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TNZIIS

This is to certify that the

thesis entitled

PROLACTIN REGULATION OF
BIOCHEMICAL AND CYTOLOGICAL DIFFERENTIATION
OF MAMMARY EPITHELIAL CELLS AND
MILK SECRETION IN PERIPARTURIENT COWS

presented by

Robert Michael Akers

has been accepted towards fulfillment
of the requirements for

 

 

Ph.D. degree in Dairx Science

34 mag/We

Major professor

Date February 29, 1980

0-7639

 

  

     

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LIBRARY

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OVERDUE FINES:
25¢ per day per item

RETUMIIE LIBRARY MATERIALS:

Place in book return to remove
charge from ct rculat‘lon records

 

 

 

    
  
   
   
   
  
   

PROLACTIN REGULATION OF

BIOCHEMICAL AND CYTOLOGICAL DIFFERENTIATION
OF FMAMMARY EPITEELIAL CELLS AND

NILE SECRETION IN PERIPARTURIENT COWS

By , l

Robert Michael Akers

:
_\.‘—_’ “A A __ _

I

A DISSERTATION ‘ 1

l;‘— . ‘ 1‘

Michigan State University h .r ;. -

In partiai fuliillmeht or the requirements _$kgl

.n prenattn: ‘01°’.t§9 degrog.gt ‘ . -~ '*11

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DOCTOR OI PHILOSOPHY
y1«ce4 syntjfi its- 7

.YBthe‘fi. my: 81' -' ‘.( (vi-ifs.”
‘ (J * -J“lnepsreueusrer Dairy Science

.435. .

.zur 7 19304‘

 

 

 

/7

 

ABSTRACT
PROLACTIN REGULATION OF
BIOCHEMICAL AND CYTOLOGICAL DIFFERENTIATION
0F MAMMARY EPITHELIAL CELLS AND
MILK SECRETION IN PERIPARTURIENT cows
By

Robert Michael Akers

Prolactin involvement in periparturient mammary de-
velopment and onset of milk secretion was studied in mul-
tiparous Holstein cows. Udders were obtained 10 days pre-
partum from untreated cows and 10 days postpartum from
untreated cows and cows treated with CB154 (2-bromo-a-
ergocryptine) or CB154 plus prolactin. Changes in the
mammary gland during the interval between 10 days pre-
partum and 10 days postpartum had a marked effect on the
capacity of mammary gland to synthesize milk. Rates of
lactose synthesis, fatty acid synthesis and CO2 production
were 4 to 14—fold greater (P i .01) in lactating controls
than in prepartum controls. In association with lower
rates of fatty acid and lactose synthesis, activities of
acetyl-CoA synthetase, acetyl-COA carboxylase and fatty
acid synthetase and concentrations of a-lactalbumin were
lower (P < .001) in mammary tissue from prepartum than
postpartum udders. Udders from cows killed prepartum con-
tained 40 and 80% less (P < .001) total DNA and RNA respec-
tively, than udders from lactating cows, and the RNA/DNA

ratio was higher (P < .01) in lactating controls (1.86)

 

 

Robert Michael Akers

than in prepartum controls (.95). Mammary epithelial
cells from prepartum glands were cytologically undifferen-
tiated, but 73 and 27% of the cells from lactating control
mammary glands were fully and intermediately differen-
tiated, respectively. Furthermore, compared with alveolar
cells from prepartum mammary glands, there were 11.3 and
5.2-fold increases (P < .001) in the cellular areas con-
taining Golgi vacuoles and rough endoplasmic reticulum
(HER), respectively, in lactating controls. Consequently,
reduced biosynthetic activity of prepartum mammary tissue
resulted because the tissue contained fewer epithelial
cells, and the cells present were poorly differentiated
relative to alveolar cells from lactating mammary glands.
Treatment with C8154 reduced basal serum prolactin

concentrations 5 to 7-fold, blocked the normal surge in

vserum prolactin concentrations at parturition and pre-

vented the usual increase in prolactin concentrations in
serum after milking (P i .001). In addition, average milk
production in cows treated with CB154 was 11.4 kg/day
lower (P < .001) than in lactating control cows during 10
days of lactation. However, periparturient infusion of
exogeneous prolactin finronly 6 days into a second group of
cows treated with CB154 restored milk yields to those ob-
served in untreated controls.

Changes in periparturient serum concentrations of
growth hormone, progesterone and glucocorticoids were not

significantly affected by CB154 or CB154 plus prolactin.

 

l

 

 

Robert Michael Akers

Nor did treatment with C8154 affect feed intake of cows.
Udders from postpartum controls or from cows treated
with either 08154 or C8154 plus prolactin contained rela—
tively equal amounts of DNA (P > .05). Thus, reduced milk
yields among cows treated with C8154 apparently were
caused by reduced cellular activity rather than fewer
cells. Rates of lactose synthesis were 40% lower
(P < .01) and concentrations of a-lactalbumin 48% lower
(P < .001) in cows treated with C8154 than in lactating
controls. Mammary tissue from cows treated with C8154
also tended (P < .07) to exhibit slower rates of fatty
acid synthesis than mammary tissue from lactating controls
or cows treated with C8154 plus prolactin. Inhibition of
prolactin secretion reduced (P < .05) total RNA content of
the mammary gland 36% and decreased the RNA/DNA ratio
(1.38), relative to cows which received prolactin replace-
ment therapy (ratio = 2.17) or untreated lactating con-
trols (ratio = 1.86). Also, among cows treated with C8154,
18% of the mammary epithelial cells were undifferentiated,
65% were intermediately differentiated and only 18% were
classified as fully differentiated. In contrast, undif-
ferentiated cells were not observed in lactating controls
or in cows treated with C8154 plus prolactin, and 73 and
79% of the epithelial cells, respectively, were fully dif-
ferentiated. Electron microscopic observations confirmed
the histological data. Specifically, the RER occupied 26

and 27% of the alveolar cell area in lactating controls

 

Robert Michael Akers

  
  
 
  
 
 
  
  
  

3i afin cows treated with 08154 plus prolactin, respec- ‘
~ml 1vely; but only 16% in cows treated with C8154 (P < .001). i
'C'H fi1flilarly, the relative area occupied by Golgi membranes

. Sand vacuoles was approximately 11% lower (P < .001) in
'1‘? ;sows treated with 08154 than in lactating controls or cows
‘ that received prolactin replacement therapy.

I conclude that periparturient secretion of prolactin
induces structural and biochemical differentiation of the
mammary epithelium, and that prolactin is essential for

the onset of copious milk secretion in cattle.

 

I
_A g.‘- A

DEDICATION

    
   
  
  
   
  
 
 
  
  
  

'_.' ‘1"? ('8

L ";I‘ '*'_‘"
I dedicate this dissertation to my wife, Phyllis

qr" ‘1.!!.L."'r

w‘- ehtherine Hamby Akers. Her love and CompanionShip has 9
-‘ fin) re." v
":1 finch an ever present Source of strength and support in -4

 

e713. past and I trust fer many years to come. i '
1'0 Guiu.nr~ b' . . . '*r~xl

em. 0:11 ._ . .. . . . .

I y _ I

'3 r ' 4.11. . or“ ‘I

’mreciau: 1r. 1 ‘ WC ' sa' . .‘ Maw ".31' . as;

Ice nf ‘;)."“,r.'.-;‘. to and \..:~'..I‘.“.,'_

  
   
  
  
  
  
  
  

To til 11:, -‘C"'oxn.$'g‘ .r-. -; - f3!» - .. -I"»‘. an.
’1‘33’5": nonetheless, E wtbi: '- 3.7 11596
. To Mr. "(1:0 “km; :1-‘: - 2..., a . 7 Jaw.“
"911.2 of help Ing basis.

tmtd also like to (Fauna ‘: . . .15?
‘CapUCO and Er. Lasts -R¥; 4 ~ Hat;
3ertezion A...» e. .. .,- 7 T-,

1".)- I then: 5': int“; “‘33.; .1: ,9 _. I»: #3
 *<1 90! trot-a.cst~c

 

 

ACKNOWLEDGMENTS

Completion of any task or fulfillment of any goal
represents a stop in a continuous process. Moreover,
where we end up depends, at least partially, on where we
start and the people we meet along the way. It has been
my good fortune to have been associated with many wonder—
ful people during my journey.

I would like to extend my thanks to the members of
my Guidance Committee - Drs. S.D. Aust, E.M. Convey,

J.L. Gill, G.R. Hooper and H.A. Tucker for their hard
work in my behalf. In particular, I extend my sincere
appreciation to Dr. H. Allen Tucker. His uncompromising
dedication to hard work, loyalty and honesty has been a
source of inspiration and admiration.

To all my contemporaries - you really are ”wild and
crazy guys"; nonetheless, I wish you all the best of good
wishes. To Mr. Tim Goodman - thank you for the friendship
and pair of helping hands.

I would also like to thank Drs. Dale Bauman and
Anthony Capuco and Mr. Larry Chapin. Without their help
this dissertation might never have been completed.

Lastly, I thank my wife (Cathy) for her love and in

particular, for typing this dissertation.

iii

 

 

TABLE OF CONTENTS

Page

LIST OF TABLES ....................................... vi
LIST OF FIGURES ...................................... Vii
INTRODUCTION ......................................... 1
REVIEW OF LITERATURE ................................. 3
1.1 Lactogenesis — Definition ................. 3

1.2 Developmental Aspects of Lactogenesis ..... 3

1.3 Hormones and Lactogenesis - Monogastrics.. 13

1.4 Periparturient Secretion of Hormones ...... 19

1.5 Hormones and Lactogenesis - Ruminants ..... 22
MATERIALS AND METHODS ................................ 29
2.1 Animals ........ i .......................... 29

2.2 Treatments ................................ 30

2.3 Blood Sampling ............................ 32

2.4 Tissue Removal - General .................. 33

2.5 Chemicals ................................. 34

2.6 Tissue Incubations ........................ 34

y 2.7 Enzyme Assays ............................. 36
2.8 Hormone Assays ............................ 37

2.9 Other Assays .............................. 37

2.10 Tissue Composition ........................ 39

2.11 Histological Methods ...................... 40

2.12 Ultrastructural Methods ................... 45

2.13 Statistical Methods ....................... 46
RESULTS..... ......................................... 47
3.1 General Results ........................... 47

3.2 Periparturient Prolactin Secretion ........ 47

iv

   

Pug:

Page

3.3 Periparturient Serum Concentrations of

Glucocorticoids, Progesterone and Growth

Hormone ................................... 58
3.4 Milk Production and Milk Composition ...... 65
3.5 Effect of CB154 on Feed Intake ............ 70
3.6 Fatty Acid Synthesis and CO2 Production

in Mammary Tissue in vitro ................ 77
3.7 Lactose Synthesis and a-Lactalbumin Con-

centrations in Mammary Tissue ............. 77
3.8 Activities of Enzymes Associated With

Fatty Acid Synthesis and NADPH Generation

in Cow Mammary Tissue ..................... 80

3.9 Biochemical Constituents and Trimmed
Weight of Cow Udders ...................... 83

3.10 Qualitative Morphology of Mammary Tissue.. 87
3.11 Quantitative Morphology of Mammary Tissue. 92

3.12 Electron Microscopy of Mammary Tissue ..... 94
DISCUSSION ........................................... 107
SUMMARY AND CONCLUSIONS .............................. 126 A
BIBLIOGRAPHY ......................................... 132

 

   

LIST OF TABLES

, Page
Enzyme Assays ................................. 38

Fatty Acid Synthesis and CO2 Production in Cow
Mammary Tissue Slices ......................... 78

Lactose Synthesis in Cow Mammary Tissue and c-
Lactalbumin Concentrations .................... 79

Average (1 SEM) Activities of Enzymes Associ-
ated With NADPH Generation in Cow Mammary Tis-

sue ......... . ............................. .... 84
Activities of Enzymes Involved in Fatty Acid
Synthesis in Cow Mammary Tissue..... ........ .. 85
Biochemical Constituents and Trimmed Weight of
Cow Udders ............................. . ...... 86
Percent of Mammary Tissue Area Assigned to
Each of Five Tissue Classifications ........... 93
Percent of Alveolar Cell Area Assigned to Each
of Five Sub-Cellular Classifications ....... . .106

5.:

vi

 

 

 

 

LIST OF FIGURES

Light micrographs of epon-araldite embedded,
0.5 to 1 pm sections of mammary tissue
stained with Azure II (all 4000 X) ...........

Fully differentiated alveolar epithelial
cells characterized by presence of numerous
vacuoles (V), rounded basally positioned
nuclei (N), abundant cytoplasm, frequent
occurrence of large apically located lipid
droplets (F) and distinct polarization of
cellular organelles ..........................

Intermediately differentiated alveolar epi-
thelial cells showing fewer cellular vacu-
oles, more irregularly shaped nuclei,

greater estimated nuclear to cytoplasmic
ratio and less pronounced cellular polarity..

Undifferentiated alveolar epithelial cells
displaying relative absence of cellular
vacuoles, highly irregularly shaped nuclei,

a mixture of large and small randomly
located lipid droplets, very large esti-
mated nuclear to cytoplasmic ratio and lack
of cellular polarity .........................

Concentrations of prolactin in serum during
10 days prior to initiation of treatment and
during 6 days after the start of CB154 in-
jections but before infusion of exogeneous
prolactin in cows treated with CB154 plus
prolactin ....................................

Concentrations of prolactin in serum before,
during and after parturition in control cows
and those treated with CB154 .................

Concentrations of prolactin in serum of cows
treated with CB154 plus prolactin during in-
fusion of exogeneous prolactin. Amounts of
prolactin infused during six consecutive
days of infusion are given in the solid bars.

Page

43

43

43

43

5O

52

55

 

 

Figure
5

10

11

12

13

14

15

15-A

Average concentrations of prolactin in serum
before, during and after two PM milkings be-
tween 5 and 9 days postpartum ................

Concentrations of glucocorticoids in serum
from 5 days prepartum to 3 days postpartum...

Concentrations of progesterone in serum from
5 days prepartum to 3 days postpartum ........

Concentrations of growth hormone in serum
from 20 days prepartum to 10 days postpartum.

Average AM and PM milk yields among lacta-
ting controls and cows treated with CB154 or
CB154 plus prolactin .........................

Average concentrations of protein in milk
obtained twice daily among lactating con-
trols and cows treated with C3154 or CB154
plus prolactin ...............................

Average concentrations of lactose in milk
obtained twice daily among lactating con-
trols and cows treated with CB154 or CB154
plus prolactin ...............................

Average concentrations of a-lactalbumin in
milk obtained twice daily among lactating
controls and cows treated with C8154 or
CB154 plus prolactin .........................

Changes in feed intake (expressed as energy
intake as % of requirement) from 10 days
prepartum to 10 days postpartum among lacta-
ting controls and cows treated with CB154 or
CB154 plus prolactin .........................

Relationship between the concentration of a-
lactalbumin (a lac) in mammary cytosol and
rate of lactose synthesis by slices of mam-
mary tissue ..................................

Light micrographs of epon-araldite embedded,
0.5 to l um sections of mammary tissue
stained with Azure II (all 750 X) ............

Tissue from prepartum control. The alveolar

lumena are darkly stained and contain some
fat globules .................................

viii

Page

57

60

62

64

67

69

72

74

76

82

89

89

 

Figure Page
15-B Tissue from postpartum control ............... 89
15-C Tissue from cow treated with CB154 ........... 89
15—D Tissue from cow treated with 08154 plus pro—

lactin ....................................... 89
16 Light micrographs of epon—araldite embedded,

0.5 to 1 pm sections of mammary tissue

stained with Azure 11 (all 4000 X) ........... 91

l6-A Tissue from prepartum control. The epithe-
lial cells display irregularly shaped nuclei
and large nuclear to cytoplasmic ratio ....... 91

16-B Tissue from postpartum control. The epithe-
lial cells have rounded basally displaced
nuclei, abundant apical vacuoles (cellular
polarization) and small nuclear to cyto-
plasmic ratio ................................ 91

16-C Tissue from cow treated with CB154. Com—
pared with postpartum control or 08154 plus
prolactin, the epithelial cells display less
polarization, have fewer vacuoles and
greater nuclear to cytoplasmic ratio ......... 91

l6-D Tissue from cow treated with CB154 plus pro—
1actin. The epithelial cells are similar to
those from postpartum control. The cells
are polarized and contain numerous vacuoles
and apical lipid droplets (arrows). In
addition, darkly stained basal cytoplasm is
denoted by a bracket ......................... 91

17 Alveolar cells from a prepartum mammary
gland characterized by a relative absence of
cellular organelles. Strands of RER and ri-
bosomes attached to the outer nuclear mem-
brane are denoted by arrows (9000 X) ......... 97

18 Alveolar cells from postpartum control mam-
mary gland characterized by marked prolifera-
tion of cellular organelles. Arrows denote
close apposition of Golgi vesicles and lipid
droplets (10,060 X) .......................... 99

ix

 

 

   

. Page
Alveolar cells from cow treated with 03154
distinguished by occurrence of fewer cellu-
lar organelles than lactating control. In
particular, lack of abundant parallel
arrays of BER and abundant apical secretory
vesicles. Strands of BER are denoted by
the arrows (11,760 X) ......... ...............101

Alveolar cells from cow treated with C3154

plus prolactin. The cells are indistin- ‘
guishable from those in the lactating con-

trol (10,040 X)..............................103

 

 

INTRODUCTION

Lactation is an essential part of mammalian reproduc-
tion. In concert with parturition, specialized mammary
gland cells are induced to synthesize and secrete milk,
which is required for the nourishment of all neonatal mam-
mals. Indeed, lactation might be considered the physio-
logical process which culminates the mammalian reproduc-
tive cycle.

Development of the capacity of the mammary gland to
synthesize milk must involve at least three events: (1)
proliferation of alveolar epithelial cells, (2) differen-
tiation of the mammary epithelium, and (3) initiation and
regulation of milk synthesis. The sequence of mammary
growth during pregnancy, periparturient secretory cell
differentiation and initiation of milk synthesis and secre-
tion postpartum, depends on interactions between ovarian,
pituitary, placental, adrenal and thyroid hormones.

Prolactin, in laboratory species, has been implicated
as an important hormone during each phase of lactation, but
it is particularly critical in initiation of milk secretion
at parturition. However, the importance of prolactin in
initiation of lactation in cattle is unclear. Knowledge

of hormonal requirements for mammary secretory cell

  
    
   
  
   

¢
‘ I

.ghfifiups for manipulation of mammary function. Induction of
'fizibarly lactations in heifers and return of non-pregnant

"wpows to milk production are also reasonable expectations.
f¥.~ The objective of the research reported in this dis-

. I113” sertation was to determine the effect of periparturient
.‘;'? prolactin secretion on milk production following parturi?

2% 9‘ tion in dairy cows, and to evaluate the role of prolactin
A. K'.‘
4%,. fin‘biochemical and cytological differentiation of mammary

I"

,k g
. 5g: ..§bcretory cells.
‘. f-s ‘“
M

 

REVIEW OF LITERATURE

1.1 Lactogenesis — Definition

Hartmann (1973) suggested that initiation of lacta-
tion in cows and sheep develops in two phases. The first
phase is characterized by limited secretion of milk con-
stituents in late pregnancy, and the second phase consists
of initiation of copious secretion of milk 0 to 4 days
prior to parturition. In agreement, Fleet £3 a1. (1975)
extended this scheme to include initiation of lactation in

goats. Lactogenesis, consequently, may be defined as pro-

 

gressive cellular and biochemical differentiation of the
mammary alveolar cells during late pregnancy which ulti—

mately results in copious synthesis and secretion of milk.

1.2 Developmental Aspects of Lactogenesis

Milk production depends upon formation of alveolar
epithelial cells during pregnancy. Indeed, the number of
milk synthesizing cells in the mammary gland is one of the
basic elements which limit milk production (Tucker, 1969).

However, a successful lactation requires not only cellular

 

proliferation but also cellular differentiation. Although

the sequence of cellular modifications which result in

   

 

 

 

differentiation of mammary cells during lactogenesis is
unknown; it is reasonable to believe that proliferation

of cellular organelles, formation and activation of mam-
mary enzymes and subsequent synthesis and secretion of
milk are closely coordinated. Thus, periparturient growth
of the mammary gland, biochemical and ultrastructural dif—
ferentiation of mammary cells and cellular aspects of syn—
thesis and secretion of milk protein, lactose and lipid V
will be discussed.

Mammary development during most of pregnancy is char-
acterized by rapid cell division and formation of alveoli
which progressively replace fatty tissue throughout the
mammary gland (Tucker, 1969). Most growth of the mammary
gland occurs during pregnancy, although in some species
substantial growth also occurs during early lactation.

For example, mammary deoxyribonucleic acid (DNA — a mea-
sure of cell number) continued to increase during early
lactation in rat (Tucker and Reece, 1963) and rabbit (Lu
and Anderson, 1973) but in sheep (Anderson, 1975) and ham-
ster (Sinha gt 21., 1970) mammary growth was completed
during the last trimester of pregnancy. It is not known
if the udder continues to grow during early lactation in
cattle, although Tucker gt 31. (1973) reported that DNA
increased 89-fold between 5 months of age and 60 days of
lactation. These results suggest that in some species

continued proliferation of the mammary cells prior to the

 

peak of lactation contributes to subsequent increases in
milk production. Consequently, understanding the factors
which control cell division in the mammary gland could
result in development of techniques to increase milk pro-
duction.

Cellular differentiation and synthesis of milk de—
pends upon formation of ribonucleic acid (RNA). For ex-
ample, production of milk proteins requires that appro-
priate mRNA molecules be available for transcription. RNA
molecules are also components of the ribosomes (the cellu-
lar organelles upon which mRNA molecules are decoded and
proteins synthesized) and finally synthesis of enzymes
which catalyze the myriad of chemical reactions required
to maintain the mammary cells and to make milk each rely
upon mRNA. In most species, total mammary gland RNA con—
centrations increase coincidently and parallel with in-
creasing total mammary DNA concentrations during pregnancy.
Thus, it is probable that RNA accumulated in the mammary
gland during gestation reflects synthesis of RNA resulting
from formation of new mammary cells. The onset of
markedly increased secretory activity, however, is pre-
ceded by increased cellular RNA concentrations (RNA/DNA)
and RNA synthesis (Denamur, 1974). It is likely that in—
duction of RNA synthesis is among the first in the se-
quence of events leading to differentiation of mammary

cells. In support of this idea, accelerated rates of RNA

 

 

synthesis are evident in rats (Tucker and Reece, 1963a),
mice (Yanai and Nagasawa, 1971) and ewes (Denamur and
Gaye, 1967) immediately prior to parturition. Also,
Denamur (1969) reported that mammary RNA concentration
increased in parallel with increasing DNA concentrations
in rabbit mammary gland until day 20 of gestation
(RNA/DNA = 0.9). But on day 20 and 21 of gestation there
was a net increase in the concentration of RNA per cell
(RNA/DNA = 1.3) which preceded the ability of the mammary
tissue to synthesize lactose (first detected on day 22 of
gestation).

However, the capacity to synthesize each of the major
milk components does not necessarily evolve at the same
time during gestation. Nor is there a regular pattern in
the order of appearance of milk components during preg-
nancy. To illustrate, lactose appears only on the last
day of pregnancy in rats (Wrenn gt 31., 1965), about 8
days prepartum in rabbits (Denamur, 1969), as early as 30
days prepartum in cows (Hartmann, 1973), and 30 to 49 days
prepartum in goats (Fleet gt gt., 1975). Furthermore,
casein synthesis is evident in mammary glands of rats
prior to the appearance of lactose (Kuhn, 1972a), but co-
incident with lactose synthesis in rabbits (Bousquet
gt gt., 1969). Such results illustrate the difficulty of
defining the timing of lactogenesis.

In lactating mammary glands, it is evident that

 

intracellular synthesis of milk and subsequent secretion
of milk components from the alveolar cell into the alveo-
lar lumen are closely related. But during lactogenesis
the enzymes required for synthesis of milk may appear be-
fore proliferation of cellular organelles needed for milk
secretion. For example, in rabbit (Mellenberger gt gl.,
1974) and mouse mammary glands (Jones, 1972) the enzymes
required for lactose synthesis are detected soon after
mid-gestation; yet the mammary glands can not synthesize
and secrete lactose until about 8 days prepartum in
rabbits and about 4 days prepartum in mice. How is this
paradox explained? Since synthesis and secretion of lac-
tose requires a well-developed Golgi apparatus (Brew,
1969; Keenan gt gt., 1970), and these organelles are not
observed until very near parturition in rabbits (Bousquet
gt g1., 1969) and mice (Hollmann, 1969), it is probable
that asynchrony of enzyme activity and lactose biosynthe-
sis in these species results from asynchronous cytological
differentiation. These results support the conclusion
that copious milk production requires complete biochemical
and ultrastructural differentiation of the mammary cells.
Many investigators have studied the activities of
mammary enzymes associated with milk synthesis and cellu—
lar metabolism during pregnancy, lactogenesis and lacta-
tion (Baldwin, 1966; Baldwin and Yang, 1974; Baldwin and

Louis, 1975). From such studies, three general

 

conclusions may be drawn: (1) many enzymes are constitu-
ttzg elements of the secretory cells whose activities do
not change coincident with lactogenesis, (2) preferential
increases in the activities of regglatorx enzymes are fun—
damental to lactogenesis, and (3) synthesis and activity
of such regulatory enzymes are under hormonal influence.
Mellenberger gt gt. (1973) reported dramatic increases
in the activities of acetyl-CoA synthetase, acetyl-CoA car-
boxylase, fatty acid synthetase, NADP-isocitrate dehydro-
genase, lactose synthetase and a—lactalbumin concentration
in biopsies of bovine mammary tissue obtained at several
intervals between 30 days prepartum and 40 days postpartum.
These workers also measured rates of fatty acid and lactose
biosynthesis tg gtttg in tissue slices prepared from the
mammary biopsies. Because activities of acetyl-CoA car—
boxylase and acetyl-CoA synthetase were Correlated (r = .97
and .95, respectively) with rates of fatty acid biosynthe-
sis, these investigators suggested that these enzymes reg-
ulated fatty acid synthesis. Similarly, because the activ-
ity of lactose synthetase was correlated (r = .96) with
rates of lactose synthesis, lactose synthetase was hypoth—
esized as a regulator of lactose synthesis. Mammary tis-
sue concentrations of a-lactalbumin also increased coin-
cidently with lactose synthetase activity during lactogene-
sis. In support of these proposals, Mackall and Lane

(1977) reported that the rapid increase in acetyl—CoA

 

 

ii; ..-‘

carboxylase activity at the onset of lactation in the rat
occurs as a result of increased synthesis and decreased
degradation of the enzyme. Also periparturient initiation
of lactose synthesis in the rat mammary gland coincides
with the appearance of a-lactalbumin (Kuhn, 1969a).

As mammary cells are formed during pregnancy, they
remain quiescent until late gestation when critical endo-
crine events signal cellular modification. Such quiescent
cells are characterized by irregularly shaped nuclei, min-
imal rough endoplasmic reticulum (RER), ill-defined Golgi
membranes, few mitochondria and a high nuclear to cyto-
plasmic ratio (Heald, 1974). Comparison of quiescent pre—
lactating epithelia with lactating epithelia indicated
that cytological differentiation includes proliferation
of RER, hypertrophy of Golgi membranes with appearance of
protein-filled vesicles, increased numbers of mitochondria,
secretion of fat globules and casein micelles into the
lumen, reduced nuclear to cytoplasmic ratio and greater
cellular polarization (basal nucleus and ergastoplasm;
apical orientation of the Golgi apparatus and associated
vacuoles) (Hollmann, 1969; Heald, 1974; Wooding, 1977).
Foster (1977) also reported that the volume of mouse mam—
mary epithelial cells increased 21-fold from early preg-
nancy until just prior to weaning.

At the onset of milk secretion, in addition to the

formation of cellular protein, there occurs first the

 

10

induction and then a rapid increase in the synthesis of
proteins specific to milk (caseins, a-lactalbumin and B-
lactoglobulin). Such secretory proteins are thought to
be synthesized on membrane-bound polyribosomes (three or
more ribosomes associated with a molecule of mRNA) whereas
constitutive proteins are believed to be synthesized on
polyribosomes which remain in the cytoplasm. During lac-
togenesis the total number of polyribosomes per cell and
the proportion of membrane-bound polyribosomes to free
polyribosomes increase. For example, free polyribosomes
represent 75 to 85% of the total polyribosomes during mam-
mary growth but only 20% during lactation in the rabbit
and ewe (Gaye and Denamur, 1969; Gaye gt gt., 1973).
Debate among early investigators concerning the ori-
gin of milk proteins (Barry, 1961) was settled when
studies with radiolabeled amino acids confirmed that
casein (Barry, 1952) and a-lactalbumin (Larson and
Gillespie, 1957) were synthesized from free amino acids.
Results of autoradiographic studies (Heald and Saacke,
1972; Ollivier—Bousquet and Denamur, 1975) support the
following scheme for milk protein synthesis and secretion.
In conjunction with membrane-bound ribosomes, milk pro-
teins are synthesized from free amino acids, transported
into the cisternae of the endoplasmic reticulum, then
shunted to the Golgi apparatus where further modification

and/or condensation into membrane-bound secretory vesicles

 

11

occurs. Finally, milk proteins are released from the
secretory cell via exocytosis across the apical plasma-
lemma (Larson, 1979). Protein-like material observed in
Golgi vacuoles in lactating mammary cells appears progres-
sively more condensed:h1vacuoles located toward the apical
cell membrane. And it is thought that this progressive
aggregation of the protein represents casein micelle for-
mation (Saacke and Heald, 1974; Wooding, 1977).

Autoradiographic localization of tritiated fatty
acids (Stein and Stein, 1967) and ultrastructural studies
(Wooding, 1977) indicate that esterification of fatty
acids and initial development of lipid droplets occurs in
conjunction with the RER (Patton and Jensen, 1976). It
seems likely that gg ggzg synthesis of fatty acids of milk
occurs exclusively in the cytoplasm of the mammary secre-
tory cell (Dekay _t _1., 1976), but that RER is involved
in esterification and fat droplet formation, since the
enzymes required for esterification are located in micro—
somal cell fractions (Bauman and Davis, 1974). In con-
trast, the occurrence of fat droplets in prepartum mammary
cells, which contain almost no RER, indicates that a de-
veloped cytomembrane system is not an absolute requirement
for intracellular lipid droplet formation (Saacke and
Heald, 1974).

Secretion of milk fat droplets is characterized by

progressive movement of lipid droplets to the apical

 

 

12

surface of the lactating cell and protrusion of the drop-
let with surrounding plasma membrane into the lumen.
Droplets are then secreted into the alveolar lumen encap—
sulated in a sheath of plasma membrane (Patton and Jensen,
1976; Pitelka and Hamamoto, 1977).

In the final reaction to form the disaccharide lac—
tose, lactose synthetase catalyzes formation of a galac-
tosyl bond in a B linkage between galactose and the 4-
position of glucose. Brodbeck and Ebner (1966) first
showed that lactose synthetase was composed of two dis—
tinct proteins. A component with high molecular weight
was identified as galactosyl transferase and a smaller
subunit as the whey protein, c-lactalbumin. It was only
when these two proteins were combined that lactose syn-
thesis was possible. Subsequent studies established that
a-lactalbumin increases the affinity of the galactosyl—
transferase for glucose, such that it becomes a suitable
acceptor of the galactose moiety of UDP-galactose thus
promoting lactose synthesis. Galactosyl transferase is
common to many tissues and apparently is fundamental in
the biosynthesis of glycoproteins. Hence, it is the pres-
ence of the unique mammary protein, a—lactalbumin which
confers the ability to synthesize lactose on the mammary
gland (Jones, 1977). Brew (1969) proposed the following
cellular mechanism to explain lactose synthesis and secre-

tion. He suggested that a-lactalbumin is synthesized on

 

Ff .

13

the ribosomes, transported through the cisternae of the
rough endoplasmic reticulum to the Golgi apparatus where
it associates with membrane—bound galactosyl transferase
to produce functional lactose synthetase. It is assumed
that newly synthesized lactose is packaged into Golgi
vacuoles along with casein micelles and subsequently re-

leased via exocytosis across the apical plasma membrane.

1.3 Hormones and Lactogenesis - Monogastrics

Initial suggestions that hormones were involved in
mammary development probably arose from observations that
activity of other reproductive tissues were associated
with mammary gland growth. For example, Halban (1900)
observed that transplantation of ovarian tissues into
previously ovariectomized animals restored normal mammary
growth. Subsequently, Hammond and Marshall (1914) re-
ported close association between corpora lutea growth and
mammary growth in pseudopregnant rabbits, and suggested
that a substance from corpora lutea stimulated mammary
growth during pregnancy. Other studies indicated that
secretion of estrogen from the ovarian follicles and pro-
gesterone from the corpora lutea accounted for the mammary
growth attributed to these tissues (Erb, 1977). Turner
and Frank (1931) reported that estrogen and progesterone
synergized to promote normal mammary lobule-alveolar de-

velopment during pregnancy. Such studies confirmed

 

U.“‘_L'2-.

14

hormonal regulation of mammary growth.

Hildebrandt (1904) hypothesized that the developing
fetus suppressed lactation during pregnancy, and Halban
(1905) suggested that during pregnancy the mammary gland
was under ovarian and placental influence, which promoted
growth but inhibited secretory activity. It was not until
Grueter (1928) initiated milk secretion in pseudopregnant
rabbits with injections of anterior pituitary extracts,
that hormonal stimulation of lactogenesis was suggested.
In subsequent studies, Stricker and Grueter (1928, 1929)
induced milk secretion in dogs, cattle and swine with pi-
tuitary extracts.

Thereafter, much effort was devoted to isolation and

identification of the lactogenic hormone from the pitui-

tary (Riddle gt gt., 1933) and development of assay proce— -

dures (Gardner and Turner, 1933; McShan and Turner, 1936).
Much of the lactogenic activity in the pituitary extracts
has been attributed to prolactin. For example, Meites and
Turner (1948a) monitored changes in the pituitary content
of prolactin during gestation and lactation and reported a
marked decrease at parturition. This was interpreted to
represent increased release of prolactin, and suggested
the possible involvement of prolactin in naturally occur-
ring lactogenesis. In concert with these studies,
Drummond-Robinson and Asdell (1926) reported that removal

of the corpora lutea in pregnant goats initiated milk

 

15

secretion. This suggested that progesterone normally in-
hibited lactation during pregnancy. Based on measurements
of pituitary prolactin concentrations, Meites and Turner
(1942a,d; 1948) reported that estrogen stimulated prolac—
tin synthesis, but that progesterone blocked estrogen's
effect on prolactin synthesis. With the knowledge that
estrogen secretion increased late in gestation and proges-
terone secretion decreased prior to parturition, these
workers reasoned that estrogen stimulated prolactin syn-
thesis in concert with decreased periparturient proges—
terone secretion, and that prolactin secretion then rap-
idly increased. Since normally inhibitory concentrations
of progesterone were also reduced, prolactin could then
induce lactation.

__Lyons gt gt. (1958), using triply-operated
(hypophysectomized—ovariectomized-adrenalectomized) rats,
demonstrated that prolactin, growth hormone, estrogen,
progesterone and corticoids were necessary for histologi—
cal development of normal lobule—alveolar structure. In
triply-operated rats with mammary development similar to
that of late pregnancy, prolactin and a corticoid were the
minimal hormonal requirement for lactogenesis. Maximal
lactogenic responses (manual expression of milk from the
gland or occurrence of secretory product in histological
preparations) were observed, however, when growth hormone

was included in the replacement therapy.

 

 

Alfilk. ..

16

Wellings gt gt. (1966) were the first to use electron
microscopy in the study of hormone-induced secretion in
organ cultures of mammary tissue. These workers reported
that a combination of insulin, glucorticoid and prolactin
caused ultrastructural differentiation of mammary explants
prepared from mid-gestation mice. Such results supported
earlier suggestions (Folley, 1940) that a complex of hor—
mones promoted lactogenesis, but provided no indication of
ultrastructural effects induced by these hormones individ-
ually. Subsequent studies (Mills and Topper, 1970), also
utilizing mammary explants from mid-gestation mice, estab-
lished that insulin had no direct effect on ultrastruc-
tural differentiation of the alveolar cells, but insulin
was required for maintenance of the explants during cul—
ture. In response to the addition of hydrocortisone, al-
veolar cells in explants pretreated with insulin exhibited
an intermediate stage of differentiation, characterized by
development of BER and Golgi membranes. The subsequent
addition of prolactin induced cellular polarity and ap—
pearance of Golgi vacuoles filled with casein micelles.
Other effects of prolactin on mammary cells tt gtttg in-
clude: stimulation of milk protein synthesis (Juergens
g a_l., 1965), swelling of Golgi membranes (Ollivier-
Bousquet, 1978), synthesis of a-lactalbumin (Vonderhaar

t 1., 1973; Kleinberg gt gt., 1978) and synthesis of

lactose (Delouis and Denamur, 1972).

 

_nn.¢~'x_3

17

Understanding of the mechanisms whereby prolactin
stimulates milk protein synthesis has been enhanced by
development of methods to quantify mammary mRNA (Rosen,
1976; Rosen and Barker, 1976). Specifically, Matusik and
Rosen (1977) showed that prolactin rapidly induced the
accumulation of casein mRNA in rat mammary gland explants
pretreated with insulin and hydrocortisone. Indeed, the
number of casein mRNA molecules per alveolar cell in-
creased from 478 prior to the addition of prolactin to
641, 1022, 3308 and 6428 molecules per cell 1, 4, 24 and
48 h after addition of prolactin, respectively. No accu—
mulation of casein mRNA was observed in explants cultured
in insulin and hydrocortisone alone, although hydrocorti-
sone combined with prolactin was required for maximal
accumulation. Also, the induction of casein mRNA by pro-
lactin was inhibited in a dose-dependent fashion when
progesterone was included in the culture media. In sub-
sequent studies, Guyette gt gt. (1979) reported that pro-
lactin induced a 2 to 4—fold increase in the rate of
casein mRNA transcription and a 17 to 25—fold increase in
the half—life of casein mRNA in mammary explants from mid-
gestation rats. Prolactin also enhanced rates of casein
synthesis in parallel with increased synthesis of casein
mRNA in organ cultures of pseudopregnant rabbit mammary
gland (Devinoy gt gt., 1978).

Chatterton gt gt. (1979) reported that prolactin was

 

18

required for ultrastructural differentiation of the mam—
mary epithelium after ovariectomy of pregnant rats, and
Tomogane gt gt. (1976) observed reduced litter weight
gains in lactating rats treated with antiserum to rat pro-
lactin. Also, McNamara and Bauman (1978) reported that
suppression of prolactin secretion with C8154 (2-a—
bromoergocryptine methanosulfate) during lactogenesis in
rats inhibited mammary lipogenesis and markedly reduced
subsequent litter weight gains. These workers also hypo-
thesized that prolactin inhibited fatty acid synthesis in
adipose tissue but stimulated milk fat synthesis in mam-
mary tissue coincident with lactogenesis. Furthermore,
Graf gt gt. (1977) reported that growth and histological
development of the mammary gland (induced by estrogen and
progesterone in hypophysectomized rats bearing ectopic
pituitaries) were blocked by CB154. Since pituitaries
transplanted to locations other than sella turcica
(ectopic) secreted large amounts of prolactin, these
workers reasoned that prolactin was necessary for
estrogen- and progesterone—induced stimulation of the mam-
mary gland.

It must be recognized, however, that other hormones
can modify, inhibit or mimic effects attributed to prolac-
tin. To illustrate, thyroid hormones greatly enhanced
prolactin stimulation of a-lactalbumin synthesis in ex-

plant cultures of mouse mammary tissue (Vonderhaar, 1977),

 

19

and placental lactogen was as effective as prolactin in
stimulating histological development and casein synthesis
of mammary tissue in culture (Turkington and Topper, 1966).
Because serum progesterone concentrations are elevated
during nearly all of gestation, progesterone inhibition of
prolactin's ability to induce RNA, protein and lactose
synthesis (Turkington and Hill, 1969; Assairi gt gt.,

1974; Rosen gt gt., 1978) is perhaps most significant.

1.4 Periparturient Secretion of Hormones

Development of sensitive assays to measure concentra-
tions of hormones in blood has resulted in greater under-
standing of changes in hormone secretion coincident with
mammary development and initiation of milk secretion
(Convey, 1974). The following discussion of temporal
changes in hormone concentrations in blood will be based
on data collected from studies with cattle.

The concentration of estrogens in blood gradually in-
crease throughout most of pregnancy (Smith gt gt., 1973).
But marked increases in estrone, estradiol—l7 B and
estradiol—l7 8 occurred during the last 2 weeks of gesta-
tion, followed by rapid decreases soon after parturition
(Robertson, 1974). Although estrogen administration stim-
ulates secretion of prolactin in rats (Chen and Meites,
1970) and from bovine pituitary cells in culture

(Padmanabhan and Convey, 1979) the relationship between

 

20

increased prepartum estrogen secretion and prolactin se-
cretion at parturition in cattle is unknown. For example,
Karg and Schams (1974) reported that in lactating cows
prolactin concentrations in blood were reduced during in-
fusion of estradiol, but 2 to 5-fold increases occurred
when infusions ended. In addition, among cows induced
into lactation with estrogen and progesterone, serum pro-
lactin concentrations increase when steroid treatments are
completed (Collier gt gt., 1977).

Progesterone concentrations remained elevated during
the greater portion of pregnancy (Wettemann and Hafs,
1973), then 2 to 3 days before calving progesterone rap—
idly declined and remained low until recurrence of post-
partum estrus (Smith gt gt., 1973). It is probable that a
reduction in serum progesterone concentrations normally
must occur before initiation of lactation is possible in
cattle.

Glucocorticoids and growth hormone increase sharply
coincident with parturition (Smith gt gt., 1973; Ingalls
gt gt., 1973), which suggests that these hormones may be
released in response to the stress of calving. Although
glucocorticoids and growth hormone elicit lactogenic re-
sponses in mammary tissue tg ttttg, their importance for
initiation of milk synthesis at parturition remains to be
established.

Serum prolactin concentrations do not change during

 

 

21

most of gestation (Vines gt gt., 1977), but a surge in
prolactin secretion occurs 1 to 2 days before calving
which lasts until 1 to 2 days following birth (Johke gt
gt., 1971; Ingalls gt gt., 1973). It must be recognized,
however, that a variety of stimuli including milking
(Tucker, 1971), season (Koprowski and Tucker, 1973), am-
bient temperature (Wettemann and Tucker, 1974), photo-
period (Bourne and Tucker, 1975) and exteroreceptive
stimuli (Terkle gt gt., 1979; Goodman, Tucker and Convey,
1979) influence secretion of prolactin. For example,
Chew gt gt. (1979) reported that changes in photoperiod
were associated with the concentration of prolactin mea-
sured in plasma collected from cows before (13 to 2 days
prepartum), during (1 day prepartum to 0.5 day postpartum)
and after parturition (1.5 to 2.5 days postpartum). Spe-
cifically, plasma collected from cows in June (longest
photoperiod) had 6.4, 1.7 and 3.6-fold greater concentra-
tions of prolactin respectively during each of the three
periods around parturition than samples collected from
cows in December (shortest photoperiod). However, these
workers also reported that the magnitude of the peripar—
tum surge in prolactin concentrations (relative to prolac-
tin measured between 13 and 2 days prepartum) remained
nearly constant (77 to 93 ng/ml) throughout the year de—
spite dramatic changes in temperature and photoperiod.

Thus, it may be speculated that the relatively constant

 

H 4.....-

22

magnitude and occurrence of the surge in prolactin secre-
tion at parturition is evidence of the requirement of

prolactin for lactogenesis.

1.5 Hormones and Lactogenesis — Ruminants

Studies designed to induce lactation in cattle have
helped unravel some of the effects of hormones which pro—
mote mammary development. Sykes and Wrenn (1951) induced
lactation in heifers using various combinations of die—
thylstilbestrol, progesterone and pituitary extracts.
Treatment with diethylstilbestrol alone resulted in de-
velopment of histologically abnormal mammary tissue, made
up of distended ducts and minimal alveolar formation.
Treatment with combinations of diethylstilbestrol and pro-
gesterone or pituitary extracts resulted in normal lobule-
alveolar development. This suggested that normal mammary
development in cattle required estrogen, progesterone and
pituitary hormones. Turner gt gt. (1956) subsequently
demonstrated that long term (180 days) estrogen and pro-
gesterone treatment elicited milk secretion in heifers.
However, milk yields were variable (66 to 137% of paternal
and maternal half siblings) and only six of eight heifers
responded to the treatment.

Tucker and Meites (1965) induced lactation in preg—
nant heifers at 3.5, 4.5 and 7.5 months of gestation fol-

lowing 6 or 7 daily injections (15 mg) of

 

 

23

9-a-fluroprednisolone acetate, a synthetic glucocorticoid.
The heifers were milked twice daily starting 6 to 7 days
after injections were begun. Glucocorticoid treatment
induced milk secretion at each time interval but milk pro-
duction was greatest among heifers at 7.5 months of gesta-
tion. Although these data showed that glucocorticoids
could initiate milk secretion during pregnancy, the im—
portance of glucocorticoids in normal lactogenesis in
cattle is unknown.

Renewed interest in artificial induction of lactation
was sparked when Smith and Schanbacher (1973, 1974) in-
advertently induced lactation in non-gravid heifers.
Attempting to elicit secretion of immune proteins after
injections for 7 days of estrogen (.1 mg/kg body wt/day)
and progesterone (.25 mg/kg body wt/day), these workers
observed that certain animals continued to secrete colos-
trum, and after several days of milking, began to produce
substantial amounts of milk.

. Using a modification of the estrogen/progesterone
treatment described by Smith and Schanbacher (1974), Howe
gt gt. (1975) induced lactation in nine dairy cows.
Starting treatment 7 days post estrus and injecting dexa—
methasone on days 12, 13 and 14 after initiation of treat-
ment, these workers recovered mammary tissue on days 18,
21 and 23 for histological study. Epithelial cells ob-

served in samples from day 18 contained small lipid

24

droplets and elongated dark-staining nuclei. In contrast,
more highly differentiated epithelial cells on day 23 had
a mixture of large and small lipid droplets, spherically
shaped nuclei and decreased nuclear to cytoplasmic ratio.
Epithelial cells from day 21 were more developed than
those on day 18 but much less developed than on day 23.
Since milking was begun on day 21 of treatment and mitotic
figures were not observed on any sampling day, these
workers suggested that continued development of the mam-
mary gland at the onset of milking involved primarily
secretory cell differentiation rather than cellular pro-
liferation. Similarly, Collier gt gt. (1976) and Croom
gt gt. (1976) demonstrated close association between de-
velopment of milk component biosynthesis and cellular dif-
ferentiation during induced lactation and subsequent milk
production.

The importance of prolactin in induced lactation has
been indicated in several studies. Howe gt gt. (1975)
noted that the mammary gland continues to enlarge in size
with the onset of milking. Since milking (Tucker, 1971)
or even teat stimulation causes secretion of prolactin
(Karg and Schams, 1974), it seems reasonable to suggest
that prolactin may stimulate mammary development and/or
milk synthesis. This idea is supported by the work of Erb
gt gt. (1976). These investigators measured estrogen,

progesterone and prolactin in the plasma of cows induced

25

into lactation (Smith and Schanbacher, 1973) and concluded
that animals with higher plasma concentrations of prolac-
tin after day 7 of treatment produced the greatest amounts
of milk. Furthermore, Collier gt gt. (1975) and Bauman
gt gt. (1976) reported greater milk yields from cows
treated with reserpine (an alkaloid which stimulated pro-
lactin secretion) 6 days after the end of estrogen/
progesterone treatment in comparison with cows given only
estrogen/progesterone. Vandeputte-Van Messom gt gt. (1976)
reported that perphenazine (which stimulated prolactin
secretion) caused udder growth and induced lactation when
stereotaxically implanted into the median eminence of six
virgin female goats. Hart (1976) reported that injections
of CB154 (2-bromo-a-ergocryptine-methanosulphonate; which
blocks prolactin secretion) blocked the ability of
estrogen/progesterone treatment to induce lactation in
goats. In contrast, Peel gt gt. (1978) reported no dif-
ferences in milk yields among cows treated with either
C8154 or reserpine during concurrent estrogen/progesterone
therapy, although the number of cows successfully induced
into lactation (> 3 kg/milk/day) was increased when reser-
pine was included in the treatment scheme.

Similar to results observed with tg ttttg studies of
mammary tissue from some laboratory species, tg XlEEQ
studies with non-lactating bovine mammary tissue, indicate

that prolactin stimulates milk biosynthesis and cytological

26

differentiation when added to cultures pretreated with
insulin and hydrocortisone (Collier gt gt., 1977).
Furthermore, Nickerson gt gt. (1978) reported that in
addition to insulin and hydrocortisone, prolactin stimula-
tion of cellular differentiation of bovine mammary cells
in explant culture was enhanced when low concentrations of
estrogen (3 pg/ml) and progesterone (3 ng/ml) were in-
cluded in the culture medium. These workers also reported
that unidentified factor(s) in calf plasma enhanced dif-
ferentiation. Similarly, Skarda gt gt. (1977) reported
that prolactin markedly stimulated lipid synthesis in mam-
mary explants obtained from virgin goats previously
treated 11 to 12 days tg 2122 with estrogen, progesterone
and deprenon (an inhibitor of prolactin secretion). Pro-
lactin did not, however, stimulate lipid synthesis in mam-
mary explants prepared from placebo-treated goats or goats
treated with estrogen and progesterone alone. In a novel
experimental approach, Welsch gt gt. (1979) showed that
combinations of estrogen and progesterone, growth hormone
and prolactin, or all four combined caused 1.7, 1.6 and
3.2-fold increases in rates of DNA synthesis (BE-thymidine
labeling) in non-lactating bovine mammary tissue trans-
planted into athymic ”nude" mice. These workers also
demonstrated that a combination of prolactin, growth hor-
mone and hydrocortisone increased the a-lactalbumin con—

tent of bovine mammary tissue in mice previously treated

27

with estrogen, progesterone, growth hormone and prolactin.
These results provided the first evidence that the pitui-
tary hormones could stimulate DNA synthesis and presumably
mammary cell proliferation in cattle as well as induction
of a-lactalbumin synthesis.

The most direct 12.2122 evidence of a necessity for
prolactin innaturally occurring lactogenesis in cattle,
has been gleaned from experiments in which inhibitors of
prolactin secretion were used. Karg and Schams (1974) re-
ported that CB154 drastically lowered plasma prolactin and
that periparturient treatment with CB154 nearly abolished
milk production in the subsequent lactation. Unfortu—
nately, control treatments in which cows were treated with
C3154 plus exogeneous prolactin were not tested. Fell gt
gt. (1974) also reported reduced milk yields in two cows
treated 24 or 60 h prepartum with bromocryptine. In con-
trast to these earlier reports, Schams (1976) reported
that periparturient prolactin suppression resulted in wide
variation in inhibition (5 to 80%) of milk production fol-
lowing parturition. However, those cows treated with
CB154 through parturition exhibited the greatest inhibi-
tion of milk production. Johke and Hodate (1978) also re-
ported that suppression of prolactin secretion from 2
weeks prior until parturition resulted in a 40% reduction
in milk yield and concentration of a-lactalbumin in milk.

These studies, however, must be interpreted cautiously

28

since milk production was evaluated by comparison with
previous lactation yields; controls (to insure that re-
duced milk yields were caused by absence of prolactin)
were not included. Furthermore, these studies provide
little knowledge as to how a reduction in periparturient

prolactin secretion influenced subsequent milk production.

MATERIALS AND METHODS

2.1 Animals

Seventeen multiparous, pregnant Holstein cows were
used. All animals were kept in stanchions and maintained
under a 16 h light: 8 h dark photOperiod (lights on 0500
h and off 2100 h). Water was available at all times and
animals were fed either 15.9 or 18.1 kg of corn silage and
6.8 kg of hay daily until 14 days prior to parturition.

At that time, concentrate feeding and monitoring of feed
consumption was begun. Concentrates were fed at 0.9 kg
per day initially and increased in 0.9 kg increments every
second day until 5.4 kg were fed daily. After calving,
concentrate feeding was further increased depending upon
milk production (0.45 kg concentrate per 0.9 kg milk pro-
duced). The cows were milked twice daily (approximately
0730 h and 1930 h), the milk was weighed and samples were
saved until assayed for protein and lactose.

Based upon experimental group assignment, cows were
killed at approximately 10 days postpartum or 10 days pre-
partum. All animals were stunned with a captive bolt pis-

tol and exanguinated.

29

2.2 Treatments

To suppress periparturient prolactin secretion, ani-
mals were given C3154 (2-a-bromoergocryptine-
methanosulfate, from Sandoz Pharmaceuticals, E. Hanover,
NJ) in accordance with the schedule given below. C3154
was dissolved in 80% ethanol and administered by subcu-
taneous injection. Injections were given at 1000 h over
the dorsal aspect of the scapula, and sequential injec-
tions were alternated between right and left scapulae.

The dose of C3154 administered at each injection was 11
mg/100 kg body weight, and no more than 4 ml of ethanol
was given in a single injection.

As groups of four cows became available for study
(between December and June) the animals were randomly as-
signed one to each of the foilowing treatment groups: (1)
prepartum control, (2) postpartum control, (3) C3154 or
(4) C3154 plus prolactin. This process was continued un-
til five cows were scheduled for each treatment.

Among cows assigned to treatment with C3154 or C3154
plus prolactin, animals were injected with C3154 according
to one of the following schemes prior to parturition: one
animal to be treated with C3154 and one with C3154 plus
prolactin were injected with C3154 every 4 days prepartum.
Similarly, two animals from each group received C3154 in-
jections every 2 days and two animals were injected with

C3154 each day prepartum. The decision to increase the

31

number of C3154 injections given prepartum, resulted be—
cause in contrast to expectations (Karg and Schams, 1974)
milk production in the cow injected every 4 days prepartum
was reduced immediately following parturition but in-
creased thereafter. It was assumed that failure of C3154
to block milk production resulted because injections of
C3154 every 4 days prepartum were inadequate to inhibit
prepartum secretion of prolactin. Thus, the frequency of
C3154 injections was increased. All injections began 12
days prior to parturition (based on expected calving date).
After parturition all animals receiving C3154 were in-
jected every other day until they were killed.

Those cows assigned to treatment with C3154 plus pro-
lactin were infused with 6, 8, 8, 9.8, 22.4 and 20.8 mg
prolactin (NIH-B4 from National Institutes of Health,
Bethesda, MD) per h on days 5 through 0 before expected
parturition, respectively. These concentrations were se—
lected on the basis of previous studies in which prolactin
was infused into cows (Akers gt gt., 1979), and were cal-
culated to mimic the increase in periparturient prolactin
secretion reported by Ingalls gt gt. (1973). On the
afternoon preceding start of infusion, each cow was fitted
with a polyvinyl cannula in each jugular vein. Blood -
samples were obtained through one cannula and prolactin
infused through the contralateral cannula. Prolactin so-

lutions were infused using a constant infusion pump

32

(Harvard Apparatus Co., Cambridge, MA).

Solutions of prolactin sufficient for each day of in-
fusion were prepared (4 to 6 h prior to use) in sterile
saline (0.9% NaCl) containing 0.1% bovine serum albumin
(BSA). The pH of solutions was adjusted (10.5 to 11.0)
with 1.0 N NaOH. Before each cow was infused, the poly-
vinyl tubing connecting the pump to the cows was flushed
with 0.1% BSA in saline to minimize adsorption of prolac-
tin to the tubing. Infusions were started at 2400 h 6
days prior to expected parturition, and scheduled changes
in rates of prolactin infusion (as described above) were
made at 24-h intervals thereafter. To monitor changes in
serum prolactin concentrations, blood samples were col-
lected at 6-h intervals during the first 4 days of the in-
fusion and at 4-h intervals during the remaining 2 days of

the infusion period.

2.3 Blood Sampling

Blood was obtained daily (AM) by puncture of the coc-
cygeal vein or artery starting 21 days before the esti-
mated calving date. Five or 6 days after the start of
blood sampling, each cow was fitted with an indwelling
jugular cannula which was used thereafter to obtain blood
samples twice daily (0700 h and 1900 h). During the in-
terval between collection of blood samples, cannulae were

filled with an anticoagulant solution of 3.5% citrate and

33

50% dextrose in sterile distilled water. Daily blood
samples were collected approximately 1 h before concen-
trate feeding or milking (when applicable).

To determine the effect of C3154 on milking-induced
»prolactin release, blood samples were collected before,
during and after the PM milking on two different days be-
tween 5 and 9 days postpartum. Cows treated with C3154
were sampled on a day when a C3154 injection was given and
on a second day when C3154 was not administered. Un-
treated cows also were sampled on two different days dur-
ing the same interval postpartum. The time of the PM
milking was designated as time 0. Blood samples were col—
lected at 30, 15, 10, 5 and 0 min before milking. Approx-
imately 15 to 20 sec prior to milking the cow's udder was
washed and at time 0 the milking machine was attached to
the udder. Blood samples were then collected at 2, 4, 6,
8, 10, 15, 20, 25, 30, 45 and 60 min after milking.

Following collection, blood samples were allowed to
clot 4 to 6 h, held at 4 C for 24 to 36 h and then centri-
fuged at 2,500 x g for 20 min. OBlood serum was then de-

canted, and stored at -20 C until assayed for hormones.

2.4 Tissue Removal - General

Immediately following exsanguination, cows udders
were removed and 100 to 150 g of mammary tissue from each

half was excised and placed in ice-cold Tris-sucrose

34

buffer (0.25 M sucrose, 30 mM Trisma HCl pH 7.3, 1 mM
glutathione and 1 mM ethylene diamine tetraacetic acid,
disodium salt) until subsequent slicing or homogenization.
The remainder of the udder was then frozen (~20 C) until

assayed for DNA, RNA, hydroxyproline and lipid.

2.5 Chemicals

Cofactors used for enzyme assays and standards for
DNA and RNA assays were obtained from Sigma Chemical Co.
(St. Louis, MO) or P-L Laboratories (Milwaukee, WI). So-
dium (2-14C) acetate, sodium (14C) bicarbonate and (U-14C)
glucose were obtained from Amersham-Searle (Arlington
Heights, IL). Glutaraldehyde, osmium tetroxide, propylene
oxide, epon 812 and araldite 510 were purchased from Ladd
Research (Arlington, VT). All other compounds used were

reagent grade or as indicated for specific assay methods.

2.6 Tissue Incubations

Slices of fresh mammary tissue prepared from mammary
tissue samples previously placed in Tris—sucrose buffer
were incubated for 3 h to measure rates of fatty acid syn-
thesis, lactose synthesis and CO2 production. Mammary
tissue slices (100 to 150 mg each) were prepared with a
Stadie-Riggs hand microtome and rinsed in isotonic sucrose.
Single randomly selected slices were then transferred to

each of six 25 ml Erlenmeyer flasks containing 3 ml of

35

Krebs-Ringer bicarbonate buffer (DeLuca and Cohen, 1964)
pH 7.4, supplemented with acetate and glucose (10 mM final
concentration) and 0.3 units of insulin. In addition,
three of the flasks contained approximately 1 n Ci of

2-14C acetate and three contained approximately 1 n Ci of

U-14C glucose. Flasks were gassed with 02 + CO2 (95:5),
stoppered and incubated in a shaking water bath at 37 C
for 3 h.

To determine rates of C02 generation, the flasks
described above were equipped with a suspended plastic
bucket with a 2 x 2 cm square of filter paper. At the end
of the incubation, 0.1 ml of 25% KOH was injected through
the rubber stopper onto the filter paper and 0.25 ml of
0.5 M H2S04 into the media to liberate C02. After an
additional 30 min incubation, the filter paper was removed,
transferred to a liquid scintillation vial with 12 ml of
scintillation fluid and assayed for radioactivity in a

liquid scintillation spectrophotometer. Three flasks con-
taining each medium but no tissue were also incubated and
assayed as above to correct for non—specific liberation of
radioactivity from the media.

To estimate rates of fatty acid synthesis at the end
of the incuabtion period, tissue slices from the three
flasks containing 2-14C acetate were removed, rinsed with

isotonic sucrose, and transferred to flasks containing 4

ml of 5 M NaOH. These flasks were then refluxed on a hot

36

plate for 5 to 6 h at 85 C, cooled and acidified with 5 ml
of 2.5 M H2804. After extracting three times with 5 ml of
petroleum ether, 1 ml aliquots of the petroleum ether ex-
tracts were assayed for radioactivity with 12 ml of scin-
tillation fluid (Bauman gt gt., 1970; Mellenberger gt gt.,
1973).

Lactose synthesis by mammary slices in each of the
six flasks was determined by measurement of lactose re—
leased into the media during the 3-h incubation period.

It was expected that release of "preformed" lactose re-
tained in the tissue at the time of slaughter would in-
flate estimates of lactose synthesis by the tissue slices.
Thus, the following precaution was taken. Three addi-
tional tissue slices were incubated for 30 min then the
media collected and assayed for lactose. The concentra-
tion of lactose measured after the 30—min incubation was
taken as an estimate of "preformed" lactose and used to
adjust rates of lactose synthesis determined from the

flasks incubated for 3 h.

2.7 Enzyme Assays

Approximately 10 g of fresh mammary tissue was minced
in ice-cold Tris-Sucrose buffer (2 ml buffer per g tissue)
then homogenized on ice by 10 x 15 sec bursts with 30 sec
between bursts using a Polytron tissue homogenizer (Type

PT 10 OD; Brinkman Instruments, Westburg, NY) at maximum

37

speed. The homogenate was then centrifuged at 800 x g.
The supernatant fluid was then transferred and centrifuged
at 12,000 x g for 30 min. The resulting supernatant fluid
was subsequently centrifuged at 105,000 x g for 60 min.
All centrifugations were at 0 to 4 C. All enzyme assays
were done using the 105,000 x g supernatant fluid (cyto-
sol). The enzymes assayed, importance of the enzyme for
milk synthesis and appropriate references are given in
Table 1. Protein contents of cytosol preparations were
determined with a dye binding assay (Biorad Protein Assay;
Biorad Laboratories, Chemical Division, Richmond, CA) as
described by Bradford (1976). Bovine gamma globulin was

used as a standard.

2.8 Hormone Assays

Prolactin and growth hormone were quantified using
previously described double antibody radioimmunoassays
(Koprowski and Tucker, 1971; Purchas gt_gt., 1970). Serum
progesterone was measured by radioimmunoassay (Louis gt
gt., 1973) and total serum glucocorticoids were determined
by competitive protein binding assay as described by Smith

gtgt. (1973).

2.9 Other Assays

Milk lactose was assayed using a modification of the

picric acid method of Perry and Doan (1950). This

38

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39

modification consisted of diluting 100 pl of skimmed milk
in 9.9 ml of saturated picric acid instead of diluting l g
of milk to 100 ml with picric acid, and centrifugation
(3,000 x.g for 15 min) instead of filtration of the di-
luted milk sample for subsequent assay.

The protein content of milk was measured using the
dye binding assay described previously, except casein in-
stead of bovine gamma globulin was used as standard. The
a-lactalbumin content of skimmed milk was quantified using
the double antibody radioimmunoassay described by Beck and
Tucker (1977).

Lactose in media from tissue slice incubations was
measured using a coupled enzymatic assay as described by
Coffey and Reithel (1969). In brief, lactose is hydro-
lyzed to B-galactose and glucose by B—galactosidase. In
turn, B-galactose is oxidized by the enzyme B-galactosidase
dehydrogenase in the presence of nicotinamide adenine di-
nucleotide (NAD), which is reduced to NADH. The NADH was

measured spectrophotometrically at 340 nm.

2.10 Tissue Composition

Previously frozen udders were sawed into strips, then
teats, skin and other non-parenchymal tissue were trimmed
and discarded. The strips were then minced in a meat
grinder. The resulting mixture was weighed and a portion

saved until assayed for DNA, RNA, hydroxyproline and lipid.

40

Samples of the mixture were transferred to dry ice,
pulverized and 5 g were suspended in 100 ml of ice-cold
distilled water. This suspension was then homogenized
using a Polytron tissue homogenizer as previously de-
scribed. A portion of the homogenate was further homo-
genized 50 strokes in a Potter-Elvehjem glass homogenizer.
The DNA, RNA and lipid content of the final homogenate was
determined as described by Tucker (1964). Hydroxyproline
was measured using the method of Prockop and Udenfriend

(1960).

2.11 Histological Methods

At the time of slaughter two mammary tissue samples
from each quarter of the udder of each cow were taken for
histological analyses. One sample was taken 3 cm anterior
and the other 3 cm caudal to an imaginary axis, directly
above the point of attachment of each teat, midway between
the upper and lower boundaries of the secretory tissue of
the udder. A slice of mammary tissue approximately 1 mm
thick and weighing about 500 mg was immersed in dilute
(0.5% formaldehyde, 1.3% glutaldehyde in 0.1 phosphate,
pH 7.2) Karnovsky's fixative (Karnovsky, 1965) and cut
into approximately 1 mm3 blocks. These blocks were subse-
quently fixed 1 to 2 h at room temperature. The samples

were then washed three times and stored at 4 C in 0.1 M

phosphate buffer containing 8% sucrose. Secondary

41

fixation was accomplished by immersing tissue blocks in 1%
phosphate buffered osmium tetroxide for l h. Thereafter,
the blocks were dehydrated by washing the samples through
an ascending series of ethanol concentrations (30, 50, 70,
90 and 100%) and 100% propylene oxide. Tissue blocks were
then embedded in a 1:1 (wt/wt) mixture of epon 812 and
araldite 502 resin. Six to eight sections 0.5 to 1 pm
thick from each of two randomly selected blocks from each
tissue sample were cut on a Porter Blum MT-Zb ultramicro-
tome (Sorvall Instruments, Newtown, CT), mounted on glass
slides and stained with Azure II.

To evaluate cytological development the proportion of
fully differentiated, intermediately differentiated and
undifferentiated epithelial cells in mammary tissue sec-
tions was determined. In addition to epithelial cells,
the proportion of stromal tissue and alveolar lumen was
also determined to evaluate histological development.

Alveolar cells corresponding to each of the three
epithelial cell classifications used are shown in Figure
1. Epithelial cells classified as fully differentiated
(Figure l-A) were characterized by presence of numerous
vacuoles (V), rounded basally displaced nuclei (N), abun-
dant cytoplasm and large apical lipid droplets (F). In
comparison with fully differentiated cells, intermediately
differentiated cells (Figure l-B) had fewer cellular vacu-

oles, more irregularly shaped nuclei, greater estimated

Figure

Figure

Figure

Figure

1

l-A

l-C

42

Light micrographs of epon-araldite embedded,
0.5 to l um sections of mammary tissue stained
with Azure II (all 4000 X).

Fully differentiated alveolar epithelial cells
characterized by presence of numerous vacuoles
(V), rounded basally positioned nuclei (N),
abundant cytoplasm, frequent occurrence of
large apically located lipid droplets (F) and
distinct polarization of cellular organelles.

Intermediately differentiated alveolar epithe-
lial cells showing fewer cellular vacuoles,
more irregularly shaped nuclei, greater esti-
mated nuclear to cytoplasmic ratio and less
pronounced cellular polarity.

Undifferentiated alveolar epithelial cells
displaying relative absence of cellular vacu-
oles, highly irregularly shaped nuclei, a mix-
ture of large and small randomly located lipid
droplets, very large estimated nuclear to cy—
toplasmic ratio and lack of cellular polarity.

 

44

nuclear to cytoplasmic ratio and less pronounced cellular
polarity. Undifferentiated epithelial cells (Figure l-C)
were characterized by absence of vacuoles, highly irregu-
larly shaped nuclei, large randomly positioned lipid drop-
lets, very large estimated nuclear to cytoplasmic ratios
and lack of cellular polarity.

A modification of Chalkey's (1943) quantitiative mor—
phological analysis was used to measure the percent of
each epithelial cell type, stromal tissue and alveolar
lumenal area in mammary tissue sections. In this modifi-
cation a grid with 64 equidistant intersections was placed
in the ocular of a microscope to provide fixed contact
points (intersections). The histological structure (epi-
thelial cell, stromal tissue or alveolar lumen) underlying
each contact point was determined. Furthermore, each epi-
thelial cell counted was further classified as described
above, then the total number of contact points correspond-
ing to each structure was computed. These totals were
then expressed as a percentage of the total number of con-
tact points. The percentage of mammary tissue area occu-
pied by these structures was determined in two randomly
selected sections from each tissue block at a magnification
of 400 x and three measurements were made of each tissue
section. Random selection of sections and fields within
sections to be quantified was accomplished by randomly re—
lacating the microscope stage and using coded slides. A

total of 104,448 contact points was classified.

45

2.12 Ultrastructural Methods

Based on the results of the quantitative histological
analysis, the tissue block most closely corresponding to
the average epithelial cell classification for each animal
was selected for ultrastructural evaluation of epithelial
cells.

Selected blocks were trimmed and gold or silver sec-
tions were cut using a Porter Blum MT-2b ultramicrotome.
These sections were mounted on copper grids then stained
with uranyl acetate (Watson, 1958) and lead citrate
(Venable and Coggeshall, 1965). The grids were then ex-
amined in a Phillips 300 electron microscope operating at
60 Kv accelerating voltage, and sections having profiles
of alveolar epithelial cells were photographed at a magni-
fication of 3,900 x. Ten to 15 micrographs were obtained
from the sections prepared from each tissue block.

To evaluate ultrastructural characteristics, the per-
cent alveolar cell area occupied by the nucleus, Golgi
membranes and vacuoles, BER and lipid droplets was deter-
mined. Elements in the remaining cellular area include
mitochondria, clusters of free ribosomes, lysosomes and
unoccupied cytoplasm. To determine these percentages the
micrographs were enlarged in a standard variable condenser
photographic enlarger (Simmon Bros. Inc., Long Island City,
NY) until the enlarged cell image (approximately 50,000 x)

was slightly greater than a rectangular 16 x 22 cm grid

46

containing 96 equidistant grid line intersections (contact
points). The cellular structure underlying each contact
point was determined, then the total number of contact
points corresponding to each cellular component (as de-
scribed above) was computed. These totals were then ex-
pressed as a percentage of the total number of contact
points. Only cells bounded by an alveolar lumen, basal
lammena and plasma membrane adjacent to other alveolar
cells were evaluated. Six to 15 cells from each animal

were measured.

2.13 Statistical Methods

Data involving repeated measurements over time were
analyzed by split plot analysis of variance (Gill and Hafs,
1971). Logarithmic transformations were made where hetero-
geneous variance existed. Specific comparisons of means
were by Bonferroni t-statistics (Gill, 1978).

Biochemical, histological and ultrastructural data
were analyzed by one-way analysis of variance. To adjust
for heterogeneous variance ultrastructural data expressed
as a percentage were transformed before the analysis using
arcsin /§, where y is the original datum expressed as deci-
mal percentage. Logarithmic transformations were also made
where heterogeneous variance existed among other data anal—
yzed by one-way analysis of variance. Specific comparisons

of means were also by Bonferroni t-statistics.

RESULTS

3.1 General Results

All postpartum cows delivered a single, live calf
without assistance. In addition, all prepartum control
cows were pregnant at the time of slaughter and each cow's
uterus contained a single calf. Data from one cow in the
postpartum control group were omitted from the results be-
cause of development of a displaced abomasum. Similarly,
data from a cow treated with CB154 plus prolactin were
omitted from the study because the cow exhibited severely
suppressed feed consumption and contracted pneumonia. A
second cow initially assigned to be treated with CB154
plus prolactin was not used because sufficient C8154 could
not be obtained. 0f the three cows treated with C8154 plus
prolactin, one was injected with CB154 every 4 days,.
another every 2 days and the third was injected every day
prepartum in accordance with the previously described

treatment schedule.

3.2 Periparturient Prolactin Secretion

Serum prolactin concentrations during 10 days prior

and 6 days following initiation of treatment are presented

47

48

in Figure 2. Prolactin concentrations in serum were not
significantly different among treatment groups (P > .05)
prior to initiation of treatments. In contrast, treatment
with C8154 reduced (P < .001) serum prolactin from an
average of 29.1 i 3.0 ng/ml just prior to treatment to

6.4 i 0.8 ng/ml 24 h later. Furthermore, average concen-
tration of prolactin in serum among cows injected with
C3154 remained lower (P < .001), approximately one-third
of the level in uninjected controls during 6 days after
start of CB154 injections (Figure 2).

Among postpartum control animals, serum prolactin
averaged 30.1 i 1.5 ng/ml from 6 to 2 days before calving.
But, after day 2 prepartum, serum prolactin concentrations
rapidly increased and reached a peak of 218 i 16 ng/ml ap-
proximately .5 day prepartum with a gradual decline there-
after (Figure 3). Serum prolactin averaged 27.9 i 3.2
ng/ml from 2 to 10 days postpartum.

Treatment with CB154 markedly reduced the concentra-
tion of prolactin measured in serum before, during and
after parturition. Indeed, basal concentrations of prolac—
tin in serum (estimated from blood samples obtained from 6
to 2 days prepartum and 2 to 10 days postpartum) were lower
(P < .001) in cows treated with CB154, approximately 15 to
20% of the level in controls. Furthermore, treatment with
CB154 completely blocked the normal prolactin increase at
parturition (Figure 3).

Changes in average serum prolactin concentrations,

Figure 2

49

Concentrations of prolactin in serum during 10
days prior to initiation of treatment and dur-
ing 6 days after the start of C8154 injections
but before infusion of exogeneous prolactin in
cows treated with CB154 plus prolactin.

SERUM PROLACTIN ( ng/ml)

50

A—A CONTROL
o—o CB- 154
4o .. A H c3-154+ PRL

 

 

’( 1 1 I I I
O 2 4 6 8 IO

DAYS PRIOR TO INITIATION
OF TREATMENTS

30 — A A

20*-

IO—

 

I l l
2 4 6

DAYS FOLLOWING INITIATION
OF TREATMENTS

51

Figure 3 Concentrations of prolactin in serum before,
during and after parturition in control cows
and those treated with C8154.

52

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among cows treated with C8154 plus prolactin, during in—
fusion of exogeneous prolactin are shown in Figure 4.
Prolactin averaged 109 t 6.2, 129 i 7.4, 138 t 9.8,

187 t 12.3, 311 i 18.6 and 278 t 19.4 ng/ml of serum dur-
ing 6 consecutive days of infusion. Prolactin infusions
ended approximately 24 and 36 h prior to parturition in
two cows and 24 h postpartum in a third cow. The cows
which calved 24 and 36 h after the end of the infusion
period were milked two and three times prepartum, respec-
tively. These milkings were essential to relieve disten-
sion of the udders.

Figure 5 shows mean concentration of serum prolactin
before, during and after milking at two PM milkings be-
tween days 5 and 9 postpartum. Among control cows, fol-
lowing application of the milking machine, serum prolactin
concentrations increased to a maximum of 63.7 t 16.7 ng/ml
within 8 min (P < .001). Thereafter, prolactin concentra-
tions declined to pre-milking concentrations, averaging
27.8 i 3.5 ng/ml within 60 min post—milking. Milking time
averaged 7.2, 6.0 and 6.6 min among postpartum control
cows, cows treated with CB154 and cows treated with CB154
plus prolactin, respectively. Prolactin release in re-
sponse to the milking stimulus was greater in lactating
controls than in cows treated with C8154 (P i .001). Also,
changes in serum prolactin in response to milking were un-
affected by day of sampling (P > .05). Thus, milking-

induced release of prolactin was suppressed throughout the

Figure 4

54

Concentrations of prolactin in serum of cows
treated with CB154 plus prolactin during in-
fusion of exogeneous prolactin. Amounts of
prolactin infused during six consecutive days
of infusion are given in the solid bars.

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56

Figure 5 Average concentrations of prolactin in serum
before, during and after two PM milkings be—
tween 5 and 9 days postpartum.

SERUM PROLACTIN ( .ng/ml )

7O

60

50

4o

30

20

l O

57

H CONTROL
0—0 CB-l54+PRL
°—° CB-l54

     
 

W

l

 

 

 

16 £0 50 4'0 50 so
TIME(MIN)

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56

Figure 5 Average concentrations of prolactin in serum
before, during and after two PM milkings be-
tween 5 and 9 days postpartum.

SERUM PROLACTIN ( .ng/ml )

7O

60

50

4O

30

20

I O

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57

H CONTROL
0—0 CB-I54-l-PRL
°—° CB- l54

    
 

 

 

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TIME(MIN)

58

interval between postpartum CB154 injections.

3.3 Periparturient Serum Concentrations of Glucocorti-
coids, Progesterone and Growth Hormone

Treatments had no significant effect (P > .05) on
concentrations of glucocorticoids measured in serum be-
tween 5 days prepartum and 3 days postpartum. Although
average daily concentrations of glucocorticoids were vari-
able, serum concentrations were greatest on the day of
calving (Figure 6).

Similarly, the concentration of progesterone in serum
was not significantly different (P > .05) among treatment
groups between 5 days prepartum and 3 days postpartum.
However, serum progesterone concentrations decreased
markedly (P < .001) between 2 days prepartum when it aver-
aged 5.8 : 1.0’ng/ml and parturition when it averaged
1.5 t 0.4 ng/ml (Figure 7). Serum progesterone concentra-
tions averaged less than 1 ng/ml between days 1 and 3
postpartum.

Changes in serum growth hormone concentrations be-
tween 20 days prepartum and 10 days postpartum are shown
in Figure 8. There were no significant differences among
treatment groups (P > .05) in periparturient serum growth
hormone concentrations. The concentration of growth hor-
mone in serum was, however, significantly increased

(P < .01) at parturition.

59

Figure 6 Concentrations of glucocorticoids in serum
from 5 days prepartum to 3 days postpartum.

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61

Figure 7 Concentrations of progesterone in serum from
5 days prepartum to 3 days postpartum.

62

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Figure 8 Concentrations of growth hormone in serum from
20 days prepartum to 10 days postpartum.

64

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65

3.4 Milk Production and Milk Composition

Suppression of periparturient prolactin secretion
with CB154 significantly reduced milk production (P < .001)
during 10 days following parturition compared to controls
(Figure 9). Furthermore, prolactin replacement therapy
(CB154 plus prolactin) prevented reduced milk yields. In
addition, the reduction in average daily yield among cows
treated with C8154 was maintained during 10 days of lacta-
tion. To illustrate, lactating controls and cows which
received prolactin therapy produced an average of
11.9 i 0.5 and 15.8 t 1.6 kg more milk per day, respec-
tively than cows treated with CB154 during the first 4
days of lactation. Moreover, untreated controls and cows
treated with CB154 plus prolactin produced an average of
11.1 i 0.4 and 11.7 i 0.2 kg more milk per day, respec-
tively between days 7 and 10 of lactation than cows
treated with CB154.

The concentration of total protein in milk following
parturition is shown in Figure 10. In cows treated with
CB154 plus prolactin and in postpartum control animals the
concentration of protein in milk declined rapidly follow-
ing parturition, averaging 3.6% by 2 days postpartum. Al-
though, a slower rate of decline (P < .05) in milk protein
percent following parturition was observed in cows treated
with CB154, treatments had no lasting effect (P > .05) on

the concentration of total protein in milk.

66

Figure 9 Average AM and PM milk yields among lactating
controls and cows treated with C8154 or CB154
plus prolactin.

67

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68

Figure 10 Average concentrations of protein in milk ob-

tained twice daily among lactating controls

and cows treated with CB154 or CB154 plus pro-
lactin.

69

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The percent lactose measured in milk following par-
turition tended (P < 0.1) to be lowest in cows treated
with CB154 (Figure 11) during 10 days of lactation.
Furthermore, results from the split plot analysis of vari-
ance indicated a highly significant (P < .0005) inter—
action of treatment by time (day). This indicated that
changes in concentration of lactose in milk following par-
turition did not follow the same pattern in each treatment
group. Indeed, milk lactose concentrations were signifi-
cantly lower (P i .05) in cows treated with CB154 than in
control cows during the first 2 days postpartum, but
nearly identical concentrations were measured in milk
after day 8 postpartum.

In contrast to lactose concentrations, the a-
lactalbumin content of milk remained lower (P i .01) in
cows treated with CB154 than in controls or cows treated

with CB154 plus prolactin (Figure 12).

3.5 Effect of CB154 on Feed Intake

Prior to parturition feed consumption (expressed as
energy intake relative to requirement) was not signifi-
cantly different (P > .05) among treatment groups. How-
ever, following parturition feed intake tended to be
greater (P < .06) in cows treated with CB154 than in con-

trols (Figure 13).

71

Figure 11 Average concentrations of lactose in milk ob-
tained twice daily among lactating controls

and cows treated with CB154 or C8154 plus pro-
lactin.

72

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Figure 12 Average concentrations of a-lactalbumin in
milk obtained twice daily among lactating con-
trols and cows treated with CB154 or CB154
plus prolactin.

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intake as % of requirement) from 10 days pre-
partum to 10 days postpartum among lactating
controls and cows treated with CB154 or CB154
plus prolactin.

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77

3.6 Fatty Acid Synthesis and C02 Production in Mammary
Tissue in vitro

Rates of fatty acid synthesis from acetate and 602
production from acetate and glucose are shown in Table 2.
The capacity to synthesize fatty acids was 6.3-fold
greater (P < .001) in mammary tissue derived from post-
partum control cows than from prepartum control cows.
Also, rates of fatty acid synthesis tended to be lower

(P i .07) in mammary tissue from cows treated with C8154
than in mammary tissue from postpartum control cows or
cows treated with CB154 plus prolactin. Rates of C02
generation from either acetate or glucose were similar

(P l .05) among postpartum controls and cows treated with
CB154 or CB154 plus prolactin. Production of C02 from
either acetate or glucose was 3 to 4-fold greater

(P < .001) in mammary slices from cows killed postpartum

than in mammary slices from cows killed prepartum.

3.7 Lactose Synthesis and a-Lactalbumin Concentration in
Mammary Tissue

Lactose synthesis by mammary tissue slices was 14.2-
fold greater (P < .001) in postpartum control cows than in

prepartum control cows (Table 3). In vivo suppression of

 

prolactin secretion caused a 40% reduction (P < .01) in
subsequent ability of mammary tissue slices to synthesize
lactose. But, prolactin replacement therapy in vivo pro-

duced rates of lactose synthesis in vitro similar to those

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80

observed in mammary tissue from postpartum control cows.
The ability of mammary tissue slices to synthesize lactose
among all cows was correlated (r = .86, P < .01) with the
concentration of a-lactalbumin measured in mammary cytosol.
Concentrations of a-lactalbumin were 3.2-fold lower

(P < .001) in mammary tissue from cows killed prepartum
and 1.9-fold lower (P < .001) in cows treated with CB154
than in postpartum control cows (Table 3). Prolactin re-
placement therapy restored the concentrations of a-
lactalbumin measured in mammary cytosol. Indeed, a-
lactalbumin in cytosol was 2.7-fold greater (P < .001) in
cows treated with CB154 plus prolactin than in those
treated with C8154. The relationship between lactose syn-
thesis in mammary tissue slices and a-lactalbumin in mam-
mary cytosol among all groups of lactating cows is shown
in Figure 14. Among cows killed after parturition, the
concentration of a—lactalbumin in mammary cytosol was posi-
tively correlated with rates of lactose synthesis measured
in mammary tissue slices (r = .72, P < .01) and with aver‘
age daily milk production during the 2 days prior to
slaughter (r = .79, P < .01). However, these correlations

may differ among individual treatment groups.
3.8 Activities of Enzymes Associated With Fatty Acid
Synthesis and NADPH Generation in Cow Mammary Tissue

With the exception of glucose 6-phosphate dehydro-

genase, the activities of each of the enzymes measured were

81

Figure 14 Relationship between the concentration of a-
lactalbumin (a lac) in mammary cytosol and
rate of lactose synthesis by slices of mammary
tissue.

82

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o=CBl54
o=CBI54 + PrL A

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83

greater (P i .01) in mammary cytosol prepared from post-
partum control cows than in mammary cytosol from prepartum
control cows (Tables 4 and 5). Suppression of prolactin
secretion, however, had no significant effect (P > .05) on
the activities of glucose 6-phosphate dehydrogenase, 6-
phosphogluconate dehydrogenase or NADP-isocitrate dehrdro-
genase. Thus, mammary tissue from all lactating cows was
presumably equally capable of synthesizing the NADPH re-
quired for milk fat biosynthesis. In contrast, the activ—
ity of mammary fatty acid synthetase was lower (P < .05) in
cows treated with C8154 than in postpartum controls or cows
treated with CB154 plus prolactin. Mammary acetyl CoA car-
boxylase activity also tended to be lower (P i .08) in

cows treated with CB154 than in postpartum controls or cows
treated with CB154 plus prolactin. Acetyl CoA synthetase
activity was, however, relatively unaffected (P > .05) by

reduced prolactin secretion (Table 5).

3.9 Biochemical Constituents and Trimmed Weight of Cow
Udders

Udders from prepartum control cows weighed signifi-
cantly less (P < .01) than udders from lactating control
cows (Table 6). Total mammary gland content of DNA, RNA
and lipid also was lower (P i .01) in udders from cows
killed prepartum. The ratio of RNA/DNA also was lower
(P < .001) in mammary glands from prepartum control cows,

but total mammary gland content of hydroxyproline was not

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significantly different in cows killed prepartum and lacta-
ting controls. Inhibition of prolactin secretion reduced
(P < .05) RNA content of the mammary gland 36% and de-
creased (P < .05) the RNA/DNA ratio, relative to cows which
received prolactin replacement therapy or to untreated con-
trol cows. None of the other biochemical constituents or
weight of the trimmed mammary glands differed significantly

(P > .05) among groups of cows killed postpartum (Table 6).

3.10 Qualitative Morphology of Mammary Tissue

Subjective light microscopic evaluation of mammary
tissue sections suggested that neither total number of
alveolar epithelial cells, amount of stromal tissue nor
alveolar lumenal area were greatly different among post-
partum treatment groups (Figure 15). However, cytological
differences among alveolar cells were evident from examina—
tion of mammary tissue sections (Figure 16). Epithelial
cells from cows killed prepartum were characterized by ir-
regularly shaped nuclei, large ratio of nucleus to cyto-
plasm and random occurrence of large and small lipid drOp-
lets within cells (Figures 15—A and l6-A). A majority of
alveolar cells from postpartum control cows and cows
treated with CB154 plus prolactin exhibited rounded, bas-
ally displaced nuclei, abundant cellular vacuoles, small
ratio of nucleus to cytoplasm and frequent occurrence of

large apically positioned lipid droplets (Figures 15-B,D

Figure

Figure

Figure

Figure

Figure

15

lS-A

15-B

15-C

15-D

88

Light micrographs of epon-araldite embedded,
0.5 to l um sections of mammary tissue stained
with Azure II (all 750 X).

Tissue from prepartum control. The alveolar
lumena are darkly stained and contain some fat
globules.

Tissue from postpartum control.

Tissue from cow treated with C3154.

Tissue from cow treated with C8154 plus pro-
lactin.

 

 

89

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Figure

Figure

16

16-A

16-B

16-D

90

Light micrographs of epon-araldite embedded,
0.5 to 1 pm sections of mammary tissue stained
with Azure II (all 4000 X).

Tissue from prepartum control. The epithelial
cells display irregularly shaped nuclei and
large nuclear to cytoplasmic ratio.

Tissue from postpartum control. The epithe-
lial cells have rounded basally displaced
nuclei, abundant apical vacuoles (cellular
polarization) and small nuclear to cytoplasmic
ratio.

Tissue from cow treated with C8154. Compared
with postpartum control or CB154 plus pro-
lactin, the epithelial cells display less
polarization, have fewer vacuoles and greater
nuclear to cytoplasmic ratio.

Tissue from cow treated with C8154 plus pro-
lactin. The epithelial cells are similar to
those from postpartum control. The cells are
polarized and contain numerous vacuoles and
apical lipid droplets (arrows). In addition,
darkly stained basal cytoplasm is denoted by a
bracket.

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staind

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91

 

92

and l6-B,D). In addition, the basal portion of these
cells were generally darkly stained in contrast to the
lightly stained apical area. In contrast, compared with
postpartum controls or cows treated with CBl54 plus pro-
lactin, most alveolar cells from cows treated with CB154
displayed more irregularly shaped nuclei, fewer cellular
vacuoles, greater ratios of nucleus to cytoplasm and a
mixture of large and small lipid droplets. These epithe-
lial cells also showed less cellular polarity (Figures

15-C and 16-C).

3.11 Quantitative Morphology of Mammary Tissue

The proprotion of stromal tissue and lumenal area in
mammary tissue were not significantly different (P > .05)
in prepartum or postpartum cows (Table 7). However, the
proportion of epithelial cells (all classifications) was
10.2% greater (P < .001) in postpartum controls than in
prepartum controls. Furthermore, all the alveolar cells
from cows killed prepartum were classified as undifferen-
tiated. In postpartum control cows 26.6% of the epithelial
cells were classified as intermediately differentiated and
73.4% were classified as fully differentiated. Suppression
of prolactin secretion did not significantly (P > .05)
affect the relative area occupied by stromal tissue, alveo-
lar lumen or total epithelial cells in mammary tissue.

Suppression of prolactin, however, markedly influenced the

93

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94

differentiation of mammary epithelial cells. For example,
among cows treated with CB154, 17.6% of the total epithe-
lial cells were undifferentiated, 65.2% were intermedi—
ately differentiated and 17.5% were classified as fully
differentiated. In contrast, none of the epithelial cells
from either lactating controls or cows treated with CB154
plus prolactin were classified as undifferentiated. In
addition, 73.0% of the epithelial cells from lactating
controls and 78.7% of the epithelial cells from cows
treated with C8154 plus prolactin were classified as fully
differentiated. And only 27.0 and 21.3% of the alveolar
cells were intermediately differentiated in lactating con-
trols and cows treated with 08154 plus prolactin, respec—
tively (Table 7). In comparison, significantly fewer

(P < .001) fully differentiated mammary cells were ob-
served in cows receiving C8154 alone and consequently, a
greater (P < .001) proportion of the epithelial cells were
intermediately differentiated. The proportion of fully
differentiated epithelial cells among all cows killed post-
partum was positively correlated with average daily milk
production during the 2 days prior to slaughter (r = .85,

P < .01).

3.12 Electron Microscopy of Mammary Tissue

Electron micrographs of alveolar cells from a prepar-

tum control, a postpartum control, a cow treated with C8154

95

and a cow treated with CB154 plus prolactin are given in
Figures 17, 18, 19 and 20, respectively. Alveolar cells
from cows killed prepartum were characterized by a rela-
tive absence of cellular organelles. In most prepartum
mammary cells the nucleus and lipid droplets were the pre-
dominant cellular structures (Figure 17). The size, loca-
tion and number of lipid droplets per cell were, however,
variable. In some cells large lipid droplets dominated
the cell or protruded into the alveolar lumen. Other
cells contained several small lipid droplets scattered
throughout the cell or no lipid droplets at all. The cyto-
plasm of such undifferentiated cells contained mitochon-
dria but few strands of BBB and only a limited number of
Golgi membranes. Clusters of ribosomes were observed
throughout the cytoplasm and ribosomes were also observed
attached to the outer nuclear membrane.

In contrast to prepartum mammary cells, alveolar
cells from lactating controls were distinguished by a
marked proliferation of cellular organelles. These cells
generally displayed abundant basally located RER, perinu-
clear hypertrophy of Golgi membranes and clusters of secre-
tory vesicles throughout the apical cytoplasm. In addi-
tion, large and small lipid droplets were noted and large
lipid droplets were often observed protruding into the
alveolar lumen, presumably fixed just prior to secretion

from the cell. Secretory vesicles were routinely in close

Figures 17-
20
Figure 17

96

Electron micrographs of alveolar epithelial
cells from bovine mammary gland. Symbols used
are: alveolar lumen, L; capillary, C; lipid
droplets, F; nucleus, N; Golgi vesicles, G;
rough endoplasmic reticulum, E.

Alveolar cells from a prepartum mammary gland
characterized by a relative absence of cellu-
lar organelles. Strands of RER and ribosomes
attached to the outer nuclear membrane are
denoted by arrows (9000 X).

 

 

98

Figure 18 Alveolar cells from postpartum control mammary

gland characterized by marked proliferation of
cellular organelles. Arrows denote close ap-

position of Golgi vesicles and lipid droplets
(10,060 X).

Figure 19

100

Alveolar cells from cow treated with CB154
distinguished by occurrence of fewer cellular
organelles than lactating control. In par-
ticular, lack of abundant parallel arrays of
RER and abundant apical secretory vesicles.
Strands of RER are denoted by the arrows
(11,760 X).

 

Figure 20

102

Alveolar cells from cow treated with CB154
plus prolactin. The cells are indistinguish-

able from those in the lactating control
(10,040 X).

 

 

104

apposition to such apically located lipid droplets (Figure
18). Mitochondria appeared to be located primarily near
the nucleus and in the basal cytoplasm. In comparison
with prepartum mammary cells, few clusters of free ribo-
somes were seen in the cytoplasm and greatly increased
numbers of ribosomes were attached to membranes in plenti-
ful parallel stacks of BER. Additionally, microvilli were
larger and more abundant in alveolar cells from lactating
controls than from prepartum controls (Figure 18).
Alveolar cells from cows treated with C8154 had all
of the organelles observed in lactating controls, but the
cells contained less BER and fewer secretory vesicles
(Figure 19). It should be noted, however, that other
cells structurally indistinguishable from the undeveloped
cells in prepartum mammary glands or the fully differen-
tiated cells in mammary glands from postpartum control
cows also were observed. Generally, alveolar cells from
cows treated with CB154 lacked the pronounced cellular
polarity attributed to a majority of the cells in lacta-
ting controls or cows treated with C8154 plus prolactin.
In particular, cells from cows treated with C8154 con-
tained numerous strands of RER, but the strands often
appeared scattered throughout the cytoplasm rather than in
parallel arrays in the basal cytoplasm. Also, such cells
lacked abundant apical secretory vesicles characteristic

of fully differentiated cells (Figure 19).

105

Ultrastructurally, alveolar cells from cows treated
with CB154 plus prolactin were indistinguishable from the
alveolar cells observed in normal lactating mammary glands.
In contrast with cows treated with CB154, structurally un—
differentiated alveolar cells were not observed in cows
which received prolactin replacement therapy (Figure 20).

Results of the quantitative ultrastructural analysis
are presented in Table 8. Compared with alveolar cells
from prepartum mammary glands there were 11.3 and 5.2-fold
increases (P < .001) in the cellular areas containing
Golgi vacuoles and RER, respectively, in lactating con-
trols. Furthermore, the nucleus occupied 43.3% of the
cellular area in prepartum mammary cells. A greater
(P < .01) proportion of the cellular area also contained
lipid droplets and other cellular organelles (Table 8) in
prepartum mammary cells. Relative proportions of cellular
area containing the nucleus, Golgi elements, lipid, RER or
other organelles were not significantly different (P > .05)
in postpartum controls compared with cows treated with
CB154 plus prolactin. The relative amounts of RER and
Golgi elements, however, were significantly lower
(P < .001) in cows treated with CB154 than in either lacta-
ting controls or in cows treated with CB154 plus prolactin.
In cows treated with CB154 the nucleus occupied 11.2 and
14.0% greater (P < .01) proportion of the mammary cell
than in postpartum controls or cows treated with C8154,

respectively.

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DISCUSSION

Results of numerous experiments with rabbits and ro-
dents indicate that growth of the mammary gland and subse-
quent biochemical and cytological differentiation of the
alveolar epithelial cells are prerequisites for the onset
of milk secretion at parturition (Kuhn, 1977; Wooding,
1977; Tucker, 1979). Differences observed between mammary
glands from cows killed prepartum and lactating controls
in the present study indicate that similar events occur
with the onset of lactation in cattle. In particular,
proliferation of mammary cells, appearance of specific
mammary enzymes, accumulation of cellular RNA and prolif-
eration of cellular organelles takes place during the in-
terval between 10 days prepartum and 10 days postpartum,
presumably just prior to or in concert with onset of milk
secretion. For example, Mellenberger 33 El- (1973) re-
ported that marked increases in the biosynthetic capacity
of cow mammary tissue occurred with the initiation of lac-
tation. Approximately 4 to 14-fold greater rates of lac-

tose synthesis (Table 3), CO production and fatty acid

2
synthesis (Table 2) in mammary tissue from lactating con-
trols compared with prepartum controls in the present study

support their findings. In addition, activities of

107

108

acetyl-CoA synthetase, acetyl—CoA carboxylase and fatty
acid synthetase were much greater (Table 5) in lactating
controls than in prepartum controls. Furthermore, in-
creased activities of these enzymes were associated with
increased rates of fatty acid synthesis (Table 2) which
supports suggestions that these enzymes regulate fatty
acid synthesis in bovine mammary gland (Mellenberger £3
91., 1973). Synthesis of milk fatty acids also requires
NADPH. Hence, it is not surprising that the activities of
enzymes which generate cytoplasmic NADPH increase with the
onset of milk synthesis. For example, Shirley 23 El-
(1973) reported 18 to 44-fold increases in the activities
of isocitrate dehydrogenase, glucose—6 phosphate dehydro-
genase and 6-phosphogluconate dehydrogenase in the mammary
glands of primiparous heifers induced into lactation as a
result of Caesarean section. These relative changes in
the activities of the NADP dependent dehydrogenases are
substantially greater than those reported by Mellenberger
gt al.'(1973) (1.5 to 2.8-fold increases between 30 days
prepartum and 7 days postpartum) or those in the present
study (1.6 to 3.4-fold). The discrepancy between these
data may be due to use of primiparous heifers by Shirley
23 a1. (1973) in contrast with the multiparous cows used
by Mellenberger gt El- (1973) and in the present study.
For lactose synthesis to occur mammary cells must syn-
thesize a-lactalbumin (Jones, 1977). Mellenberger 33 al.,

(1973) observed that synthesis of a-lactalbumin during

109

lactogenesis in cows occurred coincidently with the onset
of lactose synthetase activity and biosynthesis of lac-
tose. Similarly, increased concentrations of a-
lactalbumin (Table 3) were associated with increased rates
of lactose synthesis (Table 3) in mammary tissue from
postpartum controls compared with prepartum controls.
Concentrations of a-lactalbumin in mammary cytosol among
all cows in the present study were correlated (r = .86)
with rates of lactose synthesis in mammary tissue slices.
These results support suggestions that lactose synthesis
is regulated by lactose synthetase, and that synthesis of
a-lactalbumin controls formation of lactose synthetase
(Brew, 1969).

Specific quantitative comparisons of histological
structures in mammary glands of prepartum and postpartum
cows have not been previously reported. However, relative
proportions of stromal tissue, epithelial cells and lu-
menal area in mammary tissue from prepartum cows (Table 7)
were nearly identical to those reported by Xinsella and
Heald (1972) for mammary tissue taken from a single cow 2
days prepartum. When compared with mammary tissue from
cows killed prepartum, mammary tissue from lactating con-
trols contained a greater proportion of epithelial cells
but the relative areas occupied by connective tissue or
alveolar lumena were not significantly different (Table 7).
These comparisons, however, must be made cautiously since

lactating cows were milked .5 to l h before slaughter but

110

no attempt was made to remove variable quantities of
colostral-like mammary secretions accumulated in the mam-
mary glands of cows killed prepartum. Thus, the effect of
accumulated mammary secretions on the histological appear-
ance of mammary tissue from prepartum controls can not be
evaluated. Nonetheless, these results suggest that the
amount of connective tissue in the mammary gland is rela—
tively unchanged during the periparturient period. Lack
of a significant difference in total mammary gland content
of hydroxyproline (a measure of connective tissue - colla-
gen) in cows killed prepartum compared with lactating con-
trols (Table 6) support this conclusion. Moreover,
Harkness and Harkness (1956) reported that pregnancy and
lactation had no effect on the collagen content of the rat
mammary gland. In contrast, Paape and Tucker (1969) found
that the total mammary gland content of hydroxyproline de-
creased during involution in rats.

Results of the histological analysis (Table 7) indi-
cated that the relative number of epithelial cells was
greater in mammary tissue from lactating control cows than
in mammary tissue from prepartum controls. An increase in
total mammary gland DNA in lactating control cows compared
with prepartum control cows (Table 6) also indicates that
lactating mammary glands contained a greater number of
epithelial cells. Specifically, based on the histological
estimate there was a 1.4-fold increase in the relative num-

ber of epithelial cells in postpartum mammary gland

111

compared with prepartum glands (Table 7) and a 1.6-fold
increase in total numbers of mammary cells estimated from
DNA content of the mammary gland. Similarly, Mumford
(1964) reported good correlation (r = .83) between esti-
mates of numbers of epithelial cells in the mammary gland
determined by quantitative histological methods and esti-
mates based on mammary gland DNA. 'In addition, the histo-
logical evaluation offers two distinct advantages over the
DNA method. First, epithelial cells may be distinguished
from other types of cells found in the mammary gland and
secondly, cytological characteristics of the epithelial
cells can be determined. This is important because in-
creases in the proportion of differentiated mammary cells
have been associated with increased milk production in cows
(Croom gt gt., 1976; Akers gt gt., 1977). The histological
evaluation does not, however, provide a direct measure of
total mammary content of cells or other histological struc-
tures. Consequently, both methods are required for ade—
quate assessment of mammary gland development (Sud gt gt.,
1968).

Not only was the total DNA content increased but also
there was a 3.6-fold increase in total mammary gland con-
tent of RNA in udders from lactating controls compared with
udders from prepartum controls (Table 6). Similarly,
Anderson gt gt. (1974) found that the total RNA content of
udders from cows in late lactation was 2.6-fold greater

than the total RNA content of udders obtained from

112

non-lactating cows. The RNA/DNA ratio increased from 0.95
in prepartum mammary glands to 1.86 in postpartum mammary
glands. This observation is similar to results reported
for a variety of laboratory species (Mumford, 1964) as well
as sheep (Anderson, 1975), goats (Anderson, 1979) and cows
(Tucker gt gt., 1973) and support the general conclusion
that the RNA/DNA ratio remains less that unity during preg-
nancy but rapidly increases with the onset of lactation.
Consequently, the RNA/DNA ratio has been used as an indi—
cator of metabolic activity (Tucker, 1969). The RNA/DNA
ratio also appears to reflect variation in milk production.
For example, Anderson gt gt. (1974) reported a RNA/DNA
ratio of 1.15 in late lactating cows producing 10.1 kg/day
whereas in the present study lactating controls producing
29.2 kg/day (during 2 days prior to slaughter) had a
RNA/DNA ratio of 1.86.

Cytological changes in mammary cells which occur with
the onset of milk secretion have been reviewed (Heald, 1974;
Wooding, 1977). Comparison of alveolar cells from prepar-
tum mammary glands with alveolar cells from postpartum
control mammary glands, indicated that a majority of the
cells were fully differentiated by 10 days after parturi-
tion (Table 7), and the cells exhibited a marked prolifera-
tion of cellular organelles (Figures 17, 18 and Table 8).
In contrast, at 2 days postpartum most alveolar cells in
bovine mammary gland were poorly differentiated (Akers gt

gt., 1977) and were characterized by minimal proliferation

113

of cellular organelles (Akers and Heald, 1978). This sug-
gests that cellular differentiation is not completed im-
mediately following parturition but continues into early
lactation. Perhaps the period of time after parturition
when milk yields reach a maximum corresponds to a period of
time when completion of cellular differentiation occurs.

Treatment of cows with CB154 reduced basal concentra-
tions of prolactin in serum (Figure 2), blocked the normal
surge in serum prolactin concentrations at parturition
(Figure 3) and prevented the usual increase in serum pro-
lactin concentrations which occur at milking (Figure 4).
These results illustrate the potent effect of C3154 on
suppression of prolactin secretion in the cow and confirm
reports by others (Karg and Schams, 1972; Smith gt gt.,
1974; Beck gt gt., 1979). In addition, treatment with
CB154 markedly reduced milk production during 10 days post-
partum. This result suggests that increased secretion of
prolactin at parturition is a primary stimulator of milk
production in cows. Furthermore, periparturient infusion
of exogeneous prolactin for only 6 days into a second group
of cows treated with CB154 produced milk yields comparable
to those observed in untreated controls (Figure 9). This
provides conclusive evidence that reduced milk yields
attributed to the relative absence of prolactin in cows
treated with CB154 was indeed caused by lack of prolactin
at parturition.

Changes in periparturient concentrations of growth

114

hormone, progesterone and glucocorticoids were not signif-
icantly affected by treatment with CB154 or CB154 plus
prolactin (Figures 6, 7, 8). This is important because
growth hormone, progesterone and glucocorticoids each in-
fluence initiation of milk synthesis at parturition (Tucker,
1979). Lack of effect by C8154 on serum concentrations of
growth hormone and glucocorticoids supports a previous re-
port (Smith gt gl., 1974) which showed that C3154 had no
effect on serum concentrations of either hormone in lacta-
ting cows, and that CB154 reduced release of prolactin but
not growth hormone from bovine pituitary cells cultured
Treatment with CB154 did not adversly affect feed in-
take in cows from 10 days before through 10 days after par-
turition (Figure 13). An adequate supply of nutrients is
essential for the mammary gland to synthesize milk, and re-
ductions in feed intake markedly reduce milk production in
cows (Reid gt gt., 1977). Thus, lower milk yields in cows
treated with CB154 were not caused by reduced feed intake.
Indeed, relative to controls, cows treated with CB154
tended to consume more feed after parturition, so that they
had sufficient energy available to produce quantities of
milk greater than those observed. These results are simi-
lar to those of Stern (1977) who found no effect of CB154
on periparturient feed intake in rats. Measurements of
feed intake in cows during concurrent treatment with CB154

have not been reported, although Johke and Hodate (1978)

115

observed no apparent effect of CB154 on feed intake of
cows just before and after parturition.

Based on DNA measurements (Table 6) and histological
analysis (Table 7) the data indicate that proliferation of
mammary cells occurs during the interval between 10 days
prepartum and 10 days postpartum. Furthermore, since the
number of mammary cells were not significantly different
in udders among groups of cows killed postpartum (Table 7),
this suggests that relative absence of prolactin does not
limit periparturient growth of the mammary gland in cows.
Reports that reduced secretion of prolactin had no effect
on udder growth during pregnancy in goats (Buttle gt gt.,
1978) and sheep (Djiane gt gt., 1975) support this sugges—
tion. From the data it can not be determined whether the
observed growth of the mammary gland occurred only prepar—
tum or continued into early lactation. If the cow is simi-
lar to other ruminants, it may be expected that this growth
occurs primarily prepartum. For example, in sheep
(Anderson, 1975) and goats (Anderson, 1979) approximately
98% of mammary growth was completed at the time of parturi-
tion.

Because udders from cows treated with CB154 and from
controls or cows treated with CB154 plus prolactin had
equivalent numbers of mammary cells, it may be concluded
that lower rates of milk production among cows treated with
CB154 (Figure 9) resulted because cellular function was im-

paired in cows with reduced concentrations of serum

116

prolactin. Reduced milk yields among cows treated with
CB154 were reflected in lower rates of lactose synthesis
(Table 3) and a tendency toward lower rates of fatty acid
synthesis (Table 2) in mammary tissue slices as well as
reduced activities of enzymes specific for fatty acid syn-
thesis (Table 5) and markedly reduced concentrations of a-
lactalbumin in mammary cytosol (Table 3). Results of the
histological analysis (Table 7) indicated that mammary
tissue from cows treated with CB154 contained a smaller
proportion of fully differentiated epithelial cells, than
either cows treated with CB154 plus prolactin or lactating
controls. Also, in contrast with lactating controls or
cows which received prolactin replacement therapy, a rela-
tively large proportion of undifferentiated mammary cells
were observed in cows treated with CB154. Akers gt gt.
(1977) reported that synthesis of lactose, fat and casein
were negatively correlated (-.80, -.63 and -.83, respec-
tively) with the relative number of undifferentiated epi-
thelial cells in mammary glands from cows, suggesting that
occurrence of undifferentiated alveolar cells in cows
treated with CB154 contributed to lower milk yields. In
addition, Croom gt_gl. (1977) reported close association
between the appearance of structurally differentiated cells
in mammary tissue from cows induced into lactation and sub-
sequent milk production. Thus, lower milk yields among
cows treated with CB154 may be attributed to the occurrence

of undifferentiated mammary cells and relative lack of

117

fully differentiated mammary cells. This also indicates
that prolactin may induce differentiation of the mammary
epithelium in cows.

Suppression of prolactin secretion had no significant
effect on concentrations of total protein (Figure 10) or
lactose (Figure 11) measured in milk, and the concentra-
tions determined 1 to 5 days before slaughter were similar
to those reported for other Holstein cows (Cerbulis and
Farrell, 1975). These results seem in conflict with the
observation that rates of lactose synthesis in mammary tis-
sue from cows treated with CB154 were much lower than in
controls (Table 3). It is assumed that lactose is produced
in Golgi vacuoles (Brew, 1969), that water and ions then
enter the secretory vacuoles (Linzell and Peaker, 1971) and
that vacuole contents form the aqueous portion of normal
milk after release from the secretory cells (Foster, 1978).
Because Golgi membranes are impermeable to lactose it is
the lactose concentration within the vacuoles that creates
the osmotic gradient which draws water and ions into the
vacuole (Jones, 1977). Thus, if vacuoles were formed with
a lower than usual lactose concentration, then less total
amounts of water and ions would be secreted (lower milk
production) but the relative concentration of lactose would
be unaltered. Alternatively, lower rates of lactose syn—
thesis might simply result in formation of fewer secretory
vacuoles and consequently lower milk production. This

seems to be a likely possibility because mammary glands

118

from cows treated with CB154 had fewer fully differentiated
cells (partially characterized by a larger number of cellu-
lar vacuoles, Table 7), and mammary cells from cows treated
with CB154 had fewer Golgi membranes and vacuoles (Table 8)
than controls or cows treated with CB154 plus prolactin.

Although, a slower rate of decline in milk protein
percent (Figure 10) following parturition was observed in
cows treated with CB154, the treatment had no lasting
effect on the concentration of total protein in milk. In
contrast, the a-lactalbumin content of milk (Figure 12) re-
mained approximately 50% lower in cows treated with CB154
than in controls or cows treated with CB154 plus prolactin.
Presumably, this is a consequence of low concentrations of
serum prolactin in cows treated with CB154, since prolactin
stimulates synthesis of a-lactalbumin proportional to dose
when added to cultures of mammary tissue from primate
(Kleinberg gt gt., 1978), mouse (Vonderhaar, 1978) and cow
(Goodman gt gt., unpublished). Beck and Tucker (1977) also
reported that the prolactin and a-lactalbumin content of
milk were correlated (r = .46) during the first month of
lactation in cows.

Prolactin has multiple effects upon the mammary gland
(Cowie and Forsyth, 1975). For example, prolactin stimu-
lates formation of endoplasmic reticulum and appearance of
secretory vesicles in the mammary cells of rabbits (Devinoy
gt gt., 1979), mice (Mills and Topper, 1970), rats

(Chatterton gt gt., 1979) and cows (Collier gt gt., 1977).

119

Results of the present experiment indicate that structural
differentiation of mammary cells in cows at parturition is
at least partially dependent on prolactin. Prolactin has
also been shown to stimulate lactose and a-lactalbumin syn-
thesis in the mammary gland (Turkington and Hill, 1969).
Similarly, mammary tissue from lactating controls and cows
treated with CB154 plus prolactin synthesized greater quan-
tities of lactose and contained greater amounts of a-
lactalbumin than mammary tissue from cows with reduced pro-
lactin secretion. Indeed, the ability of mammary tissue
slices to synthesize lactose was highly correlated with the
concentrations of a—lactalbumin in mammary cytosol. This
supports suggestions that prolactin stimulates synthesis of
a-lactalbumin and that a-lactalbumin controls synthesis of
lactose.

Results of this study provide the first conclusive
evidence that periparturient secretion of prolactin stimu-
lates milk production during early lactation in cattle.
Furthermore, the data indicate that this effect on milk
production occurs because prolactin promotes biochemical
and cytological differentiation of the mammary epithelial
cells. These data not only confirm suggestions by others
that prolactin is involved in the initiation of milk secre-
tion in cattle but also provide an indication as to how
prolactin influences synthesis of milk.

Prolactin has long been implicated in the initiation

of milk secretion in rodents and rabbits (Tucker, 1979),

120

however, Karg E£.El° (1972) were apparently the first to
demonstrate that cows treated with CB154 (a synthetic ergot
alkaloid which inhibits secretion of prolactin) immediately
before parturition produced less milk than expected during
early lactation. Johke and Hodate (1978) also reported
that CB154 caused reduced milk yields in cows treated from
2 weeks prepartum until parturition. Because treatment
with C3154 also reduced basal concentrations of prolactin
in serum and prevented the usual surge in serum prolactin
concentrations at parturition, these investigators inter-
preted lowered milk yields to indicate that prolactin was
necessary for initiation of milk synthesis at parturition
in cattle. Yet this conclusion had to be considered tenta-
tive because these data could not rule out the possibility
that lower milk yields in cows treated with CB154 were
caused by effects of CB154 unrelated to the drug's ability
to suppress prolactin secretion. These results are also
difficult to reconcile with reports that C8154 does not
effect milk production during established lactation in
dairy cows (Karg gt gt., 1972; Smith gt gt., 1974).

Other reports, however, suggest that prolactin is pos-
itively related to milk secretion in cattle. For example,
pituitaries from early lactating, high milk-producing cows
contain greater amounts of prolactin than pituitaries from
late lactating low milk—producing cows (Reece and Turner,

1937). In addition, cows in early lactation release

121

greater quantities of prolactin to the milking stimulus
than cows during late lactation (Koprowski and Tucker,
1973; Peters, 1980) and estimated mammary uptake of prolac-
tin following milking or injection of TRH (thyrotropin re-
leasing hormone) was greatest during early lactation (Beck
gt gt., 1979). These data suggest that greater concentra—
tions of prolactin in serum during early lactation may be
associated with greater lactational intensity. In support
of this idea, Convey gt gt. (1973) found that TRH stimu-
lated release of prolactin from the pituitary and increased
milk yields in cows. Furthermore, prolactin secretion
rates were greater in early lactation than late lactation
in rats (Grosvenor §£.El-: 1977; Grosvenor and Whitworth,
1979), sheep (David and Borger, 1973) and cows (Akers gt
gt., 1979). Moreover, secretion rates of prolactin were
positively correlated with daily milk production in early
lactating (r = .64) and late lactating (r = .60) cows
(Akers gt gt., 1979). Also, milk yields from cows induced
into lactation with estrogen, progesterone and reserpine
injections (Collier gt gt., 1977) were greater when trials
were conducted during the spring (coincident with greater
basal concentrations of serum prolactin and increased se-
cretion of prolactin in response to reserpine) than during
the winter (coincident with lower serum prolactin concen-
trations and reduced response to reserpine) (Kensinger gt
gt., 1979). Indeed, correlations between serum concentra-

tions of prolactin after reserpine and maximum milk yields

122

or first 100 day milk yields were r = .65 and r = .64,
respectively (Kensinger gt gt., 1979). Collectively, these
data support the conclusion that prolactin is involved in
maintenance of milk production in cattle.

While results of the present study clearly show that
periparturient secretion of prolactin stimulates milk pro-
duction, it is also apparent that suppression of basal
serum prolactin concentrations and prevention of the surge
in serum prolactin concentrations at parturition did not
completely prevent initiation of lactation. This indicates
that only minimal quantities of prolactin are absolutely
required for lactogenesis in cattle but that increased con-
centrations of prolactin stimulate onset of copious milk
secretion just after parturition. Since it is recognized
that a complex of hormones affect the mammary gland to pro-
mote lactogenesis (Tucker, 1979), perhaps increased concen-
trations of growth hormone, glucocorticoids or other hor-
mones at parturition synergize with the low concentrations
of prolactin resulting from CB154 treatment to initiate
lactation.

Treatment of cows with CB154 suppressed serum concen-
trations of prolactin, but did not completely abolish se-
cretion of prolactin. Even in the face of relatively lower
concentrations of serum prolactin (m 6 to 10 ng/ml) udders
of cows treated with CB154 would have been exposed to con-
siderable quantities of prolactin each day. If it is as-

sumed that prolactin averaged 10 ng/ml of blood during day 1

123

of lactation and that cows treated with CB154 produced an
average of 3.5 kg of milk during the first day of lacta-
tion, mammary blood flow (MBF) may be calculated using the
equation (Y = 1.0 + 0.42X, where Y = MBF in liters/min and
X = daily milk yield in liters) of Knonfeld gt gt. (1968).
The calculated blood flow for cows treated with CB154 using
these assumptions would be 3.94 liters/min. Thus, during
the first day of lactation, udders from cows treated with
CB154 would have been exposed to approximately 57 mg of
prolactin. Also, because milk production increased follow-
ing parturition, total quantities of prolactin available to
the mammary gland would also increase as a result of in—
creased blood flow. Milk production in cows treated with
CB154 progressively increased despite continued suppression
of prolactin secretion. How may increased milk production
be explained? Results of related experiments with prolac-
tin receptors provide the basis for a possible answer. The
number of prolactin receptors in the mammary glands of rab-
bits (Djiane gt gt., 1977), rats (Hayden gt gt., 1979) and
mice (Sakai et a1., 1978) remain low during pregnancy but
increase dramatically in close association with the onset
of milk secretion. In addition, antisera prepared against
solubilized prolactin receptors from rabbit mammary gland
blocked the stimulatory action of prolactin on casein syn-
thesis and amino acid transport by rabbit mammary gland tg
vitro (Shiu and Friesen, 1976). These observations support

the hypothesis that at least a portion of prolactin's

124

effect on the mammary gland is mediated via receptors, and
that increasing numbers of prolactin receptors at the onset
of lactation could amplify prolactin stimulation of milk
synthesis. Related to the hormonal shifts at parturition,
progesterone blocks the induction of prolactin receptors
and prolactin stimulates formation of its own receptor in
the rabbit mammary gland (Djiane and Durand, 1977). If the
mammary gland of the cow behaves in a similar fashion, the
following scenario may explain the progressive increase in
milk production following parturition in cows treated with
CBl54. In controls and cows treated with CB154 plus pro-
lactin, prolactin concentrations would have been increased
when progesterone declined at parturition. Therefore,
large quantities of prolactin would have been available to
stimulate formation of new prolactin receptors which were
subsequently involved in increased milk synthesis. In con-
trast, in cows treated with 08154 relatively less prolactin
would have been available at parturition; consequently,
formation of prolactin receptors and stimulation of milk
synthesis immediately after parturition would have been
minimal and/or delayed. Therefore, the progressive in-
crease in milk production in cows treated with CB154 may
reflect the ability of small amounts of prolactin to stimu-
late milk production because its influence is amplified by
progressive increases in the number of prolactin receptors
in the mammary gland. Thus, it may be speculated that the

periparturient surge in prolactin secretion evolved to

125

insure that c0pious amounts of milk would be produced soon
after parturition. In contrast, the author is unaware of
any reports which demonstrate the complete suppression of
prolactin secretion in cattle. Consequently, whether lac-
togenesis would occur in cows in the complete absence of
prolactin is unknown. Nonetheless, the data presented in
this dissertation clearly show that secretion of prolactin
at parturition stimulates subsequent milk production and
that prolactin promotes biochemical and cytological dif-
ferentiation of the mammary epithelium in cattle. Further-
more, markedly reduced secretion of milk following parturi-
tion in cows treated with CB154 underscores stimulation of
milk synthesis usually elicited by prolactin. Therefore,
based on the results in this study and survey of the perti-
nent literature, I conclude that prolactin is essential for

onset of copious milk secretion in cattle.

SUMMARY AND CONCLUSIONS

The role of prolactin in mammary development during
late pregnancy and stimulation of milk secretion during
early lactation was studied in multiparous Holstein cows.
Specifically, the biosynthetic capacity, activities of en-
zymes associated with milk synthesis, nucleic acid content,
histological appearance and ultrastructure of alveolar epi-
thelial cells were evaluated in mammary tissue obtained
from cows killed 10 days prepartum or 10 days postpartum.
To study the effect of prolactin on periparturient changes
in the mammary gland and subsequent milk production, cows
were treated with 2-bromo-a-ergocryptine (CB154) to sup—
press periparturient secretion of prolactin. As controls,
a second group of cows treated with CB154 were infused for
6 days with exogeneous prolactin beginning 5 days before
expected parturition. Cows treated with CB154 or CB154
plus prolactin were killed 10 days postpartum, the udders
were removed and variables described previously were used
to evaluate mammary function. In addition, concentrations
of prolactin, growth hormone, glucocorticoids and proges-
terone were measured in selected serum samples collected
before, during and after parturition.

Changes in the mammary gland during the interval

126

127

between 10 days prepartum and 10 days postpartum had a
marked effect on the capacity of mammary tissue to synthe-
size milk. Rates of lactose synthesis, fatty acid synthe-
sis and CO2 production were 4 to 14-fold greater (P i .01)
in mammary tissue from lactating controls than in prepar-
tum controls. Similarly, activities of acetyl-CoA synthe-
tase, acetyl-CoA carboxylase and fatty acid synthetase
were much greater (P < .001) in mammary tissue from lacta-
ting cows than in mammary tissue taken from cows killed
prepartum. Thus, greater rates of fatty acid synthesis in
lactating mammary tissue corresponded to greater activi-
ties of enzymes associated with milk fat synthesis.
Markedly lower (P < .001) concentrations of a-lactalbumin
in prepartum mammary tissue paralleled the relative in-
ability of mammary tissue slices from cows killed prepar- ”
tum to synthesize lactose.

Lower biosynthetic activity in prepartum mammary
glands was also associated with the occurrence of propor-
tionally fewer epithelial cells (P < .001) compared with
postpartum mammary tissue. Furthermore, all of the mam-
mary epithelial cells examined from prepartum glands were
cytologically undifferentiated whereas 73.4% of the epithe-
lial cells examined from lactating mammary glands were
classified as fully differentiated. Also, udders from
cows killed prepartum contained less (P < .001) total DNA
than udders from lactating cows. These data confirmed the

histological evidence that udders obtained prepartum

128

contained fewer epithelial cells than lactating udders.

Compared with alveolar cells from prepartum mammary
glands, there was an 11.3 and 5.2-fold increase (P < .001)
in the cellular area containing Golgi vacuoles and RER,
respectively, in lactating controls. Thus, marked prolif-
eration of cellular organelles involved in milk synthesis
and secretion occurred during late pregnancy and early lac-
tation. Total RNA content of the mammary gland increased
3.6-fold and the RNA/DNA ratio increased from .95 to 1.86
in lactating compared with non-lactating mammary glands
(P g .01). Consequently, I conclude that reduced biosyn-
thetic activity of prepartum mammary tissue resulted be-
cause the tissue contained fewer epithelial cells and that
the cells were poorly differentiated relative to alveolar
celts from lactating mammary glands.

Treatment of cows with CB154 reduced basal concentra-
tions of prolactin in serum, blocked the normal surge in
serum prolactin concentrations at parturition and prevented
the usual increase in serum prolactin concentrations which
occur at milking (P i .001). Treatment with CB154 reduced
milk yields of cows during 10 days of lactation. However,
periparturient infusion of exogeneous prolactin for only 6
days into a second group of cows treated with CB154 pro-
duced milk yields comparable to those observed in untreated
controls. This provides conclusive evidence that secretion
of prolactin at parturition is a primary initiator of milk

synthesis in cows.

129

Udders from postpartum controls and cows treated with
CB154 or CB154 plus prolactin contained relatively equiva-
lent total amounts of DNA (P > .05). This indicates that
reduced milk yields among cows treated with CB154 resulted
not because cows with suppressed prolactin secretion had
fewer mammary cells but rather that cellular function was
impaired.

Rates of lactose synthesis were 40% lower (P < .01)
and concentrations of o-lactalbumin 48% lower (P < .001) in
cows treated with C8154 than in lactating controls. More-
over, prolactin replacement therapy produced rates of lac-
tose synthesis and concentrations of o-lactalbumin compa-
rable to those observed in lactating controls. These re-
sults suggest that prolactin stimulates lactose synthesis
and that synthesis of a-lactalbumin is closely associated
with the ability of the mammary gland to produce lactose.
In support of this idea, among all cows killed postpartum,
the concentration of a-lactalbumin in mammary cytosol was
positively correlated with rates of lactose synthesis mea-
sured in mammary tissue slices (r = .72, P < .01). Mammary
tissue from cows treated with CB154 also tended (P < .07)
to exhibit slower rates of fatty acid synthesis than mam-
mary tissue from lactating controls or cows treated with
CB154 plus prolactin. This tendency was complemented by
lower activity of fatty acid synthetase (P < .05) in cows
treated with CB154 than in lactating controls or cows

treated with CB154 plus prolactin. Inhibition of prolactin

130

secretion reduced (P < .05) total RNA content of the mam-
mary gland 36% and decreased the RNA/DNA ratio (1.38), rel-
ative to cows which received prolactin replacement therapy
(ratio = 2.17) or to untreated lactating controls (ratio =
1.86). These results also support the conclusion of lower
metabolic activity in cows treated with CB154.

Suppression of prolactin had a marked influence on
differentiation of mammary epithelial cells. Among cows
treated with C8154, 17.6% of the epithelial cells evaluated
were undifferentiated, 65.2% were intermediately differen-
tiated and only 17.5% were classified as fully differen-
tiated. In contrast, none of the epithelial cells charac-
terized from either lactating controls or cows treated with
CB154 plus prolactin were undifferentiated. In addition,
73.0% of the epithelial cells from lactating controls and
78.7% of the epithelial cells from cows treated with CB154
plus prolactin were classified as fully differentiated.

And only 27.0 and 21.3% of the alveolar cells were inter-
mediately differentiated in lactating controls and cows
treated with CB154 plus prolactin, respectively. It seems
likely that the degree of cellular differentiation of the
alveolar epithelium closely reflects the relative biosyn-
thetic capacity of these milk synthesizing cells. For ex-
ample, the proportion of fully differentiated epithelial
cells among all cows killed postpartum was positively cor-
related with average daily milk production during the 2

days prior to slaughter (r = .85, P < .01).

131

Relative lack of cellular differentiation in cows with
suppressed prolactin secretion was also indicated by elec-
tron microscopic examination of alveolar cells. Specifi—
cally, the RER occupied 23.5 and 26.8% of the alveolar cell
area in lactating controls and cows treated with CB154 plus
prolactin, respectively; but only 16.4% in cows treated
with CB154 (P < .001). In addition, the relative area
occupied by Golgi membranes and vacuoles was approximately
11% lower (P < .001) in cows treated with CB154 than in
lactating controls or cows which received prolactin re-
placement therapy. Since the RER and Golgi are essential
for the synthesis and secretion of milk, lower milk yields
in cows treated with C8154 may be partially attributed to
failure of complete differentiation of secretory cells in
cows with suppressed periparturient secretion of prolactin.
Consequently, it may be concluded that secretion of prolac-
tin at parturition stimulates periparturient cellular and
biochemical differentiation of the mammary epithelium, and
that prolactin is essential for the onset of copious milk

secretion in cows.

BIBLIOGRAPHY

BIBLIOGRAPHY

Akers, R.M., G.T. Goodman and H.A. Tucker. 1979. Effect
of stage of lactation on secretion and clearance
rates of prolactin in Holstein cows. In: Program
of 7lst Annual Meeting of Amer. Soc. Anim. Sci.

p. 277.

Akers, R.M. and C.W. Heald. 1978. Stimulatory effect of
prepartum milk removal on secretory cell differen-
tiation in the bovine mammary gland. J. Ultra-
structure Res. 63:316.

Akers, R.M., C.W. Heald, T.L. Bibb and M.L. McGilliard.
1977. Effect of prepartum milk removal on the
quantitative morphology of bovine lactogenesis. J.
Dairy Sci. 60:1272.

Anderson, R.R. 1975. Mammary gland growth in sheep. J.
Anim. Sci. 41:118.

Anderson, R. R. 1979. Mammary growth in dairy goats. J.
Dairy Sci. 62: Supplement #1 Program of 74th Annual
Meeting. p. 56.

Anderson, R.R., M.H. Lu, J.J. Trojanor and J.L. Clark.
1974. Milk production, wet weight, dry weight, po-
tassium, and nucleic acid measurements of cow's
udders. J. Dairy Sci. 57:1350.

Assairi, L., C. Delouis, P. Gaye, L. Houdebine, M.
Ollivier-Bousquet and R. Denamur. 1974. Inhibi-
tion by progesterone of the lactogenic effect of
prolactin the pseudopregnant rabbit. Biochem. J.
144:245.

Baldwin, R.L. 1966. Enzymic activities in mammary glands
of several species. J. Dairy Sci. 49:1533.

Baldwin, R.L. and 8. Louis. 1975. Hormonal actions on
mammary metabolism. J. Dairy Sci. 5821033.

132

133

Baldwin, R.L. and Y.T. Yang. 1974. Enzymatic and meta-
bolic changes in the development of lactation. In:
Lactation: a comprehensive treatise. B.L. Larssfi
and V.R. Smith, eds., Vol. I. Academic Press, Inc.,
New York.

Barry, J.M. 1952. The source of lysine, tyrosine and
phosphorus for casein synthesis. J. Biol. Chem.
195:795.

Barry, J.M. 1961. Protein metabolism. tg: Milk: the
mammary gland and its secretion. S.K. Kon and A.T.
Cowie, eds., Vol. II. Academic Press, Inc., New
York.

Bauman, D.E., R.E. Brown and C.L. David. 1970. Pathways
of fatty acid synthesis and reducing equivalent
generation in mammary gland of rat, sow and cow.
Arch. Biochem. Biophys. 140:237.

Bauman, D.E., R.J. Collier, H.A. Tucker and R.L. Hays.
1976. Prolactin and milk yield following reserpine.
J. Anim. Sci. 43:273.

Bauman, D.E. and C.L. Davis. 1974. Biosynthesis of milk
fat. tg: Lactation: a comprehensive treatise.
B.L. Larson and V.R. Smith, eds., Vol II. Academic
Press, Inc., New York.

Beck, N.F.G. and H.A. Tucker. 1977. Relationships be-
tween radioimmunoassays of alpha-lactalbumin and
prolactin in bovine skim milk. J. Dairy Sci. 60:542.

Beck, N.F.G., H.A. Tucker and W.D. Oxender. 1979. Mammary
arterial and venous concentrations of prolactin in
lactating cows after milking or administration of
thyrotropin-releasing hormone or ergocryptine.
Endocrinology 104:111.

Bourne, R.A. and H.A. Tucker. 1975. Serum prolactin and
LH responses to photoperiod in bull calves. Endo-
crinology 97:473.

Bousquet, M., J.E. Flechon and R. Denamur. 1969. Aspects
ultrastructuraux de la glande mammaire de lapine
pendant la lactogenese. Z. Zellforsch. mikrosk
Anat. 96:418.

Bradford, M.M. 1976. A rapid and sensitive method for
quantification of microgram quantities of protein
utilizing the principle of protein-dye binding.
Anal. Biochem. 72:248.

134

Brew, K. 1969. Secretion of alpha-lactalbumin into milk
and its relevance to the organization and control
of lactose synthetase. Nature 223:671.

Brodbeck, U. and K.E. Ebner. 1966. Resolution of a sol-
uble lactose synthetase into two protein components
and solubilization of microsomal lactose synthetase.
J. Biol. Chem. 241:762.

Buttle, B.L., A.T. Cowie, E.A. Jones and A. Turney. 1979.
Mammary growth during pregnancy in hypophysecto-
mized or bromocryptine-treated goats. J.
Endocrinol. 80:343.

Cerbulis, J. and E.M. Farrell, Jr. 1975. Composition of
milk of dairy cattle. 1. Protein, lactose, and fat
contents and distribution of protein fraction. J.
Dairy Sci. 58:817.

Chalkley, H.W. 1943. Method for the quantitative morpho-
logic analysis of tissues. J. Nat. Cancer Inst.
4:47.

Chang, H., I. Seidman, G. Teebor and M.D. Lane. 1967.
Liver acetyl-CoA carboxylase and fatty acid synthe-
tase: relative activities in the normal state and
in hereditary obseity. Biochem. Biophys. Res. Comm.
28:682.

Chatterton, Jr., R.T., J.A. Harris, W.J. King and R.M.
Wynn. 1979. Ultrastructural alterations in mam-
mary glands of pregnant rats after ovariectomy and
hysterectomy: effect of adrenal steroids and pro-
lactin. Amer. J. Obstet. Gynecol. 133:694.

Chen, C.L. and J. Meites. 1970. Effects of estrogen and
progesterone on serum and pituitary prolactin
levels in ovariectomized rats. Endocrinology 86:
503.

Chew, B.P., P.V. Malven, R.E. Erb, C.N. Zamet, M.F. D'Amico
and V.F. Colenbrander. 1979. Variables associated
with peripartum traits in dairy cows. IV. Seasonal
relationships among temperature, photoperiod, and
blood plasma prolactin. J. Dairy Sci. 62:1394.

Coffey, R.G. and F.J. Reithel. 1969. An enzymatic deter-
mination of lactose. Anal. Biochem. 32:229.

Collier, R.J., D.E. Bauman and R.L. Hays. 1977. Effect of
reserpine on milk production and serum prolactin of

cows hormonally induced into lactation. J. Dairy
Sci. 60:89.

135

Collier, R.J., D.E. Bauman and R.L. Hays. 1977. Lacto-
genesis in explant cultures of mammary tissue from
pregnant cows. Endocrinology 10021192.

Collier, R.J., W.J. Croom, D.E. Bauman and R.L. Hays.
1975. Efficacy of reserpine during hormonal induc-
tion of lactation in cows. J. Dairy Sci. 58:740.

Collier, R.J., W.J. Croom, D.E. Bauman and R.L. Hays.
1976. Cellular studies of mammary tissue from cows
hormonally induced into lactation: lactose and
fatty acid synthesis. J. Dairy Sci. 59:1226.

Convey, E.M. 1974. Serum hormone concentrations in rumi—
nants during mammary growth, lactogenesis, and lac-
tation: a review. J. Dairy Sci. 57:905.

Convey, E.M., J.W. Thomas, H.A. Tucker and J.L. Gill.
1973b. Effect of thyrotropin releasing hormone on
yield and composition of bovine milk. J. Dairy
Sci. 56:484.

Cook, R.M., S.C. Lui and S. Quraishi. 1969. Utilization
of volatile fatty acids in ruminants. III. Com-
parison of mitochondrial acyl coenzyme A synthetase
activity in different tissues. Biochem. 8:2966.

Cowie, A.T. and I.A. Forsyth. 1975. Biology of prolactin.
Pharmac. Therap. B. 1:437.

Croom, W.J., R.J. Collier, D.E. Bauman and R.L. Hays.
1976. Cellular studies of mammary tissue from cows
hormonally induced into lactation: histology and
ultrastructure. J. Dairy Sci. 59:1232.

Davis, S.L. and M.L. Borger. 1973. Metabolic clearance
rates and secretion rates of prolactin in sheep.
Endocrinology 92:1414.

DeKay, D.E., D.E. Bauman and C.L. Davis. 1976. Charac-
terization of fatty acid synthesis by cow mammary
subcellular fraction. J. Dairy Sci. 59:1513.

Delouis, C. and R. Denamur. 1972. Induction of lactose
synthesis by prolactin in rabbit mammary gland ex-
plants. J. Endocrinol. 52:311.

Deluca, H.F. and P.P. Cohen. 1964. tg: Manometric Tech-
niques. W.W. Umbreit, R.H. Burris and J.F. Stauffer,
eds. Burgess Publishing Co., Minneapolis, MN.

 

 

136

Denamur, R. 1969. Changes in the ribonucleic acids of
mammary cells at lactogenesis. t2; Lactogenesis:
the initiation of milk secretion at parturition.
M. Reynolds and S.J. Folley, eds. University of
Pennsylvania Press, Philadelphia.

Denamur, R. 1974. Ribonucleic acids and ribonucleopro-
tein particles of the mammary gland. tg: Lacta-
tion: a comprehensive treatise. B.L. Larson and
V.R. smith, eds., Vol. 1. Academic Press, New
York.

Denamur, R. and P. Gaye. 1967. Les acides ribonucleiques
de la glande mammaire au cours de l'induction et de
l'entretien de la secretion lactee. Arch. Anat.
Microscopique Morphol. Exp. 56:596.

Devinoy, E., L. Houdebine and C. Delouis. 1978. Role of
prolactin and glucocorticoids in the expression of
casein genes in rabbit mammary gland organ culture--
quantification of casein mRNA. Biochim. Biophys.
Acta 517:360.

Devinoy, E., L. Houdebine and M. Ollivier-Bousquet. 1979.
Role of glucocorticoids and progesterone in the de-
velopment of rough endoplasmic reticulum involved
in casein biosynthesis. Biochimie. 61:453.

Djiane, J. and P. Durand. 1977. Prolactin-progesterone
“‘ antagonism in self regulation of prolactin recep-
tors in the mammary gland. Nature 266:641.

Djiane, J., P. Durand and P.A. Kelly. 1977. Evolution of
prolactin receptors in rabbit mammary gland during
pregnancy and lactation. Endocrinology 100:1348.

Djiane, J., G. Kann, C. Delouis and J. Martal. 1975.
Placental lactogenic hormone: plasma in ruminants;
study of its role on mammogenesis and lactogenesis
in the ewe. Intern. Symp. Growth Hormone and Re-
lated Peptides 1:128.

Drummond-Robinson, G. and S.A. Asdell. 1926. The rela-
tion between the corpus luteum and the mammary
gland. J. Physiol. 61:608.

Erb, R.E. 1977. Hormonal control of mammogenesis and on-
set of lactation in cows--a review. J. Dairy Sci.
60:155.

137

Erb, R.E., P.V. Malven, B.L. Monk, T.A. Mollett, K.L.
Smith, F.L. Schanbacher and L.B. Willett. 1976.
Hormone induced lactation in the cow. IV. Rela-
tionships between lactational performance and hor—
-mone concentrations in blood plasma. J. Dairy
Sci. 59:1420.

Fell, L.R., N.J. Chandler and J.R. Goding. 1974. Effects
of inhibition of prolactin secretion by br-alpha-
ergocryptine at parturition and during lactation in
cows. J. Reprod. Fert. 36:419.

Fleet, I.R., J.A. Goode, M.H. Hamon, M.S. Laurie, J.L.
Linzell and M. Peaker. 1975. Secretory activity
of goat mammary glands during pregnancy and the on-
set of lactation. J. Physiol. Lond. 251:763.

Folley, S.J. 1940. Lactation. Biol. Rev. 15:421.

Foster, R.G. 1977. Changes in mouse mammary epithelial
cell size during mammary gland development. Cell
Differentiation 6:1.

Foster, R.G. 1978. Lactose synthesis and secretion by
mammary epithelial cell in vitro, a comparative
study. Comp. Biochem. Physiol. 61:253.

Gardner, W.U. and C.W. Turner. 1933. The function, assay
and preparation of galactin, a lactation stimula-
ting hormone of the anterior pituitary and an in-
vestigation of the factors responsible for the con-
trol of normal lactation. Mo. Agr. Exp. Sta. Res.
Bull. 196.

Gaye, P. and R. Denamur. 1969. Acides ribonucleiques et
polyribosomes de la glande mammaire de la lapine an
course de la lactogenese indaite par la prolactine.
Biochim. Biophys. Acta. 186:99.

Gaye, P., L. Houdebine, G. Petrissant and R. Denamur.
1973. Protein synthesis in mammary gland. Acta
Endocrinologica (Kbh.), Supplement #180.

Gill, J.L. 1978a. Design and analysis of experiments in
the animal and medical sciences. Vol. I. The Iowa
State University Press, Ames, IA.

Gill, J.L. 1978b. Design and analysis of experiments in
the animal and medical sciences. Vol. II. The
Iowa State University Press, Ames, IA.

138

Gill, J.L. and H.D. Hafs. 1971. Analysis of repeated
measurements of animals. J. Anim. Sci. 33:331.

Glock, G.E. and P. Maclean. 1953. Further studies on the
properties and assay of glucose 6-phosphate dehy-
drogenase and 6-phophogluconate dehydrogenase of
rat liver. Biochem. J. 55:499.

Goodman, G.T., R.M. Akers and H.A. Tucker. 1980. Effects
of prolactin, growth hormone and progesterone on
synthesis and secretion of alpha—lactalbumin in ex-
plants of bovine mammary tissue. Unpublished obser-
vations.

Goodman, G.T., H.A. Tucker and E.M. Convey. 1979. Pres—
ence of the calf affects secretion of prolactin in
cows. Proc. Soc. Exp. Biol. Med. 1692421.

Graf, K.J., R. Horowski and M.F. El Eltreby. 1977.
Effect of prolactin inhibitory agents on the
ectopic anterior pituitary and the mammary gland in
rats. Acta Endocrinologica 852267.

Grosvenor, C.E., F. Mena and N.S. Whitworth. 1977. Meta-
bolic clearnace of rat prolactin in the lactating
and non-lactating rat. J. Endocrinol. 7321.

Grosvenor, G.E. and N.S. Whitworth. 1979. Secretion rate
and metabolic clearnace rate of prolactin in the
rat during mid- and late-lactation. J. Endocrinol.
822409.

Grueter, F. 1928. Contribution a l'etude du fonctionne-
ment de lobe anterieur de l'hypophyse. Compt. Rend.
Soc. de Biol. 9821215.

Guyette, W.A., R.J. Matusik and J.M. Rosen. 1979.
Prolactin-mediated transcriptional and post-
transcriptional control of casein gene expression.
Cell 1721013.

Halban, J. 1900. Uber den einfluss der ovarien auf die
enlwichlung des genitales. Monatschr. F. Geburtsh.
U. Gynak. 12:496.

Halban, J. 1905. Die innere secretion von ovarium und
placenta und ihre beduntung fur die function der
milchdruse. Arch. Gynaek. 752353.

139

Hammond, J. and F.H.A. Marshall. 1914. The functional
correlation between the ovaries, uterus, and mam-
mary glands in the rabbit with observations on the
estrous cycle. Proc. Roy. Soc. B. 87:422.

Harkness, M.L.R. and R.D. Harkness. 1956. The effect of
pregnancy and lactation on the collagen content of
the mammary gland of the rat. J. Physiol. 1322476.

Hart, I.C. 1976. Prolactin, growth hormone, insulin and
thyroxin: their possible roles in steroid-induced
mammary growth and lactation in the goat. J.
Endocrinol. 71:41.

Hartmann, P.E. 1973. Changes in the composition and
yield of the mammary secretion of coes during the
initiation of lactation. J. Endocrinol. 59:231.

Hayden, T.J., R.G. Bonney and I.A. Forsyth. 1979. Onto-
geny and control of prolactin receptors in the mam-
mary gland and liver of virgin, pregnant and lacta-
ting rats. J. Endocrinol. 80:259.

Heald, C.W. 1974. Hormonal effects on mammary cytology.
J. Dairy Sci. 572917.

Heald, C.W. and R.G. Saacke. 1972. Cytological compari-
son of milk protein synthesis of rat mammary tissue
in vivo and in vitro. J. Dairy Sci. 552621.

Hildebrandt, P. 1904. Zur lehr von der milchbildung.
Beit. Z. Chem. Physiol. U. Path. 52463.

Hollmann, K.H. 1969. Quantitative elctron microscopy of
sub-cellular organization in mammary gland cells
before and after parturition. tg: Lactogenesis:
the initiation of milk secretion at parturition.
My. Reynolds and S.J. Folley, eds. University of
Pennsylvania Press, Philadelphia.

Howe, J.E., C.W. Heald and T.L. Bibb. 1974. Histology of
induced bovine lactogenesis. J. Dairy Sci. 58:853.

Ingalls, W.G., E.M. Convey and H.D. Hafs. 1973. Bovine
serum LH, GH and prolactin during late pregnancy,
parturition and early lactation. Proc. Soc. Exp.
Biol. Med. 1432161.

Johke, T., H. Fuse and M. Oshima. 1971. Changes in plasma
prolactin level during late pregnancy and early lac-
tation in the goat and the cow. Jap. J. Zootech.
Sci. 42:173.

140

Johke, T. and K. Hodate. 1978. Effects of CB154 on serum
hormone level and lactogenesis in dairy cows.
Endocrinol. Japon. 25:67.

Jones, E.A. 1972. Studies on the particulate lactose syn-
thetase of mouse mammary gland. Biochem. J. 126267.

Jones, E.A. 1977; Synthesis and secretion of milk sugars.
Symp. Zool. Soc. Lond. 41:77.

Juergens, W. F.E. Stockdale, Y.J. Topper and J.J. Elias.
1965. Hormone-dependent differentiation of mammary
gland in vitro. Proc. Nat. Acad. Sci. 542629.

Karg, H. and D. Schams. 1972. Effects of 2-br-a-
ergocryptine on plasma prolactin level and milk
yield in cows. Experientia 28:574.

Karg, H. and D. Schams. 1974. Prolactin release in cattle.
J. Reprod. Fert. 392463.

Keenan, T.W., D.J. Morre and R.D. Cheetam. 1970. Lactose
synthesis by a Golgi apparatus fraction from rat
mammary gland. Nature 228:1105.

Kensinger, R.S., D.E. Bauman and R.J. Collier. 1979.
Season and treatment effects on serum prolactin and
milk yield during induced lactation. J. Dairy Sci.
6221880.

Kinsella, J.E. and C.W. Heald. 1972. Na 1-14C stearate
and Na 2-14C acetate metabolism and morphological
analysis of late prepartum bovine mammary tissue.
J. Dairy Sci. 5521085.

Kleinberg, D.L. J. Todd and W. Niemann. 1978. Prolactin
stimulation of alpha-lactalbumin in normal primate
mammary gland. J. Clin. Endocrinol. Metab.

Koprowski, J.A. and H.A. Tucker. 1971. Failure of oxyto-
cin to initiate prolactin or luteinizing hormone
release in lactating dairy cows. J. Dairy Sci.
5421675.

Koprowski, J.A. and H.A. Tucker. 1973. Serum prolactin
during various physiological states and its rela—
tionship to milk production in the bovine. Endo-
crinology 9221480.

Kronfeld, D.S., F. Raggi and C.F. Ramberg. 1968. Mammary
blood flow and ketone body metabolism in normal,
fasted, and ketotic cows. Amer. J. Physiol. 2152218.

141

Kuhn, N.J. 1969a. Progesterone withdrawal as the lacto-
genic trigger in the rat. Biochem. J. 44239.

Kuhn, N.J. 1972a. Changes in the protein phosphorous
content of rat mammary tissue during lactogenesis.
J. Endocrinol. 552219.

Kuhn, N.J. 1977. Lactogenesis: the search for trigger
mechanisms in different species. Sym. Zool. Soc.
Lond. 41:165.

Larson, B.L. 1979. Biosynthesis and secretion of milk
protein: a review. J. Dairy Res. 46:161.

Larson, L.L. and D.C. Gillespi. 1957. Origin of the
major specific proteins in milk. J. Biol. Chem.
227:565.

Linzell, J.R. and M. Peaker. 1971. Mechanism of milk
secretion. Physiol. Rev. 51:564.

Louis, T.M., H.D. Hafs and B.E. Seguin. 1973. Proges-
terone, LH, estrus and ovulation after prosto-
glandin an in heifers. Proc. Soc. Exp. Biol. Med.
1432152.

Lu, M.H. and R.R. Anderson. 1973. Growth of the mammary
gland during pregnancy and lactation in the rabbit.
Biol. Reprod. 9:538.

Lyons, W.R., C.H. Li and R.E. Johnson. 1958. The hor-
monal control of mammary growth and lactation.
Rec. Prog. Horm. Res. 14:219.

Mackall, J.C. and M.D. Lane. 1977. Changes in mammary-
gland acetyl-coenzyme A carboxylase associated with
lactogenic differentiation. Biochem. J. 1622635.

Martin, D.E., M.G. Horning and P.R. Vagelos. 1961. Fatty
acid synthesis in adipose tissue. 1. Purification
and properties of a long chain fatty acid synthe-
sizing system. J. Biol. Chem. 2362663.

Matusik, R.J. and J.M. Rosen. 1978. Prolactin induction
of casein mRNA in organ culture--a model system for
studying peptide hormonal regulation of gene expres-
sion. J. Biol. Chem. 25322342.

McNamara, J.P. and D.E. Bauman. 1978. Partitioning of
nutrients between mammary and adipose tissue of the
rat during lactogenesis. J. Dairy Sci. 612Supple-
ment #1 Program of 73rd Annual Meeting. p.156.

142

McShan, W.H. and C.W. Turner. 1936. Bioassay of galactin,
the lactogenic hormone. Proc. Soc. Exp. Biol. Med.
34:50.

Meites, J. and C.W. Turner. 1942a. I. Can estrogen sup-
press the lactogenic hormone of the pituitary?
Endocrinology 302711.

Meites, J. and C.W. Turner. 1942d. II. Why lactation is
not initiated during pregnancy. Endocrinology
30:719.

Meites, J. and C.W. Turner. 1948a. Studies concerning
the idunction and maintenance of lactation. I. The
mechanism controlling the initiation of lactation
at parturition. Mo Agr. Exp. Sta. Res. Bull. 415.

Mellenberger, R.W. and D.E. Bauman. 1974. Metabolic
adaptations during lactogenesis: lactose synthesis
in rabbit mammary tissue during pregnancy and lac-
tation. Biochem. J. 1422659.

Mellenberger, R.W., D.E. Bauman and D.R. Nelson. 1973.
Metabolic adaptations during lactogenesis: fatty
acid and lactose synthesis in cow mammary tissue.
Biochem. J. 1362741.

Mills, E.S. and Y.J. Topper. 1970. Some ultrastructural
effects of insulin, hydrocortisone and prolactin on
mammary gland explants. J. Cell Biol. 44:310.

Munford, R.E. 1964. A review of anatomical and bio-
chemical changes in the mammary gland with particu-
lar reference to quantitative methods of assessing
mammary development. Dairy Sci. Abst. 26:293.

Nickerson, S.C., C.W. Heald, T.L. Bibb and M.L. McGilliard.
1978. Cytological effects of hormones and plasma
on bovine mammary tissue in vitro. J. Endocrinol.
792363.

Ollivier—Bousquet, M. 1978. Early effects of prolactin.
on lactating rabbit mammary gland--ultrastructural
changes and stimulation of casein secretion. Cell
Tiss. Res. 187225.

Ollivier-Bousquet, M. and R. Denamur. 1975. Effet de
l'etat physiologique et du 3'5' adenosine mono-
phosphate cyclique sur le transit intracellulaire
et l'ercretion des proteines du lait etude auto-
adiographique en microscopie electronique. J.
Microscopie Biol. Cell. 23:63.

143

Paape, M.J. and H.A. Tucker. 1969. Mammary nucleic acid,
‘ hydroxyproline and hexosamine of pregnant rats dur-
ing lactation and post-lactional involution. J.
Dairy Sci. 52:380.

Padmanabhan, V and E.M. Convey. 1979. Estradiol-l78
stimulates basal and thyrotropin releasing hormone
induced prolactin secretion by bovine pituitary
cells in primary culture. Mole. Cell. Endocrinol.
14:103.

Patton, S. and R. Jensen. 1976. Biomedical aspects of
lactation. Pergamon Press, Oxford.

Peel, C.J., J.W. Taylor, I.B. Robinson, A.A. McGowan, R.D.
Hooley and J.R. Findlay. 1978. The importance of
prolactin and the milking stimulus with artificial
induction of lactation in cows. Aust. J. Biol. Sci.
312187.

Perry, N.A. and F.J. Doan. 1950. A picric acid method
for the simultaneous determination of lactose and
sucrose in dairy products. J. Dairy Sci. 33:176.

Peters, R.R. 1980. Photoperiod regulation of serum hor-
mones, body growth and lactation in dairy cattle.
Ph.D. Thesis, Michigan State Univ., E. Lansing.

Pitelka, D.R. and S.T. Hamamoto. 1977. Form and function
in mammary epithelium: the interprelation of ultra-
structure. J. Dairy Sci. 60:643.

Plaut, G.W.E. 1962. Isocitric dehydrogenase (TPN-linked)
from pig heart (revised procedure). tg: Methods
in Enzymology. S.P. Colowick and N.O. Kaplan, eds.,
Vol. V. Academic Press, Inc., New York.

Prockop, D.J. and S. Udenfriend. 1960. A specific method
for the analysis of hydroxyproline in tissues and
urine. Anal. Biochem. 12228.

Purchas, R.W., K.L. Macmillan and H.D. Hafs. 1970. Pitui-
tary and plasma growth hormone levels in bull from
birth to one year of age. J. Anim. Sci. 31:358.

Reece, R.P. and C.W. Turner. 1937. The lactogenic and
thyrotopic content of the anterior lobe of the
pituitary gland. Mo. Agr. Exp. Sta. Res. Bull. 226.

 

144

Reid, I.M., A.J. Stark and R.N. Isenor. 1977. Fasting
and refeeding in the lactating dairy cow. 1. The
recovery of milk yield and blood chemistry follow-
ing a six-day fast. J. Comp. Path. 87:241.

Riddle, 0., R.W. Bates and S.W. Dykshorn. 1933. The pre-
paration, identification and assay of prolactin--a
hormone of the anterior pituitary. Amer. J.
Physiol. 1052191.

Robertson, H.A. 1974. Changes in the concentration of un-
conjugated oestrone, oestradiol-l7a and oestradiol-
178 in the maternal plasma of the pregnant cow in
relation to the initiation of parturition and lacta-
tion. J. Reprod. Fert. 36:1.

Rosen, J.M. 1976. Isolation and characterization of pur-
ified rat casein messenger ribonucleic acids.
Biochemistry 1525263.

Rosen, J.M. and S.W. Barker. 1976. Quantitation of
casein messenger ribonucleic acid sequences using
a specific complementary DNA hybridization probe.
Biochemistry 1525272.

Rosen, J.M., D.L. O'Neal, J.E. McHugh and J.P. Comstock.
1978. Progesterone-mediated inhibition of casein
mRNA and polysomal casein synthesis in the rat mam-
mary gland during pregnancy. Biochemistry 172290.

Saacke, R.G. and C.W. Heald. 1974. Cytological aspects
of milk formation and secretion. tg; Lactation:
a comprehensive treatise. B.L. Larson and V.R.
Smith, eds., Vol 11. Academic Press, Inc., New
York.

Sakai, S. J. Enami, S. Naudi and M.H. Banerjee.‘ 1978.
Prolactin receptor on dissociated mammary epithe-
lial cells at different stages of development.
Mole. Cell. Endocrinol. 12:285.

Schams, D. 1976. Hormonal control of lactation. tg:
Breast-feeding the the mother. Vol. 45. Ciba
Foundation Symposium.

Schams, D., V. Reinhardt and H. Karg. 1973. Effects of
2-br-a-ergocryptine on plasma prolactin level during
parturition and onset of lactation in cows.
Experientia 28:697.

 

145

Shirley, J.E., R.S. Emery, E.M. Convey and W.D. Oxender.

1973. Enzymic changes in bovine adipose and mam-
mary tissue, serum and mammary tissue hormonal
changes with initiation of lactation. J. Dairy
Sci. 562569.

Shiu, R.P.C. and H.G. Friesen. 1976. Blockade of prolac-

Sinha,

Skarda,

Smith,

Smith,

Smith,

Smith,

Stein,

Stern,

tin action by an antiserum to its receptors.
Science 1922259.

K.N., R.R. Anderson and C.W. Turner. 1970.
Growth of the mammary glands of the golden hamster,
mesocricetus auratus. Biol. Reprod. 22185.

J., E. Urbanova and J. Bilek. 1977. Effect of
estradiol and progesterone on udder growth in goats
in vivo and lipid synthesis in goat mammary tissue
in vitro. Endocrinologia Experimentalis 11:183.

K.L. and F.L. Schanbacher. 1973. Hormone induced
lactation in the bovine. l. Lactational perfor-
mance following injections of 17 beta-estradiol and
progesterone. J. Dairy Sci. 562738.

K.L. and F.L. Schanbacher. 1974. Hormone induced
lactation in the bovine. 2. Response of nulli~
gravida heifers to modified estrogen-progesterone
treatment. J. Dairy Sci. 57:296.

V.G., T.W. Beck, E.M. Convey and H.A. Tucker. 1974.
Bovine serum prolactin, growth hormone, cortisol
and milk yield after ergocryptine. Neuroendocrinol.
15:172.

V.G., L.A. Edgerton, H.D. Hafs and E.M. Convey.
1973. Bovine serum estrogens, progestins and
glucocorticoids during late pregnancy parturition
and early lactation. J. Anim. Sci. 362391.

0. and Y. Stein. 1967. Lipid synthesis, intra-
cellular storage and secretion. 2. Electron-
microscope autoradiographic study of the mouse lac-
tating mammary gland. J. Cell Biol. 34:251.

J.M. 1977. Effects of ergocryptine on postpartum
maternal behavior, ovarian cyclicity and food in-
take in rats. Beh. Biol. 212134.

Stricker, P. and F. Grueter. 1928. Action du lobe an-

terior de l'hypophyse sur la montee laiteuse.
Comp. Rend. Soc. de Biol. 9921978.

 

146

Stricker, P. and F. Grueter. 1929. Recherches experi-

Sud, S.

Sykes,

Terkel,

mentales sue les fonctions du lobe anterior sur
l'appareil genital de la lapine et sur la montee
laiteuse. Presse. Med. 3721268.

C., H.A. Tucker and J. Meites. 1968. Estrogen-
progesterone requirements for udder development in
ovariectomized heifers. J. Dairy Sci. 5121.

J.F. and T.R. Wrenn. 1951. Hormonal development
of mammary tissue in dairy heifers. J. Dairy Sci.
3421174.

J., D.A. Damassa and C.H. Sawyer. 1979. Ultra-
sonic cries from infant rats stimulate prolactin
release in lactating mothers. Hor. Beh. 12:95.

Tomogane, H., K. Ota and A. Yokoyama. 1976. Decrease in

Tucker,

Tucker,

Tucker,

Tucker,

Tucker,

Tucker,

Tucker,

litter weight gain and in progesterone secretion in
lactating rats treated with antiserum to rat pro-
lactin. J. Reprod. Fert. 47:347.

H.A. 1964. Influence of number of suckling young
on nucleic acid content of lactating rat mammary
gland. Proc. Soc. Exp. Biol. Med. 116:218.

H.A. 1969. Factors affecting mammary gland cell
numbers.“ J. Dairy Sci. 52:721.

H.A. 1971. Hormonal response to milking. J.
Anim. Sci. 322Supplement #1.

H.A. 1979. Endocrinology of lactation. Seminars
in Perinatology 3:199.

H.A., J.A. Koprowski and W.D. Oxender. 1973.
Relationships among mammary nucleic acids, milk
yield, serum prolactin, and growth hormone in
heifers from 3 months of age to lactation. J.
Dairy Sci. 562184.

H.A. and J. Meites. 1965. Induction of lactation
in pregnant heifers with 9-fluoroprednisolone ace-
tate. J. Dairy Sci. 48:403.

H.A. and R.P. Reece. 1963. Nucleic acid content
of mammary glands of lactating rats. Proc. Soc.
Exp. Biol. Med. 1122409.

 

147

Turkington, R.W. and Y.J. Topper. 1966. Stimulation of
casein synthesis and histological development of
mammary gland by human placental lactogen in vitro.
Endocrinology 79:175.

Turkington, R.W. and R.L. Hill. 1969. Lactose synthetase:
progesterone inhibition of lactose synthetase in
the developing mouse mammary gland. Science 163:
1458.

Turner, C.W. and A.H. Frank. 1931. The relation between
the estrus producing hormone and a corpus luteum

extract on the growth of the mammary gland. Science
72:295.

Turner, C.W., H. Yamamoto and H.L. Ruppert, Jr. 1956.
The experimental induction of growth of the cow's
udder and the initiation of milk secretion. J.
Dairy Sci. 3921717.

Vandeputte—VanMesson, G., G. Peeters and H. Lauwers. 1976.
Artifical induction of lactation by hypothalamic
implantation of perphenazine in virgin goats. J.
Dairy Res. 4329.

Venable, J.H. and R. Coggeshall. 1965. A simplified lead
citrate stain for use in electron microscopy. J.
Cell Biol. 252407.

Vines, D.T., E.M. Convey and H.A. Tucker. 1977. Serum
prolactin and growth hormone responses to thytropin
releasing hormone in postpubertal cattle. J. Dairy
Sci. 6011949.

Vonderhaar, B.K. 1977. Studies on the mechanism by which
thyroid hormones enhance a-lactalbumin activity in
explants from mouse mammary gland. Endocrinology
10021423.

Vonderhaar, B.K. 1979. Lactose synthetase activity in
mouse mammary glands is controlled by thyroid hor-
mones. J. Cell Biol. 82 675.

Vonderhaar, B.K., I.S. Owens and Y.J. Topper. 1973. An
early effect of prolactin on the formation of
alpha-lactalbumin by mouse mammary epithelial cells.
J. Biol. Chem. 2482467.

Watson, M.L. 1958. Staining of tissue sections for elec-
tron microscopy with heavy metals. J. Biophys.
Biochem. Cytol. 4:475.

148

Wellings, S.R., R.A. Cooper and E.M. Rivera. 1966.
Electron microscopy of induced secretion in organ
cultures of mammary tissue of the C3H/Crgl mouse.
J. Nat. Cancer Inst. 36:657.

Welsch, C.W., M.J. McManus, J.V. DeHoog, G.T. Goodman and
H.A. Tucker. 1979. Hormone-induced growth and
lactogenesis of grafts of bovine mammary gland
maintained in the athymic "nude" mouse. Cancer
Res. 3922046.

Wetteman, R.P. and H.D. Hafs. 1973. LH, prolactin, es-
tradiol and progesterone in bovine blood serum dur-
ing early pregnancy. J. Anim. Sci. 36237.

Wetteman, R.P. and H.A. Tucker. 1974. Relationship of
ambient temperature to serum prolactin in heifers.
Proc. Soc. Exp. Biol. Med. 149:908.

Wooding, F.B.P. 1977. Comparative mammary fine structure.
Sym. Zool. Soc. Lond. 4121.

Wrenn, T.R., W.R. Delander and J. Bitman. 1965. Rat mam-
mary gland composition during pregnancy and lacta-
tion. J. Dairy Sci. 4821517.

Yanai, R. and H. Nagasawa. 1971. Mammary growth and
placental mammotropin during pregnancy in mice with
high or low lactational performance. J. Dairy Sci.
542906.

 

 

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