Protoplasma (1990) 159:96-111

OROTO0 $MA
9 by Springer-Verlag 1990

Lactation physiology: a ruminant animal perspective

R. M. Akers*

Lactation Physiology Laboratory, Department of Dairy Science, Virginia Polytechnic Institute and State University, Blacksburg, Virginia

Received August 7, 1990

Accepted August 17, 1990

It has been a pleasure for me to have the opportunity to prepare this paper in honor of Dr. Stuart Patton on his 70th birthday. Dr. Patton,
one of the founders of the Gordon Conference on Mammary Gland Biology, had the vision to bring together the variety of workers interested
in the mammary gland and drive to foster wide ranging collaborations.

Summary. Importance of peripubertal mammary development as a
foundation for subsequent mammary growth and milk production
was discussed. Morphological differences in peripubertal mammary
growth in rodents and ruminants were described. The relevance of
tissue interactions and association with hormones and growth factors
in mammary development were delineated. Data from specificstudies
with ruminant mammary parenchyma were outlined for comparison
with rodent studies. It is concluded that the wholesale extrapolation
of data from rodent studies to explain udder development is inap-
propriate. Lastly, recent data from experiments with culture of mam-
mary explants from bulls is described. Pragmatically, these data
suggest that responses of mammary tissue from bulls might provide
a means for early selection of superior sires or provide a unique
model to study tissue interactions in udder development.
Keywords: Mammary growth; Ruminants; Hormones; Growth fac-
tors; Mammary cell culture; Morphogenesis.
Introduction
It is apparent from recent volumes and reviews (Rich
etal. 1986, Neville and Daniel 1987, Tucker 1987, For-
syth 1989) that the m a m m a r y gland, its growth, dif-
ferentiation, secretions and dysfunction provide an ex-
ceedingly rich substrate for a multitude o f scientists.
T u m o r biologists, immunologists, and micro-biologists
seek to unravel the puzzle o f breast cancer and mastitis.
Cell biologists, endocrinologists, and the "new com-
ers" - the molecular biologists - scurry for the Rosetta
Stone to explain the language o f the chemical milieu
* Correspondence and reprints: Department of Dairy Science, Litton
Reaves Hall, Virginia Polytechnic Institute, Blacksburg, VA 24061,
U.S.A.
that drives the phases o f m a m m a r y development.
Chemical engineers and venture capitalists seek a nipple
that yields an exotic soup o f precious polypeptides.
Finally, agricultural scientists p o n d e r the relevance o f
the rodent as a model for udders, dream o f glands that
stop producing for the weekend, and aid the production
o f the milk, cheese, yogurt, and ice cream all the others
pick up at the store on the way h o m e f r o m the lab.
M y goal is to consider several aspects o f lactation that
have particular interest for dairy scientists, but which
also address ideas which m a y influence thinking o f
other m a m m a r y gland biologists.
The cow a calf becomes
While individual variation is often n o t as apparent
when studying lactation o f highly inbred laboratory
animals, a visit to a milking parlor makes it clear that
there are m a r k e d differences in milk p r o d u c t i o n a m o n g
cows (or sheep or goats for that matter). Even after
differences due to parity, stage o f lactation, nutritional
program, and reproductive status are considered dif-
ferences between individuals can be marked. While dif-
ferences in p r o d u c t i o n potential likely do not change
biological fundamentals, the economic impact o f better
selection o f superior animals is enormous. F o r example,
a m o n g Holstein dairy cows average yearly p r o d u c t i o n
has been estimated to have increased 75 kg per year
between 1960 to 1975 due to genetic selection. Available
estimates indicate Holsteins produce an average of
R. M. Akers: Lactation physiology:a ruminant animal perspective
7,195 kg milk/year but half of these animals produce
between 3,810kg/year and the mean (Powell etal.
1977). Such variation is both a curse when designing
experiments related to milk production and a source
of relevant research material. It may be that differ-
entials in milk production result from failure of the
potential milk synthesizing cells to proliferate in low
compared with high milk producing animals or that
secretory cells in low producing animals fail to achieve
an equivalent degree of biochemical differentiation.
I believe that the tissue foundation created during the
peripubertal period in ruminants, modulates capacity
of the mammary gland to produce alveoli, and thus
future milk production. Alterations in animal man-
agement during this period clearly modify udder de-
velopment. Declining milk price and increasing feed
costs frequently dictate a desire for more rapid rearing
of heifers to gain the benefits of more economic meat
and milk production, higher weaning weights and in-
creased numbers of offspring (due to earlier attainment
of puberty). Modern labor-efficient heifer rearing sys-
tems have increased rates of feed utilization such that
animals may gain 1 kg or more per day. However, as
outlined in recent reviews (Foldager and Sejrsen 1987,
Johnson t988), rapid gain during the prepubertal pe-
riod can profoundly reduce future milk production
(--~25% loss). Thus, the prepubertal period of allo-
metric udder growth is sensitive to effects of overfeed-
ing, but rapid rearing during later stages likely enhance
udder development and subsequent milk yields. To
what extent variation in prepubertal mammary growth
is responsible for observed milk yield differences in the
cattle population is unknown. Use of uniformly reared,
homogenous rodents minimize such effects in experi-
mental animals, but certainly heterogeneous rearing of
humans may have important consequences for lacta-
tion success of future mtrsing mothers or propensity
to develop tumors.
Hormones, growth factors, and tissue interactions in
mammary growth
In rodents, neonatal mammary growth involves pro-
liferation and branching of a primary ductuIar network
within the mammary fat pad. During the altometric
growth phase, ducts elongate and fill the fat pad with
subsequent secondary branching to form this mam-
mary duct network. Duct elongation involves penetra-
tion of the fat pad by a highly-specialized structure,
the terminal endbud. These 0.1 to 0.8ram diameter
structures form at the proliferating end of ducts and
97
extend throughout, to the margin of the fat pad (Wil-
liams and Daniel 1983). From about 4 to 8 wks of age,
endbuds grow through the fat pad and occasionally
bifurcate to form new growth points. Elongation con-
tinues until the endbud reaches the margin of the fat
pad when elongation stops and the structure regresses
(Silberstein and Daniel 1987). The situation in rumi-
nants is much less clear. There is no compelling evidence
that endbuds exist. Furthermore as ducts elongate in
the rodent gland, epithelial cells are in physical contact
with adipocytes. Figure 1 illustrates the histological
appearance of mammary tissue taken from prepubertal
ewe lambs. The lambs were approximately 22 wks of
age at the time of slaughter, when explants of mammary
parenchyma were taken for incubation in medium 199
with tritiated thymidine (2 h at 37 ~ then fixed, sec-
tioned and processed for autoradiography as described
(Smith et al. 1989). Specifics related to nutritional treat-
ments applied during the growth period (beginning at
7 wks of age) and details of other udder measurements
are given in McFadden et al. (1990 a, b). The data are
unpublished micrographs from the same study. Nutri-
tional treatments had no consistent effect on histolog-
ical appearance, thus differences indicate between and
within animal variation. Figure 1 A and B shows cross
sections of ducts with distinct areas of dense stromal
tissue between ducts but the absence of adipocytes.
Figure 1 A shows that labeled cells are most often (but
not exclusively) located at the periphery of groups of
ducts. Panel B illustrates the predominance of dense
fibroblast-like cells surrounding ductular profiles. Fig-
ure 1 C and D demonstrates the presence of adipocytes
near some areas of ducts. But it is evident that in the
limited areas where there are adipocytes scattered in
the parenchyma, that there is consistently a sheath of
fibroblast-like cells separating the ducts and the adi-
pocytes. Figure 1 E shows that even in the prepubertal
gland there are groups of epithelial cells that show
evidence of limited secretory activity (presence of lipid
droplets and darkly stained luminal spaces) but that
DNA synthesis continues among the cells in such struc-
tures. Figure 1 F shows the predominant histological
appearance of the tissue. The ductular cells are densely
packed with little evidence of secretory activity and
labeled epithelial cells appear randomly placed within
individual ductular structures. Histological study of
mammary tissue from prepubertal heifers (Sejrsen et al.
1986, Smith et al. 1986, Woodward and Akers unpubl.)
indicate a similar appearance.
An experiment to determine the role of the ovaries in
Prl and GH stimulation of mammary development in
98 R . M . Akers: Lactation physiology: a ruminant animal perspective

Fig. 1. Autoradiograms of mammary tissue from prepubertal ewe Iambs. Unpublished micrographs from study by McFadden etal. (1990).
A Illustration of frequently observed distribution of tritium labeled epithelial cells near periphery of clusters of ducts and presence of dense
arrays of fibroblast-like cells surrounding profiles of individual ducts. B Area with abundance of stromal cells around ducts and illustration
of irregular outline of cross sectioned ducts. C Area of tissue showing limited occurrence of adipocytes near ducts and D enlargement of
similar area. E Area of mammary parenchyma from prepubertal lamb with evidence of secretory activity and DNA synthesis. F Section of
tissue corresponding with usual appearance of mammary tissue from prepubertal cattle and sheep. S Stromal tissue; d ductular lumena; a
adipocytes; I~ tritiated thymidine labeled nuclei
R. M. Akers: Lactationphysiology:a ruminantanimal perspective
prepubertal mice (Wells and Akers unpubl.) had as a
goal to determine if prepubertal mice exhibit increased
mammary growth in response to GH - as do ruminants
(Sejrsen etal. 1986)-with the hope that mice might
serve as a model to study GH induced development.
Histologically, the appearance ofmurine mammary tis-
sue shows some rather striking contrasts with the ru-
minant. Ducts are much more widely scattered and those
cut in cross-section usually present a rounded profile
indicating a section through a simple tubular structure
and the entire structure is suspended in a sea of adi-
pocytes interwoven with blood vessels and various leu-
cocytic cells. As shown in Fig. 1, cross-sectioned ducts
from the prepubertal ruminant gland usually demon-
strated a scalloped appearance, suggesting a more com-
plex tubular structure with abundant outpocketing of
epithelium along the course of the ducts. This suggests
that in the ruminant, in contrast with rodents, the gland
is not filled in with elongated ducts during the pre-
pubertal period waiting for subsequent development of
side branches. Furthermore, the periphery of the rum-
inant gland is devoid of epithelium. These morpholog-
ical features suggest that the rodent model should not
be assumed to accurately describe ruminant mammary
development. It is also evident that many hypotheses
related to mammary development have arisen from
observations from rodent studies. It remains to be seen
if these ideas apply equally well to other species.
Culture experiments
Even a casual observer is quick to realize that exper-
iments with large ruminants are more difficult and
costly than those that might be done with laboratory
animals or with strict in vitro procedures. Therefore,
it should not be surprising that the majority of mam-
mary related experiments have not been done with dairy
animals. This means that most hypotheses concerned
with udder development are derived from model system
and extrapolated to dairy species. While many fun-
damentals of mammary development likely apply
across all mammals, I believe wholesale extrapolation
is unwarranted.
Simple histological observations of mammary tissue
confirm the fact that the tissue is composed of a variety
of cell types. Simplistically, mammary growth involves
an invasion of the underlying mesenchyme by surface
epithelium. The critical nature of interactions between
the epithelium and the mesenchyme in fetal mammary
development has been elegantly studied by Kratochwil
99
and his colleagues and recently reviewed (Kratochwil
1986). These tissue recombination and receptor studies
confirm that sexual dimorphism of mammary devel-
opment (in rodents) requires the appearance of andro-
gen receptors in the mesenchyme surrounding the de-
veloping mammary rudiment but that receptors occur
only in the presence of the epithelial structure. Further
studies showed that newly formed mammary buds in-
duced surrounding mesenchymal cells to elaborate an-
drogen and E2 receptors. In the male, the production
of testosterone results in activation of the androgen
receptors and destruction of the epithelial bud (male
mice have no nipples) whereas in the absence of tes-
tosterone, subsequent development follows a female
pattern. These studies have led to wide spread recog-
nition that tissue and cell type interactions are likely
critical in all phases of mammary development (Shef-
field 1988). Since it is the epithelial portion of the gland
that determines milk production, it is easy to under-
stand research emphasis on epithelial proliferation and
differentiation but it is also clear that alveolar devel-
opment is intimately intertwined with stromal tissue
metabolism.
As reviewed (Forsyth 1989), early results with growth
of isolated mammary cells were disappointing in that
growth of cells on plastic was minimal and not always
associated with expected responses to known mam-
mogenic hormones. Moreover, serum addition (with
its unknown complement of growth factors and nu-
trients) was usually needed to maintain growth of cells.
Development of cell lines have allowed elucidation of
some specific biochemical features of cell metabolism
but have been largely of unknown relevance since the
cells frequently exhibit "non-epithelial" and/or heter-
ogenous morpholbgies and most often only limited ca-
pacity to synthesize and secrete milk constituents. In-
troduction of culture methods utilizing collagen and
other extracellular matrix proteins has revitalized cul-
ture experiments particularly with primary cultures
(Bissell and Hall 1987), since such cultures exhibit
growth and subsequent mammary specific differentia-
tion in serum-free media preparations. These systems
also promise the opportunity to better evaluate cell
type interactions since co-cultures composed of various
proportions of cells can easily be prepared, In the case
of bovine mammary gland Hung and Turner (1990)
have produced a clonal line of SV40 large T antigen
transformed bovine mammary cells which grow well in
culture but differentiate when subsequently transferred
to floating collagen gels. Studies with these cells may
well provide a unique model to study regulation of
100
ruminant mammary cell function without the "nui-
sance" of large animal variation associated with prep-
aration of primary cultures.
Importance of substratum
The critical nature of the extraceUular matrix for ep-
ithelial cell differentiation (Aggeler et al. 1988) has been
repeatedly confirmed and biochemical details eluci-
dated as a consequence of the report of Emerman and
Pitelka (1977) describing maintenance of lactating
mammary cell function when the cells were cultured
on floating collagen gets. Parry et al. (1990) have dem-
onstrated that establishment of membrane domains, to
create polarity of epithelial cells in non-confluent cul-
tures, also depends on composition of the substratum
and have suggested that elaboration of vitronectin re-
ceptors in the basolateral membranes is necessary. Le-
vay-Young et al. (1990) have shown epithelial cells from
virgin mice induced to grow by either hormonal or
nonhormonal stimulation in collagen gels produce
daughter cells capable of synthesizing milk constituents
with subsequent exposure to lactogenic hormones.
However, they suggest that the culture systems appar-
ently favor the proliferation of lumenal cells and/or
selectively promote evolution of cells with lumenal ep-
ithelial characteristics rather than ductular (based on
absence of secretory capability). This is relevant since
starting material for the cultures is tissue from virgin
mammary glands and should yield primarily ductular
cells.
McGrath (1987) has described use of the collagen gel
culture system with attached gels to allow growth
(within the collagen matrix) of epithelial structures iso-
lated from pregnant, non-lactating cows, and prolif-
erative responses of epithelial structures isolated from
udders of prepubertal calves Shamay et al. (1988). Since
the stromal cells elaborate at least a portion of the
extracellular material surrounding the ducts it is rea-
sonable to associate changing stromal cell metabolism
with epithelial proliferation.
Role of non-epithelial cells
Halsam (1988 a) recently reviewed a series of in vitro
experiments to study interactions between mammary
fibroblasts and response of mammary epithelial cells
with respect to induction of proliferation and proges-
terone receptors. Data from these co-culture experi-
ments suggest that presence of fibroblasts enhance es-
trogen-dependent increases in epithelial cell progester-
one receptor via fibroblast production of a collagen
R.M. Akers: Lactation physiology:a ruminant animal perspective
substratum and that presence of metabolically active
fibroblasts in close contact with the epithelium is re-
quired for estrogen induction of epithelial proliferation.
Levine and Stockdale (1984) reported growth of iso-
lated mammary cells was stimulated if cultured on a
feeder layer of adipocytes. Subsequently, Weins et al.
(1987) observed that mammary epithelial cells cultured
on adipocytes from the 3T3-Li cell line grew, formed
branching ductular systems and secreted caseins and
alpha-lactalbumin if the cultures were exposed to lac-
togenic hormones. In a related experiment, Beck and
Hosick (1988) reported that growth of mammary ep-
ikhelial cells on or in collagen was stimulated if exposed
to conditioned media from cultures of isolated adi-
pocytes or explants of epithelial free mammary fat
pads. Since it has been shown that dietary fats can alter
mammary tumor growth (Ip 1986) as well as normal
development (Carrington and Hosick 1985, Welsch and
O'Connor 1989) and that cells can be stimulated by
certain free fatty #cids (Imagawa et al. 1986), it might
be suspected that effects of adipocyte co-cultures reflect
lipolysis and availability of growth promoting fatty
acids. Further characterization of conditioned media
(Beck et al. 1989) showed that both saturated and un-
saturated fatty acids were present in the media. Further
experiments confirmed that oleate-, arachadonate-, or
tinoleate-induced mammary cell proliferation. These
authors suggested that the data support a rote for fatty
acids in proliferation but do not rule out the possibility
that other growth factors or elements important for
biomatrix formation are also produced by the adipose
tissue surrounding mammary ducts.
Of course the question is how do these observations
apply to mammary growth in ruminants? Although
there is very little data, as cited above, mammary frag-
ments from prepubertal calves and pregnant heifers
grow when cultured inside collagen gels. Moreover,
Shamay et al. (1988) showed that fragments from heifer
calves grew in response to IGF-I or pharmacological
concentrations of insulin but not in response to Prl or
GH in serum free media. However, maximal responses
were only 25-40% of that in medium supplemented
with 10% fetal calf serum. We (Furumura et al. 1990)
also have observed marked proliferation of mammary
organoids prepared from udders of midpregnant Hol-
stein and Angus heifers over nine days of culture in
collagen gels in the presence of serum. Examples of
autoradiograms of sectioned profiles of mammary or-
ganoids exposed to tritiated thymidine during the last
24 h of culture (harvested on day 9 of culture) are shown
in Fig. 2. Tissues were prepared from udders of preg-
R. M. Akers: Lactation physiology: a ruminant animal perspective 101

Fig. 2. Autoradiograms of sections of mammary organoids from pregnant Holstein heifers following culture for nine days in rat tail collagen
in the presence of 10% fetal calf serum and 1 gCi tritiated thymidine for the final 24 h of culture. In each panel arrowheads illustrate labeled
epithelial cells. A Portion of a profile through an alveolar-like structure, labeled epithelial cells are numerous and an apically positioned lipid
droplet is indicated (fwith arrow). B Section through profile with ductular and alveolar-likeregions. C Section through alveolar-likestructure
with bud-like, epithelial structures appearing to branch from the periphery (b with arrows). 1 Lumenal space. D Enlargement of area with
bud-like structures. Unpublished micrographs from study by Fumumura et al. (1990)

n a n t Holstein a n d A n g u s heifers a n d cultured essen- ithelium); duct-like with l o n g i t u d i n a l a p p e a r a n c e or

tially as described by M c G r a t h (1987). Profiles o f sec- solid a n d bud-like. C o u n t i n g o f labeled epithelial cells
tioned organoids were: alveolar-like (as evidenced from in profiles o f alveolar or duct-like structures showed
a p p a r e n t l u m e n a l space, a n d generally single layer ep- that 42 to 58% of the cells were labeled a m o n g cultures
102
maintained in presence of 10% fetal calf serum. Figure
2A shows an enlarged area of a single alveolar-like
structure, labeled cells are abudant and an apical lipid
droplet is indicated. Figure 2 B and C shows larger por-
tions of single structures. In particular, Fig. 2 C shows
what appears as buds branching from the outer rim of
a single layered epithelial structure and Fig. 2 D an
enlargement of the area. These data demonstrate that
organoids from mammary parenchyma of pregnant
cattle can be maintained and proliferative responses
induced following culture in collagen gels. The data
also suggest that growth responses of organoids from
Angus heifers (a beef breed) is reduced when compared
with those from dairy heifers (Holstein). Thus, part of
the genetic potential for milk production in cattle may
depend on differences in high and low production an-
imals' capacity to response to growth promoters during
mammogenesis. The data are unpublished micrographs
from Furumura et al. (1990).
Winder et al. (1989) have also observed dose response
related proliferation of mammary organoids from non-
pregnant and pregnant ewes in response to IGF-I and
insulin following culture on collagen gels. These data
indicate that ruminant mammary epithelial cell frag-
ments grow in or on collagen gels in a manner anal-
ogous to rodent mammary cells. Thus, in this respect,
rodent derived data suggesting the importance of the
extracellular matrix for morphogenesis would seem to
also apply to ruminants. But what about specifics with
regard to cellular interactions?
As deduced from autoradiograms oftritiated thymidine
uptake by mammary tissue (Shyamala and Ferenczy
1984) it has been suggested that steriod induction of
mammary epithelial proliferation in virgin mice de-
pends on a wave of proliferation in the stromal tissue
which precedes epithelial growth. This supports the
idea that E2 stimulates the epithelium in a paracrine
fashion and thus the notion that the stromal tissue
modulates epithelial proliferation. We (Woodward,
Akers, and Beal unpublished) have recently completed
a similar experiment with prepubertal heifers. In brief,
each of three heifers were treated with E2 (0.1 mg/kg),
P4 (0.25mg/kg) or the combination for four days.
Mammary biopsies were taken 24 h before then, 24,
48, and 96 h after start of treatments. Mammary tissue
explants were incubated for 2 h in M 199 supplemented
with 1 [sCi/ml tritiated thymidine. Thereafter tissue
samples were fixed, embedded, and serial sections pre-
pared for autoradiography. Proportions of labeled cells
were determined for epithelial cells located in the ap-
parent blunt end of ducts or along the longitudinal axis
of ducts. Fibroblasts immediately adjacent to ducts or
R.M. Akers: Lactationphysiology:a ruminantanimalperspective
those surrounded by stromal tissue were evaluated as
were adipocytes (rarely observed and never in close
apposition to the epithelium) and endothelial cells. Re-
sults are illustrated in Fig. 3. The essential finding is
that E2 acutely increased the labeling of epithelium and
that the increase in labeling preceded changes in fibro-
blast labeling. It is also clear that effects of E2 were
dramatic but that P4 had no effect and that the com-
bination was less effective than E2 alone. These data
suggest that mechanisms for early proliferation of
mammary ducts in the prepubertal mouse do not mirror
those in ruminants and thus emphasize the need for
further studies directly with ruminants.
A role for adipocytes in mammary proliferation in rum-
inants is very difficult to support with apparent mor-
phological differences between species. This makes it
unlikely that acute associations between epithelial cells
and adipocytes are critical in ruminants. This would
of course not rule off the possibility that production
of specific growth factors or fatty acids produced by
the mammary or other fat cells could influence mam-
mary development. However, once the rumen becomes
functional, biohydrogenation would certainly limit the
potential effects that unsaturated fatty acids in the ra-
tion could have on udder development. However, given
that factors early in the neonatal period can alter sub-
sequent development, it is interesting to speculate that
supplemental dietary lipids might stimulate udder de-
velopment if given to suckling calves. In fact, we have
recently reported that feeding protected unsaturated
fat to ewe lambs stimulates growth of the mammary
parenchyma as well as the mammary fat pad
(McFadden et al. 1990 a) and increases numbers of GH
receptors in the liver and Prl receptors in the mammary
parenchyma (McFadden et al. 1990 b).
While classic endocrine ablation experiments have es-
tablished the need for pituitary, ovarian, and adrenal
hormones in mammary growth and differentiation in
whole animals (Akers 1985) it is only with the use of
cell culture (particularly the collagen methodology)
that it has been possible to determine if these hormones
have direct effects on mammary tissue. The techniques
also have been critical in identification of a variety of
peptides capable of altering mammary function. In the
next sections I will review recent results related to clas-
sic hormones and growth factors.
Estrogens
It has been known for years that estrogens are potent
mammary mitogens when injected, but direct effects
R. M. Akers: Lactation physiology: a ruminant animal perspective 103

5, 5 84
r--1 PRETREA"IMENT I'--I PRETREA'IMENT
24 HOURS 24 HOURS
1 48 HOURS 48 HOURS
4, I ~ 96 HOURS 4 84 96 HOUR~
a D
w W
._J _.1
W 3 W
m m
3 3
b~
2-

HI
ESTROGEN
n~IF~
PROGESTERONE
nil
COMBINATION
A
ESTROGEN PROGESTERONE COMBINATION

5-
F"-I pRE~EATMENT
24 HOURS
1 48 HOURS
4- Fig. 3. Summary of labeling percentages of mammary ductal epi-
96 HOURS
a thelial cells and fibroblast adjacent or non-adjacent to ducts in mam-
W mary tissue of prepubertal heifers before, then 24, 48, and 96 h after
..3
W 3- exogenous estradiol, progesterone or both. A Labeling index for
8O
5 ductal epithelial cells. Effects of estrogen are pronounced (P < 0.01).
The combination of moderate influence (P < 0.05) and progesterone
2-
without effect on labeling index (compared with pretreatment). B
Labeling index of fibroblasts adjacent (within a radius of 50 gin) to
mammary ducts. Responses are small relative to epithelial cells but
follow similar tends. Importantly, there is no significant change in
~_|B~ n~__ ~_|~ labeling with exogenous estradiol before 96 h. C Labeling index of
fibroblasts not adjacent to ducts, Only minimal iabeling observed,
ESTROGEN PROGESTERONE COMBINATION
no time or treatment effects. Data from unpublished study by
C Woodard et al.

were usually difficult to demonstrate even with the
collagen cultures (Imagawa etal. 1986). On the other
hand E2 directly stimulates some breast cancer cell lines
and there is evidence that E2 induces such cells to
elaborate factors which act as "second messengers" for
the mitogenic effect (Dickson et al. 1986). Winder and
Forsyth (unpublished, cited in Forsyth 1989) have also
shown direct effects of E2 on DNA synthesis in ovine
mammary explants if cultured in phenol red free me-
dium. This indicates that failure to demonstrate E2
effects in culture may in some cases be due to effects
being masked by presence of phenol red. Haslam (1988)
also demonstrated that local implantation of E2 in the
mouse mammary gland induced localized proliferation
of endbuds in the immature virgin gland independent
of any systemic effects. However, local implantation
had no effect on formation of side branches in glands
of mature virgin mice but systemic increases in E2
stimulated proliferation. These interesting results in-
dicate that there are at least two modes of action for
estrogen(s) on the mammary gland (local vs systemic)
and that mechanisms are variably operative depending
on physiological state. Of course demonstration of local
stimulation (in the gland or in explants) does not rule
out the possibility that effects are autocrine or paracrine
in nature. Considering the variety of effects, perhaps
it is prudent to suggest that any of a number of pos-
sibilities could explain E2 action in a particular cir-
cumstances. Current proposals are: (1) E2 acts directly
as a mitogen on target cells, (2) E2 acts indirectly via
local (paracrine or autocrine) or systemic production
of growth factors (the estomedin hypothesis; Sirbasku
1978) or (3) that E2 induces a release from inhibiton
(the estrocolyone hypothesis; Soto and Sonnenschein
1987).
There are limited cellular data for effects of E2 in pre-
pubertal ruminants but our data (Akers and Purup
unpubl.) suggest E2 has little direct effect in mammary
explant cultures, although administration of E2 in vivo
induces acute specific increases in DNA synthesis in
104
mammary ducts (Fig. 3) (Woodward et al. unpubl.).
Historically, it has been suggested that E2 effects on
the mammary gland were mediated by Prl since E2 is
implicated in the control of Prl synthesis and secretion
in a variety of species (Labrie et al. 1980). However, in
pubertal heifers Beck et al. (1976) found no differences
in serum Prl in untreated, ovariectomized heifers com-
pared with ovariectomized heifers given E2, P4, or the
combination. Furthermore, Haslam (1988 b) cited un-
published data indicating that local implantation of Prl
had no effect on mammary ducts in rodents and Sha-
may etal. (1988) showed that Prl had no effect on
proliferation of mammary organoids cultured in col-
lagen gels.
Growth hormone
Older endocrine ablation/replacement experiments
(Forsyth 1989) demonstrated clearly that E2 and GH
were the essential hormonal requirements to stimulate
mammary ductular growth. However, a host of recent
papers provide compelling evidence that GH effects on
mammary function are indirectly mediated via IGF-I.
First there are no detectable somatotrophic receptors
in mammary tissue from prepubertal (McFadden et al.
1990 b), pregnant (Ename et al. 1986, Smith et al. 1989)
or lactating (Gertler et al. 1984, Akers and Keys 1985,
Kazmer et al. 1986) ruminants. Secondly, addition of
GH to explant cultures (in contrast with stimulation
of milk production in vivo) has no effect on milk con-
stituent biosynthesis (Gertler et al. 1983). Third, IGF-
I induces dose response increases in DNA synthesis for
mammary tissue from prepubertal heifers (Shamay
etal. 1988, 1990), pregnant cows and sheep (Baum-
rucker and Stemberger 1989, Winder and Forsyth
1989), and lactating cows (Baumrucker and Stemberger
1989).
These data and characterization of IGF-I and II bind-
ing sites in mammary membranes from cattle (Dehoff
etal. 1988, Hasdell etal. 1990) and sheep (Disenhaus
et al. 1988, Winder and Forsyth 1987) also support the
contention that mitogenic effects attributed to addition
of pharmacological concentrations of insulin in mam-
mary tissue cultures are essentially explained by the
capacity of insulin to cross react with IGF receptors.
It is also likely relevant that mitogenic effects of IGF-
I are induced at concentrations similar to those ob-
served for blood levels of IGF.
On the other hand, if confirmed, the data obtained by
Kleinberg et al. (1990) may require some modification
of the notion that GH has no direct effects on mare-
R.M. Akers: Lactation physiology:a ruminant animal perspective
mary growth as they show that local implantation of
GH in male rat mammary glands induces morphoge-
nesis of mammary ducts. They also cite a paper by
Lincoln et al. (1990) purporting the expression of GH
receptor in the proliferating rat mammary gland. To
my knowledge this is the only report of a true soma-
totrophic receptor in the mammary gland; i.e., binding
of hGH clearly may be to lactogenic receptors (Akers
and Keys 1985). Regardless, these data would not rule
out the possibility that GH effects may be paracrine
in nature, perhaps mediated by local production of
IGFs (see Funder 1990, for a view of descriptions of
cellular communication). Relevance to ruminants is
also unknown as there is no evidence for direct effects
of GH or presence of somatotrophic receptors (see
above).
Growth factors
The number of reviews in the past few years (Oka and
Yoshimura 1986, Salomon et al. 1986, Dembinski and
Shiu 1987, Forsyth t989) serve to illustrate the explo-
sion in information regarding mammary active growth
regulators. As suggested above, revitalization of culture
methods for mammary tissue, particularly successful
growth in serum free media, has been critical in iden-
tification of these factors. A second critical boost has
been production of some of these factors via recom-
binant DNA technology such that materials formally
available only as extracts or in very limited quantities
to selected researchers are easier to obtain. It is not my
goal to extensively review the plethora of mammary
active growth factors but rather to emphasize data rel-
ative to ruminants.
Insulin-like growth factor I and II
There is growing evidence (see section above') that IGFs
are important regulators (perhaps primary) of both
mammogenesis and galactopoiesis in cattle and sheep.
It seems apparent that elucidation of gene structures
and molecular biology of the IGFs (Daughaday and
Rotwin 1989) will eventually lead to a comprehensive
view of secretion, synthesis, and mechanisms of action
for the entire family of insulin and insulin-like growth
factors.
Epidermal growth factor (EGF)
First isolated from submaxillary glands of male mice,
EGF effects on mammary tissue were apparently first
reported by Turkington (1969), and since then a variety
of experiments have suggested EGF can modulate
mammary growth and lactogenesis in rodents. For ex-
R. M. Akers: Lactation physiology:a ruminant animal perspective
ample, Edery etal. (1985) demonstrated the presence
of specific receptors for EGF in mouse mammary gland
and that receptor content appeared to change in cor-
respondence with proliferative activity. Imagawa et al.
(1985) showed direct proliferative effects of EGF on
mm~ne mammary cells grown in collagen and Coleman
etal. (1988) showed that local implantation of EGF
induced formation of endbuds, increased ductual di-
ameters and reinitiated epithelial cell DNA synthesis
in growth quiescent mammary glands of ovariectom-
ized mice. However, EGF effects are subject to complex
regulation, since Coleman and Daniel (1990) have re-
cently reported that EGF induced reversible inhibition
of ductal growth in endocrine intact virgin mice. Thus,
EGF effects apparently are subject to extreme modu-
lation by physiological state of the treated animal. Re-
lationships between EGF receptor and oncogene
expression (Velu 1990) further hint at the complexity
of EGF modulation of proliferation.
It is unclear whether EGF might be normally important
in ruminants. I am unaware of any reports of isolation
or characterization of specific ovine or bovine EGFs.
However, specific receptors for EGF have been de-
scribed for plasma membranes prepared from mam-
mary tissue from pregnant and lactating cows (Spitzer
and Gross 1987) and we (Akers and McFadden un-
publ.) have detected specific binding of EGF in mem-
branes from udders of prepubertal ewes and observed
apparent EGF activity in homogenates of ovine sali-
vary glands based on competition of binding of iodin-
ated murine EGF with its specific antibody. In addi-
tion, Shamay etal. (1988, 1990) reported that EGF
stimulated DNA synthesis of mammary organoids
from calves and that EGF produced a synergistic in-
crease when added with IGF-I. We (Furumura et al.
1990) also have observed that EGF appears to enhance
the capacity of the combination of insulin, E2, P4,
hydrocortisone, and Prl in serum free media to stim-
ulate DNA synthesis in mammary organoids from
pregnant heifers. Sheffield and Yuh (1988) demon-
strated that E2 + P4-induced DNA synthesis of bovine
mammary tissue xenografted into athymic nude mice
was reduced in sialodectomized (a major source of
EGF) mice but was restored in mice given exogenous
EGF. Furthermore, Collier and McGrath (1988)
showed that intramammary injections of human EGF
stimulated local udder development. Thus, it is un-
known if EGF plays a normal role in udder develop-
ment in ruminants but mouse or human EGF is capable
of stimulating udder development in vivo and in vitro.
If EGF becomes available in sufficient quantities it
t05
could be practical to determine effects of EGF on udder
development and subsequent milk production.
Transforming growth factors (TGFs)
As described (Salomon et al. 1986), transformation of
epithelial cells usually reduces the requirements of the
cells for specific growth factors and cause loss of an-
chorage-dependent proliferation. TGFs make up a
family of peptides which have been isolated from con-
ditioned media and cellular extracts of tumors. Sub-
sequent studies have shown that these peptides also are
found in a variety of normal tissue including bovine
mammary gland (Eckert et al. 1985). Two variants have
been described. TGF-(x has been shown to induce acute
local increases in mammary duct growth in mice (Von-
derhaar 1987) although the interaction of TGF-a with
EGF receptors make it difficult to distinguish effects
of the two peptides. However, it is reasonable to spec-
ulate that local tissue production of TGF-a could elicit
EGF effects. This nicely would explain the relevance
of EGF receptors in the bovine mammary gland in the
presumptive absence of EGF (see above). TGF-[3 has
been shown to be a reversible inhibitor of epithelial
proliferation in mammary ducts as demonstrated fol-
lowing local implantation in murine mammary glands
(Silberstein and Daniel 1987) but to have little effect
on proliferation of lobulo-alveolar structures during
pregnancy (Daniel etal. 1989). Rasmussen and Ra-
praeger (1988) have also shown that TGF-~3 alters the
proteoglycan strucuture of the surface of mammary
cells, suggesting that TGF associated inhibition may
involve blocking of surface receptors for positive stim-
ulators.
The presence of TGFs in normal tissues suggest the
possibility that they may have a role irrespective of
carcinogenesis. The endocrine hypothesis of estrogen
action-based on the finding of increased growth of
mammary tumor cells when incubated with other tis-
sues (pituitary, kidney, and uterus) previously exposed
to E 2 - m a y ultimately be linked to estrogen induced
production of TGFs, FGF or close relatives (see be-
low). Liscia et al. (1990) have shown that during preg-
nancy and lactation, levels of TGF-a mRNA in mam-
mary ducts and alveolar cells increase two- to three-
fold. This supports a possible role for TGF in normal
mammary development as well as in the growth and
transformation of human mammary cells (Bates et al.
1990).
Fibroblast growth factors (FGFs)
As outlined (Gospodarowicz et al. 1987), two types of
FGFs have been isolated. A basic form (bFGF) was
106
first identified by its capacity to stimulate fibroblast
proliferation as the name would imply. A second va-
riety, with an acidic isoelectric point (aFGF), was
shown to stimulate myoblasts and later rediscovered
as a consequence of its ability to stimulate endothelial
cells. The FGFs share similar structures such that they
interact with the same receptor. However, aFGF has
a restricted cellular distribution in comparison with
bFGF. Following structural characterization of aFGF
and bFGF and cloning of the gene, it became clear
that many of the growth factor activities associated
with fractions isolated from many tissues were one of
the FGFs. For example, tumor angiogenesis factor,
macrophage growth factor, and uterine derived growth
factor are among the more than 16 synonyms which
have existed for bFGF. Similarly, more than 8 syn-
onyms exist for aFGF. The FGFs apparently have been
very welt conserved through evolution in that bovine
and human bFGF differ in only 2 of 146 amino acids
and avian and bovine forms have the same amino acid
composition and likely equivalent sequences, bFGF is
an especially potent mitogen for mesoderm-derived
cells as illustrated by the finding that it triggers pro-
liferation of endothelial cells at concentrations as low
as 2pg/ml, with maximal effects at about 140pg/ml.
Given the intimate association between mammary ep-
ithelial growth and the surrounding mesoderm derived
stromal tissue, it is reasonable to speculate on a role
for the FGFs in preparation of the mammary stromat
for invasion by growing epithelium. It may be that
estrogen induced mammary growth may be linked to
paracrine production of FGFs. As discussed above, we
recently have found that exogenous E2 not only pro-
motes an acute increase in tritiated thymidine labeling
of mammary ductular cells in prepubertal heifers but
also elicits ,-~20-fold increase in labeling of endothelial
cells by 48 h after injection. Progesterone had no effect
on endothelial cell labeling and the combination of
E2 + P4 only a moderate effect (Woodward et al. un-
publ.), bFGF has been shown to stimulate growth of
human breast cancer cells (Dembinski and Shui 1986,
for review) and Levay-Young et al. (1989) have shown
that bFGF stimulates growth of virgin mouse epithelial
cells cultured in collagen.
Growth inhibitors
Ultimately, cell proliferation is most likely controlled
by a balance of positive and negative regulators. It is
also possible that growth occurs simply as a result or
release from inhibition. This is in fact a simple view of
R.M. Akers: Lactationphysiology:a ruminantanimalperspective
the action of tissue (but not species) specific growth
inhibitors referred to as chalones. The essential feature
being that a tissue chalone would act to control the
mature size of an organ. This idea is perhaps most
easily envisioned for the liver. Removal of a section of
liver, for example, wotfld lower tissue concentration of
the chalone allowing replacement of lost tissue until
size (and presumably concentration of the chalone)
returned to pre-surgery levels. A number of years ago,
Gonzaley and Verly (1976) reported the isolation of a
presumptive peptide from lactating bovine mammary
gland that exhibited many of the characteristics of a
mammary chalone and Knight and Peaker (1982) dis-
cussed the concept relative to the mammary gland.
Ervin etal. (1989) have isolated and characterized a
tissue specific mammary growth inhibitor from con-
ditioned media from cultures of normal human mam-
mary cells. Mammastatin as it is called, inhibits the
proliferation of a wide variety of human breast cancer
cell lines, and normal mammary cells but has no effect
on proliferation of at least 11 different nonmammary
cell lines. The tissue specific nature of the inhibitor and
biochemical evaluations indicate the material is inde-
pendent of TGFs or FGFs. Bohmer et al. (1987) have
isolated a second tissue specific inhibitor from bovine
mammary gland which has close sequence homology
with a family of hydrophobic ligand-binding proteins,
but relationships of the hydrophobic binding site and
inhibitory action is unknown.
Tissue--tissue communication
Aside from growth factors, many experiments over the
past ten years point to the likelihood that there is com-
plex regulation of biochemical processes between tis-
sues and organs to support the predominant physio-
logical state of an animal-the homeorhesis concept
(Bauman and Currie 1980). More narrow in scope, the
synlactin hypothesis suggests that mitogenic effects of
Prl on target cells are mediated by interaction of Prl
directly and Prl induction of a somatomedin-like mol-
ecule (called synlactin) produced by the liver. Media
from incubations of liver slices from pregnant and lac-
tating rats increased DNA synthesis in mammary ex-
plants and Prl-responsive pigeon crop sac tissue but
media from liver slices of male or nonpregnant rats
were not effective (Nicoll et al. 1985, 1987). In further
experimentation these workers (English etal. 1990)
have shown that intrahepatic infusion of Prl stimulated
mammary development in hypophysectomized rats but
that infusion of the same dose of prolactin into the
R. M. Akers: Lactation physiology: a ruminant animal perspective
external jugular was ineffective. Furthen-nore, the ef-
fects appeared not to be mediated by changes in serum
IGF-I concentrations. In a similar study, Hoeffier and
Frawley (1987) identified a liver lactogenic factor
(LLF) from lactating rats that mimiced the lactogenic
action of Prl and subsequently (Frawley etal. 1988)
that the LLF was a more potent inducer of casein
secretion than native Prl and that LLF acted in a po-
sitive feedback fashion to increase pituitary cell pro-
duction of Prl mRNA and secretion of Prl.
We (Akers et al. 1986) also have observed that mam-
mectomy of pregnant primiparous heifers reduced in-
duced secretion of Prl during the periparturient period.
This suggests that the mammary gland has some ca-
pacity to modulate its endocrine environment. Further,
107
it is clear that mammary proteins are capable of en-
tering serum. For example, we showed that serum con-
centrations of cz-lactalbumin followed a pattern indi-
cating a two-stage evolution of lactogenesis, that it was
acutely increased at calving and during hormonal in-
duction of lactation (McFadden etal. 1987). Subse-
quent study showed acute increases with non-bacterial
induction of leucytosis and close correspondence with
somatic cells counts in milk (McFadden etal. 1988).
We have observed also that serum concentrations are
acutely increased during milking (Akers and Mc-
Fadden unpubl.) and appear to change in lymph (Akers
and Capuco unpubl.). Therefore it is logical to suggest
the possiblity that the mammary gland may produce
regulators that drive other organs to change their me-
Fig. 4. Autoradiograms of sections of mam-
mary parenchymal explants from Holstein
bulls after incubation for 96h in medium
199 supplemented with insulin, hydrocor-
tisone, estradiol, triiodothyronine, and pro-
lactin. A Multilayered nonsecretory epithe-
lial cells on the surface of a large duct (ar-
rowheads) and apparent invagination of the
epithelium to form pockets with evidence
of secretory activity (c with arrows). B Sec-
ond crypt region with alveolar-like struc-
ture and evidence of secretory activity
(small vacuoles - assumed to be fipid drop-
lets; and staining of lumenal spaces) also in
evidence are several tritiated thymidine la-
beled epithelial cell nuclei. Data are un-
published micrographs from a study by Fi-
lep and Akers (1990)
108 R.M. Akers: Lactation physiology:a ruminant animal perspective

tabolism to insure the success of lactation. This notion formed invaginations into the underlying mesenchymal
seems especially satisfying in an evolutionary sense. tissue. This is better illustrated at higher magnification
Taken together, this variety of data illustrates the po- in Fig. 4 B. Cells in these areas appear to from single
tential for complex regulatory interactions between tis- layer surfaces with lumenal spaces which presumably
sues and organs to determine the relative success of lead to the exterior surface. Some of these cells acquire
events during mammogenesis, lactogenesis, and gal- a secretory appearance (presence of small vacuoles,
actopoiesis to optimize milk production. At a minimum staining of lumenal spaces) especially when incubated
there is no compelling reason to think of the mammary with prolactin. These observations suggest that most
gland only as a target tissue. of the lactogenic response of mammary tissue from
bulls depends on this isolated population of cells in
these crypts. In selected cases these areas take on a
Lactation in males distinct alveolar-like appearance. This suggests not
In a final section, I would like to discuss some novel only a potential practical application of such cultures
data concerned with lactogenic responses in mammary (sire selection) but also that these tissues may provide
tissue from bulls. The practical background for such an interesting model for morphogenesis.
study concerns attempts to identify and select animals
with superior genetic potential for milk production. Future prospects
Sire selection based on production records of daughters Without doubt the continued application of molecular
is the foundation of the marked genetic progress biology techniques to problems of mammary gland
achieved through artificial insemination in dairy cows. proliferation and function will fuel an information ex-
However, procedures for proving young sires require plosion. Much of the lead will come from basic research
several years and are costly. Thus, a mechanism to concerned with breast cancer, largely as a consequence
select potential sires earlier in life would be of economic of funding differentials to study problems perceived as
benefit. Our initial study on this topic considered three health oriented compared with milk secretion and dairy
questions: (1) do explants from bulls secrete milk pro- production. There is also likely to be a shortage of new
teins in culture, (2) are responses altered by lactogenic researchers capable or willing to apply these technol-
hormones, and (3) do beef compared with dairy bulls
exhibit differential responses? Results of this study with
tissue from prepubertal bulls primed with estrogen and
progesterone (McFadden etal. 1988) were encourag-
ing. Explants secreted casein and a-lactalbumin, re-
sponses were increased in the presence of prolactin and
beef and dairy bulls exhibited significantly differei~t
responses. In a subsequent experiment, reported in ab-
stract form, we (Filep and Akers 1990 a, b) studied the
ability of mammary tissue from mature bulls to exhibit
lactogenic responses in culture. The experiment in-
volved bulls whose daughters were high yielding com-
pared with bulls whose daughters were low production
but all bulls were Holsteins from the same herd. These
data demonstrated that tissue from bulls in the high
line secreted significantly more casein than tissue of
low line, that pretreatment of the bulls with E2 + P4
enhanced responses in culture but was not necessary
t o distinguish genetic lines. Surprisingly, tissues pro-
duced only minimal amounts of a-lactalbumin. The
histological appearance of mammary explants after
96 h of culture in the presence of prolactin is illustrated
in Fig. 4. Figure 4 A shows an area with a multilayered Fig. 5. Electron micrograph from section of mammary tissue from
epithelium, typical of the gland cistern in the udder, as a lactating Holstein cow. Shown is a surprisingly novel profile
well as areas where the epithelium appears to have through a cytoplasmicdroplet in the alveolarlumena, x 24,500
R. M. Akers: Lactation physiology: a ruminant animal perspective
ogies to u d d e r d e v e l o p m e n t . N o n e t h e l e s s , s p i n o f f s w i t h
r e l e v a n c e to r u m i n a n t s a r e likely, b u t d i r e c t e x t r a p o -
l a t i o n to d a i r y p r o d u c t i o n will o n l y s l o w l y be realized.
Fundamental understanding of cellular and molecular
e v e n t s in u d d e r g r o w t h a n d m o r p h o g e n e s i s will surely
lag b e h i n d s i m i l a r u n d e r s t a n d i n g o f m a m m a r y devel-
o p m e n t in e c o n o m i c a l l y u n i n t e r e s t i n g rats a n d mice.
R e g a r d l e s s , l a c t a t i o n r e s e a r c h is sure to c o n t i n u e to
yield surprises, as i l l u s t r a t e d in the m i c r o g r a p h I t o o k
a n u m b e r o f y e a r s a g o o f m a m m a r y tissue f r o m a lac-
t a t i n g c o w (Fig. 5).
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