 1990.
Evidence of enzymatic degradation of insulin-like grth factor-binding proteins in the
150K complex during pregnancy. J. Clin. Endocrinol. Metab. 71:797-805.

Houseknecht, K. L., C. A. Baile, R. L. Matteri, and M. E. Spurlock. 1998. The biology of
leptin: a review. J. Anim. Sci. 76:1405-1420.

Houseknecht, K. L., C. P. Portocarrero, S. Ji, R. Lemenager, and M. E. Spurlock. 2000.
Growth hormone regulates leptin gene expression in bovine adipose tissue: correlation
with adipose IGF-I expression. J. Endocrinol. 164:51-57.

Hovey, R. 1996. The role of the mammary fat pad during mammogenesis. PhD Thesis,
Massey Univer., New Zealand.

Hovey, R. C., D. D. S. MacKenzie, and T. B. McFadden. 1998a. The proliferation of
mouse mammary epithelial cells in response to specific mitogens is modulated by the
mammary fat pad in vitro. In Vitro Cell Dev. Biol. (Animal) 34:385-392.

Hovey, R. C., H. W. Davey, D. D. S. Mackenzie, and T. B. McFadden. 1998b. Ontogeny
and epithelial-stromal interactions regulate IGF expression in the ovine mammary gland.
Mol. Cell. Endocrinol. 136: 139-144.

Hovey, R. C., H. W. Davey, B. K. Vonderhaar, D. D. S. Mackenzie, and T. B.
McFadden. 2001. Paracrine action of keratinocyte growth factor (KGF) during ruminant
mammogenesis. Mol. Cell. Endocrinol. 181:47-56.

Huynh, H. T., G. Robitaille, and J. D. Turner. 1991. Establishment of bovine mammary
epithelial cells (MAC-T): an in vitro model for bovine lactation. Exp. Cell Res. 197:191-
199.

Hwa, V., Y. Oh, R. G. Rosenfeld. 1999. The insulin-like growth factor-binding protein
(IGFBP) superfamily. Endocrine Rev. 20:761-787.

79

Jensen, M. D., N. Moller, K. S. Nair, P. Eisenberg, M. Landt, and S. Klein. 1999.
Regional leptin kinetics in humans. Am. J. Clin. Nutr. 69:18-21.

Keifer, M. D., F. R. Masiarz, D. Bauer, and J. Zapf. 1991. Identification and molecular
cloning of two new 30 kDa insulin-like growth factor binding proteins isolated from adult
human serum. J. Biol. Chem. 266:9043-9049.

Kleinberg, D. L. 1997. Early mammary development: growth hormone and IGF-1. J.
Mam. Gland Biol. Neoplasia 2:49-57.

Knight, C. H. and M. Peaker. 1982. Development of the mammary gland. J. Reprod.
Fertil. 65:521-536.

Knight, C. H., and C. J. Wilde. 1987. Mammary growth during lactation: implications
for increasing milk yield. J. Dairy Sci. 70:1991-2000.

Knight, C. H. 2000. The importance of cell division in udder development and lactation.
Livest. Prod. Sci. 66: 169-176.

Kraetzl, W. D., C. Zimmer, D. Schneider, and D. Schams. 1998. Secretion patterns of
growth hormone, prolactin, insulin and insulin-like growth factor-1 in the periparturient
sow depending on the metabolic state during lactation. J. Anim. Sci. 67:339-347.

Labarca, C., and K. Paigen. 1980. A simple, rapid, and sensitive DNA assay procedure.
Analytical Biochem. 102:344-352.

Lametsch, R., J. T. Rasmussen, L. B. Johnsen, S. Purup, K. Sejrsen, T. E. Petersen, and
C. W. Heegaard. 2000. Structural characterization of the fibroblast growth factor-binding

protein purified from bovine prepartum mammary gland secretion. J. Biol. Chem.
275:19469-19474.

Lammers, B. P., A. J. Heinrichs, and R. S. Kensinger. 1999. The effects of accelerated
growth rates and estrogen implants in prepubertal holstein heifers on estimates of

mammary development and subsequent reproduction and milk production. J. Dairy Sci.
82:1753-1764.

Lemozy, S., J. B. Pucilowska, and L. E. Underwood. 1994. Reduction of insulin-like
growth factor-I (IGF-I) in protein-restricted rats is associated with differential regulation
of IGF-binding protein in liver and serum. Endocrinology 135:617-623.

Lewitt, M. S., G. S. Denyer, G. J. Cooney, and R. C. Baxter. 1991. Insulin-like growth
factor-binding protein-1 modulates blood glucose levels. Endocrinology 129:2254-2256.

Maier, R., P. Schmid, D. Cox, G. Bilbe, and G. McCaster. 1991. Localization of
transforming growth factor-Bl, 432 and -[33 gene expression in bovine mammary gland.
Mol. Cell. Endocrinol. 82:191-198.

80

McCusker, R. H. 1998. Controlling insulin-like growth factor activity and the modulation
of insulin-like growth factor binding protein and receptor binding. J. Dairy Sci. 81:1790—
1 800.

McDowell, G. H. and 1. C. Hart. 1984. Responses to infusion of growth hormone into the
mammary arteries of lactating sheep. Can. J. Anim. Sci. 64(Suppl.):306-307.

McFadden, T. B., T. E. Daniel, and R. M. Akers. 1990. Effects of plane of nutrition,
growth hormone and unsaturated fat on growth hormone, insulin and prolactin receptors
in prepubertal lambs. J. Anim. Sci. 68:3180-3189.

McFadden, T. B., and D. L. Cockrell. 1993. Regulation of growth in cultured mammary
epithelimn from beef and dairy heifers. Proc. New Zealand Soc. Anim. Prod. 53: 143-145.

McGrath, C. M. 1983. Augmentation of the response of normal mammary epithelial cells
to estradiol by mammary stroma. Cancer Research 43:1355-1360.

McGrath, M. F ., R. J. Collier, D. R. Clemmons, W. H. Busby, C. A. Sweeny, and G. G.
Krivi. 1991. The direct in vitro effect of insulin-like growth factors (IGFs) on normal

bovine mammary cell proliferation and production of IGF binding proteins.
Endocrinology 129:671-678.

McGuire, M., D. Dwyer, D. Bauman, and D. Smith. 1998. Insulin-like growth factors in
plasma and afferent mammary lymph of lactating cows deprived of feed or treated with
bovine somatotrophin. J. Dairy Sci. 81 :950-957.

Mohan, S. and D. J. Baylink. 1995. Development of a simple valid method for the
complete removal of insulin-like growth factor (IGF)-binding proteins from IGFs in
human serum and other biological fluids: comparison with acid-ethanol treatment and C13
sep-pak separation. J. Clin. Endocrinol. Metab. 80:637-647.

Nandi, S. 1958. Endocrine control of marnmary-gland development and function in the
C3H/He Crgl mouse. J. Natl. Cancer Inst. 21:1039-1055.

Oh, Y. 1998. IGF-independent regulation of breast cancer growth by IGF binding
proteins. Breast Cancer Res. Treat. 47:283-293.

Oh, Y., H. Muller, L. Ng, and R. Rosenfeld. 1995. Transforming growth factor B-induced
cell grth inhibition in human breast cancer cells is mediated through insulin-like
growth factor-binding protein-3 action. J. Biol. Chem. 270:13589-13592.

Oka, T., M. Yoshimura, S. Lavandero, K. Wada, and Y. Oka. 1991. Control of growth
and differentiation of the mammary gland by growth factors. J. Dairy Sci. 74:2788-2800.

Peel, C. J. and D. E. Bauman. 1987. Somatotropin and lactation. J. Dairy Sci. 70:474-
486.

81

Plaut, K. 1993. Role of epidermal growth factor and transforming growth factor in
mammary development and lactation. J. Dairy Sci. 76: 1 526-1538.

Prosser, C. G., I. R. Fleet, A. N. Corps, E. R. Froesch, and R. B. Heap. 1990. Increase in
milk secretion and mammary blood flow by intra-arterial infusion of insulin-like growth
factor-I into the mammary gland of the goat. J. Endocrinol. 126:437-443.

Prosser, C. G., S. R. Davis, V. C. Farr, L. G. Moore, and P. D. Gluckman. 1994. Effects
of close-arterial (external pudic) infusion of insulin-like growth factor-II on milk yield
and mammary blood flow in lactating goats. J. Endocrinol. 142:93-99.

Pump, 8., K. Sejrsen, J. F oldager, and R. Akers. 1993. Effect of exogenous bovine
growth hormone and ovariectomy on prepubertal mammary growth, serum hormones and
acute in-vitro proliferative response of mammary explants from Holstein heifers. J.
Endocrinol. 139: 19-26.

Purup, S., M. Vestegaard, M. S. Weber, K. Plaut, R. M. Akers, and K. Sejrsen. 2000.
Local regulation of pubertal mammary grth in heifers. J. Animal Sci. 78 (Suppl. 3):36-
47.

Rajah, R., B. Valentinis, and P. Cohen. 1997. Insulin-like growth factor (IGF)-binding
protein-3 induces apoptosis and mediates the effects of transforming growth factor-[31 on

programmed cell death through a p53- and IGF-independent mechanism. J. Biol. Chem.
272:12181-12188.

Reece, R. P. 1956. The physiology of milk production. J. Dairy Sci. 39:726-734.

Reiss, M. and M. Barcellos-Hoff. 1997. Transforming growth factor-[3 in breast cancer: A
working hypothesis. Breast Cancer Res. Treat. 45:81-95.

Robinson, G. W., G. H. Smith, D. Gallahan, A. Zimmer, P. A. Furth, and L.
Hennighausen. 1996. Understanding mammary gland development through the
imbalanced expression of growth regulators. Dev. Dynamics 206: 1 59-1 68.

Rodgers, J ., L. Wiltrout, L. Nanu, and M. Fant. 1996. Developmentally regulated
expression of IGF binding protein-3 (IGFBP-3) in human placental fibroblast: effects of
exogenous IGFBP-3 on IGF-1 action. Reg. Peptides 61 :189—195.

Ronge, H., J. Blurn, C. Clement, F. Jans, H. Leuenberger, and H. Binder. 1988.
Somatomedin C in dairy cows related to energy and protein supply and to milk
production. Anim. Prod. 47:165-183.

Ross, M., G. L. Francis, L., Szabo, J. C. Wallace, and F. J. Ballard. 1989. Insulin-like

growth factor (IGF) binding proteins inhibit the biological activities of IGF-I and IGF-2
but not des(1-3) IGF-I. Biochem. J. 258:267-272.

82

Ruan, W., and D. L. Kleinberg. 1999. Insulin-like growth factor I is essential for terminal
end bud formation and ductal morphogenesis during mammary development.
Endocrinology. 140:5075-5081.

Rubin, J. S., H. Osada, P. W. Finch, W. G. Taylor, S. Rudikoff, and S. A Aaronson. 1989.
Purification and characterization of a newly identified growth factor specific for
epithelial cells. Proc. Natl. Acad. Sci. USA 86:802-806.

Sandles, L. D. and C. J. Peel. 1987. Marnmogenesis and first lactation milk yields of
identical-twin heifers following pre-pubertal administration of bovine growth hormone.
Anim. Prod. 45:349-357.

Sandowski, Y., I. Peri, and A. Gertler. 1993. Partial purification and characterization of
putative paracrine / autocrine bovine mammary epithelium grth factors. Livest. Prod.
Sci. 35:35-48.

Schillo, K. K., J. B. Hall, and S. M. Hileman. 1992. Effects of nutrition and season on the
onset of puberty in the beef heifer. J. Anim. Sci. 70:3994-4005.

Sejrsen, K., J. T. Huber, H. A. Tucker, and R. M. Akers. 1982. Influence of nutrition on
mammary development in pre- and postpubertal heifers. J. Dairy Sci. 65:793-800.

Sejrsen, K., J. T. Huber, and H. A. Tucker. 1983. Influence of amount fed on hormone
concentrations and their relationship to mammary growth in heifers. J. Dairy Sci. 66:845-
855.

Sejrsen, K., J. Foldager, M. T. Sorensen, R. M. Akers, and D. E. Bauman. 1986. Effect of
exogenous bovine somatotropin on pubertal mammary development in heifers. J. Dairy
Sci. 69:1528-1535.

Sejrsen, K. 1994. Relationships between nutrition, puberty and mammary development in
cattle. Proc. Nutr. Soc. 53:103-111.

Sharnay, A., N. Cohen, M. Niwa, and A. Gertler. 1988. Effect of insulin-like growth
factor I on deoxyribonucleic acid synthesis and galactopoiesis in bovine undifferentiated
and lactating mammary tissue in vitro. Endocrinology 123:804-809.

Sharma, B. K., M. J. VandeHaar, and N. K. Ames. 1994. Expression of insulin-like
growth factor-I in cows at different stages of lactation and in late lactation cows treated
with somatotropin. J. Dairy Sci. 77:2232-2241.

Sheffield, L. G. 1988. Organization and growth of mammary epithelia in the mammary
gland fat pad. J. Dairy Sci. 71 :2855-2874.

83

Sheffield, L. G., C. W. Welsh. 1987. Transplantation of bovine mammary tissue to
athymic nude mice: growth subcutaneously and in mammary gland-free fat pads. J. Dairy
Sci. 69:1141-1147.

Silberstein, G. B., P. Strickland, S. Coleman, and C. W. Daniel. 1990. Epithelium-

dependent extracellular matrix synthesis in transforming growth factor-Bl-growth-
inhibited mouse mammary gland. J. Cell Biol. 110:2209—2219.

Silva, L. F. P., and M. J. VandeHaar. 1999. Role of leptin in mammary cell proliferation.
S. Afr. J. Anim. Sci. 29(ISRP):300-301.

Silva, L. F. P., M. J. VandeHaar, M. S. Weber, and G. W. Smith. 2001. Leptin receptor
expression in the bovine mammary gland and other tissues. J. Dairy Sci. 84(Suppl.
1):933.

Silva, L. F. P., M. J. VandeHaar, and M. S. Weber Nielsen. 2002. Intramammary infusion
of IGF-I increases BrdU-labeling in mammary epithelial cells of prepubertal heifers. To
be presented at ADSA/ASAS Meetings in Quebec, Canada.

Sinha, Y. N. and H. A. Tucker. 1966. Mammary gland growth of rats between 10 and 100
days of age. Am. J. Physiol. 210:601-605.

Sinha, Y. and H. A. Tucker. 1969. Mammary development and pituitary prolactin level of
heifers from birth through puberty and during the estrous cycle. J. Dairy Sci. 52:507-512.

Sjogren, K., J. Liu, K. Blad, S. Skrtic, O. Vidal, V. Wallenius, D. LeRoith, J. Tomell, O.
Isaksson, J. J ansson, and C. Ohlsson. 1999. Liver-derived insulin-like growth factor I
(IGF-I) is the principal source of IGF-I in blood but is not required for postnatal body
growth in mice. Proc. Natl. Acad. Sci. USA 96:7088-7092.

Skarr, T. C., C. R. Baumrucker, D. R. Deaver, and J. W. Blum. 1994. Diet effects and
ontogeny of alterations of circulating insulin-like grth factor binding proteins in
newborn dairy calves. J. Anim. Sci. 72:421-427.

Smith, J. L., and L. G. Sheffield. 2002. Production and regulation of leptin in bovine
mammary epithelial cells. Domest. Anim. Endocrinol. 22:145-154.

Sordillo, L. M., S. P. Oliver, and R. M. Akers. 1988. Culture of bovine mammary
epithelial cells in D-valine modified medium: selective removal of contaminating
fibroblasts. Cell Biol. Int. Rep. 12:355-364.

Sorensen, M. T., K. Sejrsen, and J. Foldager. 1987. Estimation of pubertal mammary
development in heifers by computed tomography. J. Dairy Sci. 70:265-270.

84

Soriano, J ., M. Pepper, L. Orci, and R. Montesano. 1998. Roles of hepatocyte growth
factor/scatter factor and transforming growth factor-Bl in mammary gland ductal
morphogenesis. J. Mam. Gland Bio. Neoplasia 3: 133-150.

Thissen, J. P., J. M. Ketelslegers, and L. E. Underwood. 1994. Nutritional regulation of
the insulin-like growth factors. Endocrine Rev. 15:80-101.

Tomas, F. M., S. E. Knowles, C. S. Chandler, G. L. Francis, P. C. Owens, and F. J.
Ballard. 1993. Anabolic effects of insulin-like growth factor-I (IGF-I) and an IGF-I
variant in normal female rats. J. Endocrinol. 137:413-421.

Tucker, H. A. 1981. Physiological control of mammary growth, lactogenesis, and
lactation. J. Dairy Sci. 64:1403-1421.

Vander Kooi, W. K., M. J. VanderHaar, B. K. Sharma, M. Binelli, H. A. Tucker, R. M.
Akers, and W. M. Moseley. 1995. Comparison of growth hormone-releasing factor and

somatotropin: the somatotropic axis in lactating primiparous cows. J. Dairy Sci. 78:2140-
2149.

Vega, J. R., C. A. Gibson, T. C. Skaar, D. L. Hadsell, and C. R. Batunrucker. 1991.
Insulin-like growth factor (IGF)-I and —II and IGF binding proteins in serum and
mammary secretions during the dry period and early lactation in dairy cows. J. Anim. Sci.
69:2538-2547.

Vicini, J. L., F. C. Buonomo, J. J. Veenhuizen, M. A. Miller, D. R. Clemmons, and R. J.
Collier. 1991. Nutrient balance and stage of lactation affect responses of insulin, insulin-
like growth factors I and II, and insulin-like growth factor-binding protein 2 to
somatotropin administration in dairy cows. J. Nutr. 121 :1656-1664.

Waksman, M., A. Shamay, and A. Gertler. 1991. Bovine pituitary, kidney, uterine and
mammary gland extracts contain bovine mammary epithelium growth factors that
synergize with IGF-I and fetal calf serum: indication for involvement of GTP-binding
proteins. Domest. Anim. Endocrinol. 8:271-280.

Walden, P. D., W. Ruan, M. F eldrnan, and D. L. Kleinberg. 1998. Evidence that the
mammary fat pad mediates the action of growth hormone in mammary gland
development. Endocrinology 139:659-662.

Weber, M., S. Purup, M. Vestergaard, S. Ellis, J. Scndergard-Anderson, R. Akers, and K.
Sejrsen. 1999. Contribution of insulin-like growth factor (IGF)-I and IGF-binding
protein-3 to mitogenic activity in bovine mammary extracts and serum. J. Endocrinol.
161 :365-373.

Weber, M., S. Purup, M. Vestergaard, R. Akers, and K. Sejrsen. 2000a. Regulation of

local synthesis of insulin-like growth factor-1 and binding proteins in mammary tissue. J.
Dairy Sci. 83:30-37.

85

Weber, M., S. Purup, M. Vestergaard, R. Akers, and K. Sejrsen. 2000b. Nutritional and
somatotrophin regulation of the mitogenic response of mammary cells to mammary tissue
extracts. Domest. Anim. Endocrinol. 18: 159-164.

Winder, S. J ., A. Turvey, and I. A. Forsyth. 1989. Stimulation of DNA synthesis in
cultures of ovine mammary epithelial cells by insulin and insulin-like growth factors. J.
Endocrinol. 123:319—326.

Wolf, E., P. M. Jehle, M. M. Weber, H. Sauerwein, A. Daxenberger, B. H. Breier, U.
Besenfelder, L. Frenyo, and G. Brem. 1997. Human insulin-like growth factor I (IGF-I)
produced in the mammary glands of transgenic rabbits: yield, receptor binding, mitogenic
activity, and effects of IGF-binding proteins. Endocrinology 138:307-313.

Wong, E. A., S. M. Ohlsen, J. A. Godfredson, D. M. Dean, and J. E. Wheaton. 1989. t
Cloning of ovine insulin-like growth factor-I cDNAs: heterogeneity in the mRNA ‘
population. DNA 8:649-657.

Woodward, T. L., W. E. Beal, and R. M. Akers. 1993. Cell interactions in initiation of
mammary epithelial proliferation by oestradiol and progesterone in prepubertal heifers. J.
Endocrinol. 1362149-157.

 

Ilfi~

Woodward, T. L., R. M. Akers, and J. D. Turner. 1994. Lack of mitogenic response to
EGF, pituitary and ovarian hormones in bovine mammary epithelial cells. Endocrine
2:529—535.

Woodward, T. L., N. Dumont, M. O’Connor-McCourt, J. D. Turner, and A. Phillip. 1995.
Characterization of transforming growth factor-beta growth regulatory effects and
receptors on bovine mammary cells. J. Cell. Physiol. 165:339—348.

Woodward, T. L., J. D. Turner, H. T. Huynh, and X. Zhao. 1996. Inhibition of cellular
proliferation and modulation of insulin-like grth factor binding proteins by retinoids in
a bovine mammary epithelial cell line. J. Cellular Physio. 167:488-499.

Yakar, S., J. Liu, B. Stannard, A. Butler, A. Accili, B. Sauer, and D. LeRoith. 1999.
Normal growth and development in the absence of hepatic insulin-like growth factor 1.
Proc. Natl. Acad. Sci. USA 96:7324—7329.

Zapf, J. 1995. Physiology role of the insulin-like growth factor binding proteins.
Eur. J. Endocrinolgy 132:645-654.

Zhao, X., B. W. McBride, 1. Politis, H. T. Huynh, R. M. Akers, J. H. Burton, and J. D.

Turner. 1992. Receptor binding and growth-promoting activity of insulin-like growth
factor-I in a bovine mammary epithelial cell line (MAC-T3). J. Endocrinol. 134:307-312.

86

 

APPENDIX

Introduction:

The initial research proposal for this master’s project is outlined in this appendix.
Primary bovine mammary epithelial cell populations were harvested from prepubertal
heifers, however contamination of fibroblasts was observed and verified using
immunohistochemistry. Changes were made in the primary cell isolation to prevent or
lower fibroblast cell contamination, but these modifications proved unsuccessful.
Therefore, this project was amended using the MAC-T cell line and mammary tissue
extracts (see Thesis). The initial project consisted of two objectives, with each objective
tested on both the primary epithelial cell population and the MAC-T cell line. Much of
the in vitro studies of the role of IGF-I on bovine mammary development involve either
primary cells or the MAC-T cell line. In addition, another experiment was performed
which focused on the differences in mitogenic activity of mammary tissue extracts
prepared from a prepubertal heifer and a postpubertal heifer.

Insulin-like growth factor binding protein-3 is a potent regulator of the biological
actions of IGF-I in mammary tissue (see p. 21-23). The regulation of IGFBP-3 synthesis
in epithelial cells in the developing bovine mammary gland and the effect of IGFBP-3 on
cell proliferation remain unclear. TGF-01 stimulates IGFBP-3 production in various cell
systems such as breast and prostate cancer cell lines (Oh, 1998). Many studies suggest
that TGF-B. and IGFPB-3 may be interrelated with respect to how they affect cell

proliferation (Oh et al., 1995; Pump et al., 2000). TGF-B receptors have been

characterized on bovine mammary epithelial cells (Woodward et al., 1995) and TGF-Bl

87

 

mRNA has been identified in the bovine mammary gland (Maier et al., 1991; Plant,
1993). Levels of 25 to 50 pg/ml of TGF-[31 stimulated DNA synthesis, whereas levels of
greater than 100 pg/ml inhibited DNA synthesis (Ellis et al., 2000). TGF-[31 was also
effective in inhibiting proliferation of MAC-T cells (Woodward et al., 1995). Research
involving the role of TGF-Bl on bovine mammary development has only recently
surfaced.

The purpose of this project was to define under what conditions is the role of
IGFBP-3 stimulatory or inhibitory to the development of the mammary gland and
possible regulatory factors affecting this role. The first objective of this project was to
determine if IGFBP-3 regulates proliferation of primary bovine mammary epithelial cells
independently of IGF-I. The second objective was to investigate the regulation of

IGFBP-3 synthesis by TGF-[31 in mammary epithelial cells.

Primag Bovine Mammary EpimeliaLCell (PMEC) Isolation:

Primary bovine mammary epithelial cells (PMEC) were isolated and cultured
similar to Sharnay et al. (1988). Mammary parenchymal tissue from five prepubertal
heifers (184.5 i 29.5 kg body weight) was collected immediately after slaughter.
Reproductive tracts were dissected to confirm that heifers were prepubertal. Tissue was
placed in medium 199 with 2 g NaH2C03 (M-199, HEPES modified, Sigma, pH 7.4,
sterilized using 0.22-pm filters). Basal medium also contained soybean trypsin inhibitor
(Type I-S, Sigma, 1 mg/L), transfenin (ape-bovine, Sigma, 5 mg/L), glutathione (Sigma,
1 mg/L), insulin (bovine, 0.01 mg/L), sodium selenite (Sigma, 5 mg/L), antibiotic/

antimycotic(1nvitrogen, 10 ml/L), gentamicin(1nvitrogen, 2.5 ml/L), and bovine serum

88

 

albumin (BSA) (Invitrogen, 2.625 g/L). In a sterile culture hood (Biological Safety
Cabinet, Forrna Scientific, Marietta, OH), parenchymal tissue was diced into small pieces
using sterile scissors and placed into a petri dish containing medium. Medium was
aspirated and tissue was separated into 15 g quantities using tared petri dishes. Each 15 g
quantity of tissue was placed into a sterile bottle containing 150 ml of medium (M-199
with 5X antibiotics) with 1 ml of DNase (Sigma, 4 mg/ml) and 100 pl insulin (Sigma, 1
mg/ml). DNase digests nucleic acids, which leak into the medium as a result of cell
damage, without damage to the intact cell (Freshney, 1994). Bottles were placed on a
shaker (Brinkmann OrbiMix 1010, Incubator 1000, Germany) for 10 min at 37°C and
180 rpm. Tissue was allowed to settle for 5 min and medium in the middle phase was
aspirated. Tissue was transferred to 50-ml tubes and again was allowed to settle before
medium was aspirated. An enzyme digestion mixture [150 mg collagenase (Worthington
Biochemical, Type II, CLS-2, Lakewood, NJ), 150 mg hyaluronidase (Sigma), 150 ml of
medium; for 15 g of tissue] was made and sterile filtered. Collagenase degrades the
triple-helical collagen fibrils and disperses stroma, leaving epithelial cells in small
clusters. Hyaluronidase, a polysaccharidase, breaks bonds in hyaluronic acid and
chondroitin sulfate that are both found in high concentrations in connective tissue
(Freshney, 1994). The enzyme mixture was transferred to an Erlenmeyer flask and 15 g
of tissue was added. Also, 1 ml of DNase and 100 pl of insulin were added to each flask.
During the first digestion, flasks were incubated at 37°C and shaken at different times
and speeds depending on the isolation (see Table). The floating fatty layer was removed.

The remaining mixture was poured through Nitex filters (75 and 200 pm) so that

organoids between 75 and 200 pm in size were harvested. Organoids greater than 200

89

pm were transferred to the digestion solution and placed on the shaker for additional time
periods depending on the isolation (see Table, second digestion). For isolations #1, #2
and #3, only a 200-pm filter was used. The 75-pin Nitex filter was added into the
protocol for the last two isolations to filter out most of the single-cell suspension that
resulted from the digestion procedure.

Filtered cell organoids were transferred to 50-ml tubes and centrifuged at 110 x g
for 10 min. The supernatant was aspirated, cells were transferred into clean 50-ml tubes
with 45 ml of media, and tubes were centrifuged at 110 x g for 10 min. The supernatant
was aspirated and cells were transferred to clean 15-ml tubes with 10 ml of medium.
Cells were mixed with medium and then cells were allowed to settle for 6 minutes. The
supernatant was aspirated and medium was replaced to total 10 ml. This washing process
was repeated 10 times. Freezing solution [50% FBS, 44% basal medium, 6% DMSO
(dimethyl sulfoxide, J .T. Baker)] was prepared with 1 ml of a cell pellet equaling 10 vials
of cells plus media (1.8 ml/vial). The 2-ml cryogenic vials were immediately placed in
freezing containers (N algene Cryo 1°C, Rochester, NY) in an -80°C freezer and stored
overnight. The following day, cryogenic vials were transferred to liquid nitrogen until

use in cell culture experiments.

90

 

Table 3. Differences between primary bovine mammary epithelial cell isolation
procedures from five different heifers. Different times and shaker speeds were used for
each isolation. A 75-pm filter was used in two of the isolations and the enzyme, pronase,
was used in one isolation. During the last two isolations, collection of tissue near the
parenchyma/fat pad interface was less aggressive.

 

 

 

Heifer 75-um 1Sf 2'"I 3rd Pronase Total
Nitex Digest Digest Digest Tissue
1 4.5h@180rpm 14h@90rpm 75g
2 3h@180rpm 14h@90rpm 53g
3 l/a:1h@165rpm 15min@90rpm 85g
l/2:1.5 h @165 rpm, 45 min @180 rpm
1 h at 90 rpm
4 X 1h@165rpm 20min@90rpm 30min@ X 28g
90 rpm
5 X 4.75h@90rpm 14h@90rpm 59g

11‘“

 

91

 

PMEC Observations and Controllirg Fibroblast Contamination:

 

Our laboratory and other groups have dealt with problems associated with
fibroblast cell contamination in primary cell populations (Freshney, 1994). Primary cells,
harvested from tissue isolated from five heifers, were observed while growing on both
plastic and in a collagen gel matrix. On both surfaces, fibroblast cells were present along
with epithelial cell organoids. The presence of fibroblasts was verified by
irnmunohistochemistry using antibodies against proteins specific for different cell types
(see below).

Changes were made in the primary cell isolation to prevent or lower the amount
of fibroblast cell contamination. One of these modifications was the inclusion of a 75-
pm Nitex filter, so that organoids would be between 75- and 200-pm in size. Most
fibroblast cells were observed to be in a single-cell suspension; therefore, it was thought
that adding a smaller size filter to the protocol would eliminate a majority of the
fibroblast cells. However, addition of this step did not prevent fibroblast contamination.

Baffled shake flasks (Kimax, Kimble Glass Inc., Vineland, NJ) were used during
tissue digestion for the second and third isolations. The baffled flasks have indents at the
bottom to provide greater turbulance so that the tissue is more agitated during the
digestion process. However, using these baffled flasks decreases the length of the
digestion process. After personal communication with Dr. Sandy Haslarn (Michigan
State University), it was decided to use Erlenmeyer flasks instead to allow the tissue to be
in contact with the digestive enzymes for a longer period of time.

The use of pronase (Protease, Streptomyces griseus, Calbiochem) was included in

one of the PMEC isolations. Half of the tissue in this isolation was subjected to pronase

92

 

(15 mg / 150 ml of enzyme digestion mixture) while the other half was not; therefore, the
use of pronase could be compared against normal conditions. Observations of cells
showed that fibroblasts were still evident in the isolation using pronase. Cells did not
seem affected by the use of pronase as cells grew in a dose-dependent manner in response
to increasing amounts of IGF-I.

Manipulating the culture conditions by using selective medium is a standard
method in isolating specific cell types (F reshney, 1994). Sordillo et al. (1988) replaced
L-valine in the culture medium with D-valine and demonstrated that contamination of
bovine mammary epithelial cell cultures with fibroblasts could be prevented by use of the
D-valine medium. Epithelial cells, but not fibroblasts, possess the D-amino acid oxidase
and therefore grew normally. Minimum Essential Medium (MEM) D-valine (Gibco) was
chosen to use as a selective medium in our experiments. Both MEM D-valine and M-199
medium were used and cells were treated with basal medium, FBS at 10% concentration
of medium and an IGF-I dose-response series. Observations of cells grown in MEM D-
valine showed no response for IGF-I at all doses and only a minimal response to FBS
compared to M-199. The cells grown in D-valine medium yielded little proliferation and
had fewer fibroblasts compared to cells grown in M-199. To test the MEM media, both
L-valine and D-valine MEM medium were tested on PMEC. Observations of plates
yielded little proliferation of cells grown in both L-valine and D-valine. More fibroblasts
were apparent when grown in L-valine than with D-valine. In conclusion, the PMEC
isolations did not grow normally in the MEM medium and therefore this medium must

lack an essential nutrient needed for this cell type to grow. In addition, use of D-valine

93

 

did decrease the number of fibroblasts present but this procedure was discontinued due to
the lack of normal cell growth and cell response to a known mitogen.

Cell isolations (#1 and #3) were re-digested to increase the time that the epithelial
and fibroblast cells were in contact with the digestive enzymes, hyaluronidase and
collagenase. Vials of cells were thawed and washed in medium. Cells were added to a
flask containing medium, 1 ml DNase and 100 pl insulin. The flask was shaken for 10
min at 37°C and 180 rpm. A digestive enzyme solution (200 mg hyaluronidase, 200 mg
collagenase; 100 ml medium) was made and added to the flask containing cells. Cells
were digested with enzymes for 30 min at 37°C and 90 rpm. Cells were then filtered
through the two sizes of Nitex, washed and frozen as previously stated. When grown in
culture, re-digested cell isolations had approximately 5 to 10% fibroblasts.

The use of a Percoll gradient was not used as a cell dissociation method in this
project because the fibroblasts seemed to be embedded within the organoid. However,
this gradient has been used to separate mammary epithelial components from stromal
cells and blood elements (Hauser et al., 1990). Epithelial fragments sediment to 1.06-
1.07 g/ml density region, while stromal and blood elements can be collected at 1.03-1.04
g/ml density region (McGrath, 1987). The use of a Percoll gradient should be
investigated more to see if this technique could separate the epithelial cell organoids from

the fibroblast cells in these cell populations.

Immunohistochemisg:

The amount of fibroblast contamination within the primary mammary epithelial

cell population was quantified using immunohistochemistry. The Vectastain ABC kit

94

 

(Vector Labs, Burlingame, CA) and monoclonal antibodies against vimentin, Cytokeratin
Peptide 18 and Cytokeratin Peptide 17 (Sigma) specific to fibroblast, epithelial and
myoepithelial cells, respectively were used to identify cell types.

PMEC were grown overnight in a 10% FBS medium on top of round plastic cover
slips placed into 24-well plates. After 24 h in culture, the 10% FBS was aspirated, cells
were washed and medium was changed to basal medium. Medium was aspirated, cells
were washed with Dulbecco’s phosphate buffered saline (PBS, Gibco), PBS was
aspirated and cells were then air-dried, after three days in culture. Methanol (750 pl,
100%) at 4°C was added to each well, incubated for 5 min and aspirated. Hydrogen
peroxide (0.3% H202, Sigma, 750 pl) diluted with methanol was added, incubated for 30
min, and aspirated. Cells were rinsed with PBS, gently rocked for 5 min, and PBS was
aspirated. The Vectastain ABC universal kit (Vector Laboratories, Inc., Burlingame, CA)
contained the blocking serum, a secondary antibody and the ABC complex. Normal
blocking serum (15 ml PBS, 3 drops serum; 750 pl) was added, incubated for 20 min, and
aspirated. Cells were rinsed with PBS, gently rocked for 5 min, and PBS was aspirated.
Primary antibody (750 pl) was added, incubated 30 min, and aspirated. Primary
antibodies included Cytokeratin Peptide 18 (1:100), Cytokeratin Peptide 17 (1:400) and
vimentin (1:100) and were diluted using a buffer solution (100 ml PBS, 2 drops serum).
Cells were rinsed with PBS, gently rocked for 5 min, and PBS was aspirated. Secondary
antibody (10 ml PBS, 4 drops serum, 4 drops secondary antibody; 750 pl) was added,
incubated 30 min, and aspirated. Cells were rinsed with PBS, gently rocked for 5 min,
and PBS was aspirated. The ABC complex (15 ml PBS, 6 drops reagent ‘A’, 6 drops

reagent ‘B’; 750 pl) was added, incubated 30 min, and aspirated. Cells were rinsed with

95

PBS, gently rocked for 5 min, and PBS was aspirated. The PBS wash was repeated three
times. Vector NovaRed peroxidase solution (15 ml water, 9 drops reagent ‘1’, 6 drops
reagent ‘2’, 6 drops reagent ‘3’, 6 drops H202; 750 pl) was added, incubated for 1.5 to 2
min and aspirated. Binding of the peroxidase solution is indicated by the red appearance
of the cells in the figures (Figures 14, 15 and 16). Some of the slides were counterstained
with hematoxylin solution (Gill No. 2, Sigma, blue appearance) by addition of stain (750-
pl), incubated for 30 sec, and aspirated. Cells were then rinsed with water and dried
using ethanol at 95 and 100%. Cover slips were fixed to glass slides.
Immunohistochemistry images in this thesis are presented in color.

After immunohistochemical staining, it was apparent that the cultures of the
primary cell population harvested contained approximately 10% fibroblast cells (Figure
15). Primary cells were highly stained for Peptide 18 (Figure 14), and both organoids
and single scattered cells stained for vimentin. Fib-C cells, an immortalized cell line with
fibroblast-like properties, (Woodward et al., 1995) and the MAC-T cell line were also
used to test the antibodies on other bovine cell types. MAC-T cells stained positive for
Cytokeratin Peptide 18, were lightly stained for Cytokeratin Peptide 17, and were
negative for vimentin. F ib-C cells stained positive for both vimentin and Cytokeratin
Peptide 18. The Fib—C cells had fibroblast-like morphology. One reason for why the
Fib-C cells stained for Peptide 18 could be because the cell line was isolated while the

cells were differentiating.

96

 

 

Figure 14. Illustration of primary bovine mammary epithelial cells stained red using
irnmunohistochemistry: Vectastain ABC kit and monoclonal antibody against
Cytokeratin Peptide 18. This antibody is specific for epithelial cells. Hematoxylin (blue)
was used as a counterstain. Image is presented in color.

97

 

Figure 15. Illustration of primary bovine mammary epithelial cells stained red using
immunohistochemistry: Vectastain ABC kit and monoclonal antibody against Vimentin.
This antibody is specific for fibroblast cells. Hematoxylin (blue) was used as a
counterstain. Image is presented in color.

98

 

Figure 16. Illustration of primary bovine mammary epithelial cells stained red using
immunohistochemistry: Vectastain ABC kit and monoclonal antibody against
Cytokeratin Peptide 17. This antibody is specific for myoepithelial cells. Hematoxylin
(blue) was used as a counterstain. Image is presented in color.

99

PMEC Culture Experiments:

PMEC were grown in a collagen matrix to achieve the following: 1) observe the
proliferation and morphology of epithelial organoids in culture, 2) estimate the
percentage of fibroblast cells contaminating the primary epithelial cell population, 3) test
primary cells’ response to increasing doses of IGF-I and 4) determine if IGFBP-3
regulates the proliferation of epithelial cells dependently and independently of IGF-I.

Basal medium was prepared using Medium 199 (HEPES Modification, Sigma)
with 2 g NaH2C03 added and pH to 7.4. Basal medium also contained soybean trypsin
inhibitor (Type I-S, Sigma, 1 mg/L), transferrin (apo-bovine, Sigma, 5 mg/L), glutathione
(Sigma, 1 mg/L), insulin (bovine, 0.01 mg/L), sodium selenite (Sigma, 5 mg/L),
antibiotic/ antimycotic (Invitrogen, 10 ml/L), gentamicin (Invitrogen, 2.5 ml/L), and
bovine serum albtunin (BSA) (Invitrogen, 2.625 g/L) and was sterile filtered. Collagen
solution was prepared [10% 10X M-199 medium, 10% collagen (Type 1, Becton
Dickinson), 0.23% 1 N NaOH, 79.8% deionized H20] and 500 pl of solution was
pipetted into each well. This was allowed to incubate for 30 min to allow collagen to gel.
One vial of cells was thawed for each 24-well plate. Cells were washed three times in
basal medium, added to the remaining collagen solution and 500 pl of the collagen/cell

mixture was pipetted into each well on top of initial collagen gel. Basal medium (1
ml/well) was added and plates were stored 24 h in an incubator (Nuaire, US Autoflow) at
5% CO2 and 37°C.

Treatments were prepared and applied to cells after 24 h in culture. All
experiments included basal medium, F BS at 10% concentration of medium and

increasing doses of IGF-I (3.125, 6.25, 12.5, 25, 50 and 100 ng IGF-I/ml of medium).

100

Increasing doses of IGFBP-3 (1, 10, 50, 100, 250 and 500 ng/ml of medium) were
included to test the PMEC response to IGFBP-3 independently of IGF-I. Also, equimolar
concentrations of IGFBP-3 (300 ng/ml) and IGF-1 (50 ng/ml) were included to evaluate
the effects of IGFBP-3 and IGF-1 addition concurrently. After 48 h, treatments were
aspirated and replaced with fresh treatment media. Forty-eight hours later, 1 pCi (2.2 x

106 DPM) of [3 H] thymidine was added to each well and incubated for 2 h. Media was

Cal‘-

aspirated and collagen gels were transferred to eppendorf tubes. Acetic acid (150-pl, :_

‘ ‘“-_ n "

25%) was added to each tube and incubated for 30 min at 37°C to dissolve the gel.

Eppendorf tubes were centrifuged at 500 x g for 10 min and the supernatant was

 

aspirated. Perchloric acid (750 pl, 4%) was added to the cell pellet, tube was centrifirged
for 10 min at 500 x g and the supernatant was aspirated. This step was repeated with 750
pl of 80% ethanol, 750 pl of 100% ethanol and 750 pl of 4% perchloric acid. Next, 500
pl of 6% perchloric acid was added to the cell pellet. Cells were hydrolyzed in an 80°C
waterbath for l h, cooled to room temperature and centrifuged for 15 sec at 500 x g. The
supematant was transferred to a scintillation vial and 5 ml of scintillation fluid was
added. Tritiated thymidine was quantified on a beta counter (TM Analytical Mark V 32
ARI). Calculated counts per minute (CPM) were adjusted to disintegrations per minute
(DPM) assuming 40% efficiency of the counter.

Results showed that increasing doses of IGFBP-3 did not inhibit proliferation of
PMEC (Figure 16). However, this effect may have not been independent of IGF-I, since
fibroblasts were present in the cell population and stroma is known to produce IGF-I
(Hauser et al., 1990). A dose of 10 ng of IGFBP-3/ml of media stimulated cell

proliferation when compared to IGFBP-3 doses of 250 ng (P=0.04) and 500 ng' (P=0.06).

101

IGFBP-3 (300 ng/ml), added to equimolar concentrations of IGF-I (50 ng/ml), reduced

DNA synthesis of primary bovine mammary epithelial cells (P=0.03, Figure 17).

 

102

250000

 

200000

 

150000

 

DPM

100000

 

50000

 

 

0

 

 

 

IGFBP-3 (11ng

Figure 17. Effect of increasing doses of IGFBP-3 (1, 10, 50, 100, 250 and 500 ng/ml of
media) on [3 H] thymidine incorporation into DNA of primary bovine mammary epithelial
cells. Results are reported as DPM (disintegrations per minute). Data are presented as LS

means i SEM. Basal medium (0) and 10% fetal bovine serum (FBS) were included as
controls.

103

 

120000
100000
80000
60000
40000
20000
0

DPM

 

50 ng IGF-1 50 ng IGF-1 + 300 ng IGFBP-3

Treatment (nyml)

Figure 18. DNA synthesis of primary bovine mammary epithelial cells ([3H] thymidine
incorporation into DNA) in medium containing IGF-I alone (50 ng/ml) or with IGFBP-3
(300 ng/ml). IGFBP-3, added to equimolar concentrations of IGF-I, reduced DNA
synthesis of primary bovine mammary epithelial cells (P=0.03). Results are reported as
DPM (disintegrations per minute). Data are presented as LS means :1: SEM.

104

Preparation of Rat Tail Collagen:

Rat tails, which had previously been frozen at -20°C, were placed in 70% ethanol
for 30 min. Tails were dissected to collect collagen threads and threads were transferred
to a petri dish containing 70% ethanol. Five grams of collagen threads were weighed out
in a clean tared petri dish to make 1 liter of collagen. Collagen threads were incubated 30
min in 70% ethanol. In a sterile culture hood, 1 ml of glacial acetic acid and 1 l of sterile
deionized H2O were placed into an Erlenmeyer flask that contained a magnet. Collagen
threads were transferred to the Erlenmeyer flask and incubated for 72 h on a stir plate at
4°C. Collagen was centrifuged for 60 min at 4°C and 13,000 x g. The supernatant was
poured into a sterile bottle and centrifuged for 45 min at 4°C and 35,000 x g. The
supernatant from the second spin was filtered through 75-pm Nitex (Sefar America,
Kansas City, MO) and placed in a sterile bottle.

Collagen (25 ml) was added to 4 ml neutralizing solution (10 ml 10X M199, 5 ml
0.33 M NaOH). The collagen solution had a cloudy appearance before it was plated,
possibly due to protein degradation because of a pH change. The first collagen gel was
firm and attached to plates. However, the second collagen gel containing primary cells
did not attach to the first gel, but floated on top of medium. Collagen was diluted (800
ml collagen, 200 ml sterile deionized H20, 800 pl 25% acetic acid) and 25 ml of diluted
collagen was added to 4 ml of neutralizing solution. Again, the first gel was firm but
medium addition dissociated the second gel from the first gel. After these complications,
it was decided that rat tail collagen purchased from Becton Dickinson (Type 1, Bedford,

MA) would be used for future experiments.

105

MAC-T Cell Culture Experiments:

MAC-T cells were grown on a collagen monolayer to achieve the following: 1)
observe the proliferation and morphology of MAC-T cells in culture, 2) test the MAC-T
cells’ response to increasing doses of IGF-I, 3) determine if IGFBP-3 regulates the
proliferation of epithelial cells dependently and independently of IGF-I, and 4) evaluate
the regulation of IGFBP-3 synthesis by TGF-B in MAC-T cells.

MAC-T cells of a single passage were grown up for use in cell culture
experiments. Basal medium (DMEM/F12, Invitrogen) containing HEPES (Invitrogen,
Carlsbad, CA, 7.4 g) and NaH2C03 (cell culture grade, Sigma, St. Louis, MO, 2.478 g),
was titrated to a pH of 7.3 and sterile filtered. Basal medium also contained soybean
trypsin inhibitor (Type I-S, Sigma, 1 mg/L), transferrin (apo-bovine, Sigma, 5 mg/L),
glutathione (Sigma, mg/L), insulin (bovine, 0.01 mg/L), sodium selenite (Sigma, 5
mg/L), antibiotic/ antimycotic (Invitrogen, 10 ml/L), gentamicin (Invitrogen, 2.5 ml/L),
and bovine serum albumin (BSA) (Invitrogen, 2.625 g/L). Cells were plated at 1.5 X 104
cells per well in a 24-well plate. Cells were plated on top of a collagen monolayer (Type
I, Becton Dickinson, Bedford, MA) in a mixture of BM supplemented with 10% fetal
bovine serum (F BS, Invitrogen). After 24 hours in culture, medium was aspirated, cells
were washed, and 1 m1 of BM was applied to each well. For the IGFBP-3 experiments,
all cells were pre-incubated for 24 h with 80 ng IGFBP-3/ml on day 3. After 72 h in
culture, treatments were applied to MAC-T cells.

MAC-T cells were treated with increasing doses of IGFBP-3 (human
recombinant, Upstate Biotechnology, Lake Placid, NY) at 0.5, 1, 10, 20, 40 and 80 ng/ml

of medium. Equimolar concentrations of IGFBP-3 (40 ng/ml) and IGF-1 (8 ng/ml) were

106

 

included to evaluate the effects of IGFBP-3 and IGF-1 addition concurrently. Also, the
effects of increasing doses ofTGF-131 (human recombinant, Sigma) at 5, 10, 50, 100, 500
and 1000 pg/ml were tested on MAC-T cells. All experiments included BM, FBS at 10%

concentration of medium and an IGF-I dose-response curve of 0.5, 1, 2, 4, 8 and 16 ng/ml
of medium. After 24 h in treatment media, 1 pCi (2.2 x 106 DPM) of [3H] thymidine was
added to each well and incubated for 2 h. Media was aspirated and a series of washes
was conducted: Hanks’ Buffered Salts (HBSS, Sigma, 1 ml, 2X), 90% ethanol (250 pl,
2X), Trichloroacetic acid (TCA, Sigma, 250 pl, 2X), and 90% ethanol (500 pl, 2X).
Cells were then air-dried for 10 min and 250 pl of triton (0.1% NaOH) was added per
well. Plates of cells were gently rocked for 30 min and a 100 pl sample was taken from
each well and placed into scintillation vials. Scintillation fluid (5-ml) was added to each
vial and [3 H] was quantified on a beta counter (TM Analytical Mark V 32 ARI).
Calculated counts per minute (CPM) were adjusted to disintegrations per minute (DPM)
assuming 40% efficiency of the counter.

Results of the IGFBP-3 experiment showed that IGFBP-3 did not inhibit DNA
synthesis independently of IGF-I (Figure 18). An equimolar concentration of IGFBP-3
did inhibit IGF-I-stimulated DNA synthesis by 51% in MAC-T cells (Figure 19). TGF-
5! experiments showed that at levels 2 10 pg/ml, TGF-B1 reduced [3 H] thymidine

incorporation in MAC-T cells as compared to treatment with BM (Figure 20).

107

 

120000

 

100000

 

80000

 

60000
40000

 

IIPRG

20000

 

 

romp-3 (rug/ml)

Figure 19. Effect of increasing doses of IGFBP-3 (0.1, 1, 10, 20, 40 and 80 ng/ml of
media) on [3 H] thymidine incorporation into DNA of MAC-T cells. Results are reported
as DPM (disintegrations per minute). Data are presented as means 1: standard error of the
mean. Basal medium (0) and 10% fetal bovine serum (F BS) were included as controls.

108

1 50000

 

100000 _

 

DPM

50000 _

 

   

 

 

8 ng IGF-I 8 ng IGF-I + 40 ng BP3
Treatment

Figure 20. Inhibition of DNA synthesis of MAC-T cells in medium containing IGF-I
alone (8 ng/ml) or with IGFBP-3 (40 ng/ml). IGFBP-3, added to equimolar
concentrations of IGF-I, reduced DNA synthesis of MAC-T cells by 51%. Results are

reported as DPM (disintegrations per minute). Data are presented as means :l: standard
error of the mean.

109

 

120000

 

 

 

 

DPM
as
8
8

L

 

 

 

 

FBS 0 5 10 50 100 500 1000
TGF-131 (Pg/ml)

Figure 21. Effect of increasing doses of TGF 431 (5, 10, 50, 100, 500 and 1000 pg/ml of
media) on [3 H] thymidine incorporation into DNA of MAC-T cells. Results are reported

as DPM (disintegrations per minute). Data are presented as means :1: standard error of the
mean. Basal medium (0) and 10% fetal bovine serum (F BS) were included as controls.

110

Mitggenic Activity of Mammary Extracts fiom Prepubertal and Postpubertg Heifers:

From approximately 3 to 9 months of age, the rate of mammary gland growth is
3.5 times faster than the overall rate of body growth (Sinha and Tucker, 1969). This
period of development is considered the allometric phase. Puberty and maturation of the
ovaries signal the end of the allometric phase. From approximately the third estrus and
until pregnancy, the growth of the mammary gland occurs at the same rate as body
growth and is considered an isometric phase of development. Therefore, it would seem
logical that the mitogenic activity within the mammary gland would be greater during the
prepubertal period than the postpubertal period.

Mammary extracts were prepared as discussed previously (p. 30) fiom a
prepubertal heifer (232 kg body weight at slaughter) and a postpubertal heifer (347 kg
body weight at slaughter). Reproductive tracts were dissected to determine whether or
not heifers had ovulated. MAC-T cells were plated as described above, except at 1 X 104
cells per well. Mammary extracts were used as treatments at 1, 3 and 5% of BM on
MAC-T cells to evaluate the mitogenic activity of the extracts after 72 h in BM. Sixteen
hours after treatments were applied, 1 pCi (2.2 x 106 DPM) of [3 H] thymidine was added
to each well and incubated for 2 h. At 18 h, a [3 H] thymidine incorporation assay and a
total DNA assay (p. 33) were performed on two separate culture dishes. In addition, the
total DNA assay was performed on a third culture dish at 40 h after treatment addition.

Results showed that mammary extracts prepared from parenchymal tissue of a
prepubertal heifer increased total DNA at 40 h more than extracts of tissue from a

postpubertal heifer. This effect was significant at concentrations of 3% (P=0.03) and 5%

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of medium (P<0.001) and tended to be significant at concentrations of 1% of medium

(P=0.09).

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'FI-e

*IF

6 flPrepubertal
I Po stpubertal

 

 

DNA (ug/ml)

 

 

 

 

 

1% 3% 5%
Mammary Extract

Figure 22. Effect of mammary extracts (at 1, 3 and 5% concentrations) from prepubertal
and postpubertal heifers (n=1 heifer per stage) on total DNA of MAC-T cells after 40 h.
Results are reported as micrograms of DNA per well. Data are presented as LS means i
SEM. Asterisks indicate treatment with extracts from a prepubertal heifer increased DNA
more than those from a postpubertal heifer (t P=0.09; * P=0.03; ** P<0.001).

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