Reviews of Reproduction (1998) 3, 104–112

Local control of mammary development and function

Christopher H. Knight, Malcolm Peaker
and Colin J. Wilde
Hannah Research Institute, Ayr KA6 5HL, UK

For the mother, lactation represents the final stage of an investment in her genetic material.
Like any investment it is costly and, hence, it needs to be carefully controlled. To her offspring,
lactation means survival, so it must happen at any cost. This apparent conflict is rationalized
by the mother devolving some control to the offspring while retaining ultimate sanction her-
self. Part of this results from overt and more subtle influences of the presence of young on the
mother’s endocrine system, but an equally important part operates at each mammary gland to
ensure that output is appropriate to the needs of the young, and no more. The young exert
influence by removing milk, while the mother retains control by responding on an hour to
hour basis to the presence of milk in the gland. Local control is inevitably most evident where
secretion itself is concerned, but also operates to influence lactogenesis, gland development
and, eventually, gland involution. This paper will review local control of mammary function,
emphasising the important role played by an autocrine inhibitory protein, the feedback
inhibitor of lactation.

What is meant by local control? The scope for local control

The mammary gland is a target organ for a great variety of
hormones. Mammary development is driven by combinations
of steroid and polypeptide mammogens, principally oestrogen,
progesterone and placental lactogen, and lactation itself is
heavily influenced by prolactin and growth hormone (Flint and
Knight, 1997). However, it is evident that not all of these hor-
mones have a direct action on mammary epithelial cells; there
is continued debate as to whether oestrogens are directly mito-
genic (Silberstein et al., 1994) and growth hormone receptors
are absent from lactating mammary epithelial cells. The impli-
cation is that hormone action is often mediated indirectly, per-
haps by stimulation of the local production of growth factors.
For example, GH may increase insulin-like growth factor I
(IGF-I) production by stromal cells within the mammary gland
(Collier et al., 1993). In as much as this effect would not happen
without the IGF-I, it could be termed local control. However,
we do not consider it to be such. The IGF-I enables control to be
exerted by GH through a passive response as distinct from a
controlling one. This is an important distinction because the
mammary gland is capable of producing a great variety of hor-
mones and growth factors, few of which perform a genuinely
local regulatory role.
Another distinction was highlighted by Silberstein et al.
(1994), who used small implants of an anti-oestrogen inserted
into individual mammary glands of young mice. Ductal growth
was inhibited in the region of the implant but not elsewhere,
which provided evidence of local action but not of local control,
since the oestrogen is likely to emanate from the systemic circu-
lation. Local control is used to mean a mechanism that is intrin-
sic to an individual mammary gland and which, in the absence
of external stimuli, regulates some aspect of the development
or function of that gland independently of other glands in the
same animal.
© 1998 Journals of Reproduction and Fertility
1359-6004/98 $12.50
The mammary gland is a complex structure (Fig. 1) consisting
of a number of cell types and an extracellular matrix that is
recognized to have a regulatory input (for review see Knight,
1995). However, for the purposes of local control, the most sig-
nificant feature is the fact that for much of the time the gland is
full of its own secretion. This is an unusual attribute among
exocrine glands but is one shared to a greater or lesser extent
by mammary glands of all species. Even rodents, which fre-
quently spend very long periods on the nest apparently suck-
ling, only eject milk at intervals of around 20 min, so milk
accumulates during the interim. Rabbits suckle only once
per day, and tree shrews once every two days. Milk contains a
considerable number of bioactive factors (Grosvenor et al.,
1994) and, while it remains in the gland, these factors may be
interacting with the apical (‘milk-side’) membrane of the se-
cretory cells from which they came. Any influence they might
exert would clearly be local and, since the alveolar lumen is re-
mote from external stimuli such as endocrine signals, it would
constitute true control.
Evidence for local control of mammary function
Mammary glands are paired structures, located external to the
body cavity and more or less discrete one from another, de-
pending on species and lactational state. Observations of the
gross size and nature of individual glands can be made rela-
tively easily, and the secretory output of individual glands can
be collected for determination of amount and composition.
Therefore, it is a simple and logical step to compare one gland
with another within one animal, and from this to deduce
whether differences are evidence either of pathological change
or of local control. From our own experiences, two phenomena
are of particular relevance.
 Intramammary local control 105

(a) Ruminant mammary gland (c) Alveolus

Apical surface
Secretory tissue
Basal surface

Secretory cell

FIL effective here

FIL Small duct
Large duct

FIL ineffective here

Milk in lumen
Storage sinuses
(cistern)

(b) Human mammary gland (d) Secretory cell

FIL binds to
putative receptor
FIL
Secreted protein
Apical membrane

Protein vesicle
Mammary duct
FIL inhibits
Nipple protein
trafficking
through Golgi

Basal membrane

Fig. 1. (a) Diagrammatic representation of the mammary gland of a dairy species. Local feedback inhibition of milk secretion results from
the action of a secreted milk protein (FIL), which is effective in milk stored within secretory tissue but not in milk that has moved down
into the storage sinuses. (b) In rodents, man and some other species the storage sinuses are absent and milk drains through ducts directly to
the nipple. (c) Individual secretory alveoli are hollow sacs of polarized secretory epithelial cells drained by a fine duct. Thus, the FIL is an
autocrine factor, produced and secreted into milk by secretory epithelial cells from where it binds to presently uncharacterized sites on the
apical membrane of the same cells. (d) Within the cell, feedback inhibitor of lactation reduces the rate of protein secretion by disruption of
vesicle trafficking through the Golgi apparatus.

First, in pregnant goats approaching parturition, the mam-
mary gland will be in a state of readiness for secretion, will
contain a small quantity of prepartum fluid but will not yet be
actively secreting. If one gland is then ‘milked’ for several days
to remove the prepartum fluid, that gland, and only that one,
will start to secrete copious amounts of milk (Maule Walker
and Peaker, 1980).
Second, in lactating goats, if one gland is changed from the
normal twice a day milking to more frequent milking, the yield
of that gland, and again only that gland, will increase sig-
nificantly (Henderson et al., 1983). Conversely, if milking fre-
quency is reduced in one gland, yield will decline unilaterally.
These simple observations provide evidence of local control
of both lactogenesis (onset of milk secretion) and galactopoiesis
(maintenance of lactation). The most exquisite example of local
control is observed in macropod marsupials. Their lactation
consists of two very distinct stages: the first is characterized by
small volumes of a low fat, high carbohydrate milk produced
106 C. H. Knight et al.
by a poorly developed mammary gland, and the second by
much larger volumes of a fat-rich milk from a well-developed
gland. Remarkably, these two types of production can occur
simultaneously in adjacent mammae of the one individual, a
process which is termed asynchronous concurrent lactation
(Nicholas et al., 1995). As the first joey is born, it attaches per-
manently to one of the four teats. At this time, the glands are no
more developed than they would be during an oestrous cycle,
but the suckled gland then begins not only to secrete but also to
grow, so that by the time the first joey is leaving the pouch
(when a second may be born), the gland is entering the second
stage of lactation. Until this time, the other three glands have
remained quiescent, but now the second joey attaches to a dif-
ferent gland and the cycle begins in this one. Here, then, is local
control not only of lactogenesis and galactopoiesis, but also of
mammogenesis. Although it is not documented, the asyn-
chronous nature of the marsupial lactation cycle also implies
that gland involution can be controlled locally, since the older
joey will be weaned considerably earlier than the younger one.
We shall consider mammogenesis, lactogenesis and galacto-
poiesis in turn but, for reasons that will become apparent, we
shall do so in a slightly illogical order and leave mammary
development until last.
Local control of lactogenesis
The onset of copious milk secretion at or around parturition re-
quires the systemic interaction of a fall in progesterone com-
bined with increased prolactin and glucocorticoids. During
lactogenesis, the synthesis of individual milk constituents in-
creases rapidly and, shortly thereafter, the tight junctions be-
tween neighbouring secretory cells become truly tight,
preventing paracellular ionic flux. This explains why colostrum
has a considerably higher Na+:K+ ratio than mature milk. As
part of their investigations of the cellular control of ion move-
ments into milk, Linzell and Peaker (1974) started to milk a
single gland of goats during the last 3 weeks of pregnancy. The
composition of the fluid obtained gradually changed as tight
junction patency increased, but this happened only in the
milked gland; the contralateral, unmilked gland was not
affected, so that during the first few days after parturition it
was producing colostrum, while the experimental gland was
producing mature milk. It was proposed that a local chemical
feedback mechanism was operating within the gland, and that
the putative inhibitor was removed by the act of milking before
parturition.
In seeking the identity of the inhibitor, it was discovered
that the mammary gland of the late pregnant goat was produc-
ing both oestradiol (Maule Walker and Peaker, 1978) and
prostaglandin F2α (PGF2α) and its metabolites (Maule Walker
and Peaker, 1980). The concentration of oestradiol rose shortly
before parturition, the reverse of what was needed for removal
of an inhibitory substance, and the possibility that this increase
was actually stimulatory to lactogenesis was considered but
disproven. Prostaglandins appeared much more promising.
Their vasoactive properties suggested a potential mechanism
for holding the gland in check (although PGF2α has been
shown subsequently to have no effect on mammary blood flow
in lactating goats: Nielsen et al., 1995), and their extremely short
half-life (they are very largely metabolised during a single
passage through the lungs) could allow them to act locally,
despite being present in plasma. During mid- to late preg-
nancy, mammary production and secretion of PGF2α into
plasma was considerable. Shortly before parturition, two
events occurred: metabolites began to appear, indicating that
the gland was beginning to break down the PGF2α, and the
route of secretion changed from plasma to milk. If PGF2α was
indeed the inhibitor, then the parturient gland was trying to rid
itself of this inhibition first by chemical degradation and second
by placing the inhibitor where it would be removed as soon as
the neonate sucked. In apparent confirmation, infusion of a
stable analogue of PGF2α into individual mammary glands was
shown to inhibit lactogenesis (Maule Walker and Peaker, 1980).
However, an inhibitor of PGF2α production produced only a
very transient and incomplete lactogenesis when infused into
the gland, albeit some 30 days before expected parturition
(Maule Walker, 1981). Renewed interest has been shown in
mammary function around lactogenesis, centred on the poss-
ible role of mammary-derived parathyroid hormone-related
protein (PTHrP) in regulating local blood flow and calcium
transport (Thompson, 1993). Although local control of lacto-
genesis has been confirmed, PTHrP does not appear to be in-
volved and so further elucidation of the mechanism has not
occurred.
Local control of milk secretion
There is abundant evidence that the rate of milk secretion
within individuals is directly correlated with the frequency
with which milk is removed. Elliott (1959) reviewed early work
in dairy cows, and the same has recently been shown to be true
of human lactation (Daly et al., 1992) and similar conclusions
have been drawn for a number of other species. The most usual
milking frequency for dairy cattle is two times a day. Milking
three times a day increases yield by about 10% (Poole, 1982),
and milking once a day reduces it by up to 20% (Carruthers
et al., 1993). The most extreme example of frequent milking of
which we are aware is six times a day (Bar-Peled et al., 1995),
which increased the yield of Israeli dairy cows from 35 to 43 kg
per day, a 21% stimulation. Milking six times a day may appear
somewhat esoteric, but it is probably less frequent than natural
suckling and the recent introduction of fully automated robotic
milking systems makes very frequent milking a realistic option
for the dairy farmer.
A number of theories have been put forward to explain how
increased milking frequency stimulates milk secretion (for re-
view see Peaker, 1995). Experiments in goats showed it to be
a rapid phenomenon. Hourly milking produced a response
within a few milkings (Linzell and Peaker, 1971), indicating
that increased intake was not directly responsible. Crucially,
when the hourly milking was performed on only one gland, the
effect was restricted to that gland. The response was just as
great when the milked gland had previously been denervated,
but manual stimulation (to invoke the milk ejection reflex)
without milk removal failed to increase secretion rate. These
observations suggest that milk removal is the critical factor,
rather than endocrine events triggered by the act of milking.
To many, the obvious explanation for the frequent milking
response appeared to be relief of pressure within the mammary
gland. Historically, the mammary gland had been assumed to
 Intramammary local control
be inactive between suckling or milking bouts, with synthesis
of milk occurring concurrently with removal. Once the milk
ejection process was understood, it became apparent that syn-
thesis was a continuum, and intramammary pressure was in-
voked to explain its regulation. A fascination also developed
for describing ‘udder capacity’, that is, the total amount of se-
cretion that the udder was capable of either producing or hold-
ing without being milked. This could be measured relatively
simply by forcing quantified amounts of fluid into the udder or
allowing milk to accumulate for excessive periods before re-
moval. All that was required to measure pressure within the
teat was a catheter and manometer, so it is regrettable, but per-
haps hardly surprising, that two simplistic techniques con-
spired to mislead scientific thought. What was shown was that
at some point (believed to be around 36 h in the cow) milk se-
cretion ceased, and at this time pressure in the udder was high.
What was concluded was that secretion rate was regulated by
intramammary pressure. The assumed mechanism was either
restricted blood flow, physical disruption of the secretory epi-
thelium, or both. In reality, instantaneous secretion rate could
not be measured, particularly not at short milking intervals,
and so conclusions about its control were nothing more than
speculation.
Advances came when physiological measurements were
added to the simplistic ‘weight of milk’ approach (Fleet and
Peaker, 1978). Careful measurements in goats of blood flow,
oxygen consumption and substrate uptake combined with
better determination of intramammary pressure at the relevant
site (that is, within the secretory alveoli) and estimation of
secretory rate from gland volume led to the conclusion that
pressure does indeed inhibit milk secretion, but not until 24 h
after milking. Furthermore, at the pressures reached, there was
little likelihood of blood flow being directly restricted; rather,
the fall in flow was a consequence of the reduced secretory
activity. It was observed that further increases in pressure led,
after several days, to a marked loss of epithelial integrity, as
evidenced by breakdown of tight junctions and changes in
ionic composition (Fleet and Peaker, 1978). Experimental dis-
ruption of tight junctions has a variable effect on milk secretion
rate (Peaker and Neville, 1981; Stelwagen et al., 1995), and it is
possible to misinterpret changes in ionic composition (in-
creased milk [Na+] in the absence of decreased [K+] is more
likely to be due to reduced sodium pump activity than to
tight junction leakiness: D. B. Shennan, personal communi-
cation). Indeed, it is likely that tight junctions are a product of
active secretion by epithelial cells, rather than the cause of de-
creased secretory activity. Nevertheless, tight junction integrity
has received considerable attention as a possible modulator of
pressure-induced inhibition of milk secretion, with exactly the
same conclusion as before. In both goats (Stelwagen et al., 1994)
and cows (Stelwagen et al., 1997), there was evidence of re-
duced secretion rate and loss of tight junction integrity (well
characterized by a variety of measurements), but not until at
least 18 h after the last milking. There must be another expla-
nation for the acute regulation of milk secretion that leads to
the three times a day or hourly milking response.
Other experiments in vivo also pointed to the existence of a
different mechanism. When one gland of goats was milked
three times a day but at the extra milking, the volume of milk
removed was replaced by an equivalent volume of iso-osmotic
107
sucrose, to maintain intramammary pressure at its pre-milking
value, the stimulatory effect of the extra milking was still evi-
dent (Henderson and Peaker, 1984). Milk did not have to be
removed. When sucrose was infused into an unmilked gland to
dilute the stored milk (and any bioactive components within
it), the secretion rate increased (Henderson and Peaker, 1987).
Finally, when partially purified extracts of milk whey were in-
fused into individual goat mammary glands, milk secretion
rate was reversibly inhibited (Wilde et al., 1988). These results
are incompatible with the pressure hypothesis, and do not
support any involvement of local neural reflexes, as has been
proposed by Svennersten et al. (1991). However, they are in
agreement with a chemical inhibitory mechanism, as first pro-
posed by Linzell and Peaker (1971).
Autocrine control of milk secretion by feedback inhibitor
of lactation
Expensive in vivo infusions of milk fractions were not a viable
means of screening milk for one (or a few) inhibitor(s) among
many bioactive factors. An in vitro mammary culture system
capable of approximating rates of synthesis in vivo was needed
to facilitate the search, and the rabbit mammary explant system
proved to be convenient. Tissue from rabbits was cultured in
the presence of stimulatory hormones and rates of lactose and
casein synthesis were measured. Milk fractions were then
added and the degree of inhibition was determined (Wilde
et al., 1987a). In this way, it was quickly established that neither
milk fat nor milk casein had specific inhibitory activity, but that
dose-dependent inhibitory activity was present in fractions
containing milk whey proteins of nominal size (from ultra-
filtration) between 10 and 30 kDa. Alterations in milking fre-
quency affect milk yield but not milk composition, so it was
important to demonstrate that the inhibition occurred approxi-
mately equally in casein and lactose synthesis. Rapidity of
onset and reversibility of effect are good methods for discrimi-
nating between a specific inhibition and a generalized toxic
action; the effect of the 10–30 kDa fraction was both rapid and
reversible. Explants retain all of the local biological structure of
the intact gland but none of the systemic influences, so it was
important to show that the effects were not an in vitro artifact.
To this end, milk fractions were infused into individual mam-
mary glands of anaesthetized lactating rabbits and milk accu-
mulation was measured over the next 24 h. The same fractions
that had inhibited in vitro synthesis were also effective in vivo.
Subsequent purification of the 10–30 kDa fraction (Wilde et al.,
1995) used anion-exchange chromatography and chromato-
focusing to produce a single-protein fraction that was 40 000-
fold concentrated relative to milk and which retained all of the
inhibitory activities to both casein and lactose synthesis.
Structural analysis showed a small, acidic glycoprotein of Mr
7600, and a novel N-terminal amino acid sequence was deter-
mined (Wilde et al., 1995). At this time there was justification
for ascribing a name to what had previously become ‘peak 3’,
and the obvious choice was feedback inhibitor of lactation
(FIL). As with the earlier studies, confirmation of the biological
role of FIL was demonstrated by intramammary infusion. In
this case, FIL infused into one gland of goats inhibited secretion
relative to the contralateral control gland. Subsequently, immu-
noneutralization of endogenous FIL has been shown to have
108 C. H. Knight et al.
stimulatory effects on milk yield (Wilde et al., 1996), exactly as
would be expected.
Feedback inhibitor of lactation or analogous proteins have
been identified in milk from goats (Addey et al., 1991a), cows
(Addey et al., 1991b), humans (Prentice et al., 1989; C. Henderson
and C. J. Wilde, unpublished) and wallabies (Hendry et al.,
1992). Their presence in milk is not in itself proof of mammary
origin, so confirmation has been sought using the in vitro sys-
tem of mammosphere culture. Mammary cells cultured on a re-
constituted basement membrane form spherical structures very
reminiscent of intact alveoli (Fig. 2) and secrete vectorially into
the central lumen. The contents of the lumen can be obtained
for analysis by temporarily opening cell:cell tight junctions
with a chelating agent. When this was done, FIL was detected
immunologically in luminal contents at a considerably higher
concentration than in the culture medium (Wilde et al., 1995),
showing it to be a secreted product of the epithelial cells. Since
milk present in the alveolar lumen is in contact only with se-
cretory cells, it is apparent that FIL must exert its action on the
same cells that produce it, indicating that the mechanism is
truly autocrine (Fig. 1).
Mode of action of feedback inhibitor of lactation
Frequent drainage of milk from the udder cistern with avoid-
ance of the milk ejection reflex does not lead to increased milk
yield in the way that frequent milking does (Henderson and
Peaker, 1987). This confirms that the site of action of FIL is
within alveolar tissue. Since tight junctions prevent paracellular
flux of small molecules and ions during established lactation,
the precise site must be the apical surface of the secretory cell.
Mammosphere culture supports this: to be effective, FIL must
be placed within the lumen of the mammosphere, rather than
in the culture medium. The nature of the FIL receptor remains
to be determined, but preliminary data do support the notion
of specific binding sites on the apical membrane.
Within the cell, the action of FIL occurs at an early stage in
the constitutive secretory pathway for proteins (Rennison et al.,
1993). Pulse-chase experiments have shown that FIL inhibits
the trafficking of radiolabelled protein through the Golgi ap-
paratus, whereas the later stages of the secretory process are
unaffected. Within 1 h of addition of FIL to the cultures, the
morphology of the Golgi and endoplasmic reticulum were
markedly altered and protein secretion was reduced by as
much as 50% (Figs 1 and 3). Recovery was equally swift, cell
ultrastructure and protein secretion recovering to normal
within 1 h of removal of FIL. Similar effects are seen in other
cells in response to the fungal drug, brefeldin A. In this case,
inhibition of secretion results in a subsequent reduction of
synthesis and it seems highly likely that the same will be true
of FIL. In addition, accumulation of protein within the cell may
result in intracellular casein degradation, which FIL is known
to increase (Wilde et al., 1989a).
Concentration of feedback inhibitor of lactation in milk
and kinetics of action
Owing to a number of complicating factors, concentration
dependence of FIL action has been well established in vitro
but not yet completely characterized in vivo. Normal milking
does not remove all milk from the gland; in dairy species
about 10% additional (residual) milk can generally be re-
moved by stimulation of the milk ejection reflex with exog-
enous oxytocin, and in some species (humans are a good
example), the extent to which milk is removed from indi-
vidual glands at specific suckling bouts is highly variable (Daly
et al., 1992). In other words, FIL is still in the gland even
after milking or suckling. In species with little or no extra-
alveolar storage space (for example, humans and rabbits), there
can be no doubt that FIL is at its active site within the alveolar
lumen. However, the udders of dairy species comprise se-
cretory tissue and non-secretory cisternal storage tissues in
variable proportion, so the situation is less clear. Immediately
after milking, residual milk will be within secretory alveoli,
but whether all of it quickly drains away into the cistern is
uncertain. There is certainly some movement of milk within
a very short interval after milking (Stelwagen et al., 1996)
and, in goats (Peaker and Blatchford, 1988) and late-
lactation cows (Knight and Dewhurst, 1994), cisternal flux
during the first hour is theoretically sufficient to account for
all of the residual milk. During early lactation, the cow cistern
is less compliant and most residual milk probably remains in
the alveoli. Peaker and Blatchford (1988) and Knight and
Dewhurst (1994) showed that some milk was present in alveoli
at 1 h after milking, so it is safe to assume that the secretory
cell is always exposed to FIL. Therefore, how is feedback
inhibition relieved?
There is no reason to believe that production of FIL occurs at
anything other than a constant rate relative to other milk con-
stituents. However, we do know that active bovine FIL is most
concentrated in milk that has been present within the gland for
some time (C. J. Wilde, unpublished). Some form of processing
must occur to explain how the concentration of FIL decreases
in residual milk soon after milking. Either FIL is secreted in
an inactive form and is then activated within the alvolar lumen
or else preformed, active FIL is secreted and then metabolised
to inactive forms. There is evidence from anion-exchange
chromatography of the existence of multiple forms of the mol-
ecule, including some that are biologically inactive (Wilde et al.,
1995). Mathematical modelling indicates that first order acti-
vation or degradation would both result in the necessary in-
crease in concentration of FIL with time after milking.
Relevance of storage anatomy
As milk accumulates between milkings it also becomes dis-
tributed between secretory tissue storage and cisternal storage
(Peaker and Blatchford, 1988; Knight and Dewhurst, 1994;
Fig. 1). The fact that FIL is only active when in contact with se-
cretory cells means that it will be most effective when the ratio
of cisternal to secretory storage is low. This ratio is quite vari-
able among individuals, and those goats or cows that have a
large cistern would be expected to produce more milk per
gram of secretory tissue than those with a small cistern.
Furthermore, they should be more able to tolerate infrequent
milking, but be less responsive to frequent milking. All of
these predictions have been shown to be correct (Peaker and
Blatchford, 1988; Dewhurst and Knight, 1994; Knight and
Dewhurst, 1994). Adaptive changes occur when individual
glands are milked once a day. Over relatively short periods,
 Intramammary local control 109

Fig. 2. Scanning electron micrograph of mammosphere culture. In this in vitro system mammary epi-
thelial cells are correctly polarized and organized into hollow spherical structures (mammospheres) rem-
iniscent of secretory alveoli. Synthesized milk components are secreted into the hollow lumen of the
mammosphere. (Reproduced with permission from Hurley et al. (1994))

the cistern stretches, its compliance increases and the changed milking than those that had been milked twice a day through-
storage ratio favours higher milk production. In cows, half- out (C. H. Knight, unpublished). In multiparous goats, glands
udders milked once a day during 2 days of each week in milked once a day throughout the first 6 weeks and then
early lactation were then less affected by the next once daily milked twice a day produced more milk during the remainder
110 C. H. Knight et al.
Fig. 3. Transmission electron micrograph of mammary Golgi apparatus to show the disruptive effect of
feedback inhibitor of lactation (FIL). (a) Control; (b) 2 h culture with FIL; and (c) FIL removed, culture
continued for 2 h. (Reproduced with permission from Rennison et al., 1993.)
of the lactation than those that had been milked three times
daily and then twice a day (Brown and Knight, 1995).
Local control of milk fat secretion
There is good evidence for co-ordinated control of both protein
and lactose synthesis by FIL. It is less easy to be certain about
fat synthesis, which is not affected by FIL in the in vitro systems
where it has been tested. Inhibition of milk fat synthesis by milk
lipid fractions has been demonstrated (Levy, 1964), and intra-
peritoneal injection of milk fat globules appeared to inhibit milk
secretion in vivo (Sala et al., 1973). Recent research has identified
the biochemical pathways involved in inhibition of fat syn-
thesis and has shown that certain medium chain fatty acids
(MCFAs) are responsible (Williamson et al., 1995). Although
MCFAs are present in milk and would be expected to cross the
apical membrane of the secretory cell, definitive evidence re-
lating MCFAs in stored milk to local inhibition of fatty acid
synthesis and showing an effect of different milking fre-
quencies has not been forthcoming. The data produced by
intraperitoneal injection were artefactual; this treatment causes
generalized health problems rather than a specific effect on
milk synthesis (Peaker and Taylor, 1994). In an abstract from
a recent meeting, Shamay et al. (1997) suggested inhibition of
casein, lactose and fat synthesis by a whey fraction isolated
from cows’ milk. If the data proves to be reliable, the identity of
the factor responsible and its relationship to FIL will need to be
determined.
Effects of milking frequency on milk composition
Analysis of commercial farm records typically indicates that
milking three times a day has a slight depressing effect on fat
and protein content (Campos et al., 1994), whereas controlled
scientific studies, particularly those using within-animal de-
signs, have generally shown no effect (Henderson et al., 1983).
We suggest that the difference lies in the accuracy of the
sampling and compounding effects of altered nutritional
management, and we have confidence in the experimental
studies; milking frequency does not usually alter gross milk
composition. However, this is not always the case. In one
experiment, a small but significant change in milk protein
content was observed during milking four times a day of half-
udders of cows, but it was an increase, not a decrease (Hillerton
et al., 1990). The effect was evident within 1–2 weeks of treat-
ment, so was relatively rapid in onset, and was localized to the
treated glands. Klei et al. (1997) have reported that milking
three times a day decreased both crude protein (significantly)
and casein protein (significantly only in early lactation), but
tended to prevent the decrease in casein number (casein as
a proportion of total milk protein) that occurs over the course
of lactation. They suggest that casein proteolysis occurs be-
tween milkings to an extent directly related to the interval
between milkings, such that more frequent removal of milk
decreases proteolysis. In ongoing experiments, we have ob-
served localized stimulatory effects of frequent milking on
both milk casein content and casein number (C. H. Knight
and D. D. Muir, unpublished). These changes develop gradu-
ally over time, and the hypothesis that they arise through an
effect of milking frequency on mammary gland development
and regression is being investigated.
Local control of mammary development and regression
Changes in the number and activity of mammary secretory
cells during ruminant lactation have been reviewed (Knight
and Wilde, 1993). Cell proliferation occurs to a modest extent
during early lactation and can be stimulated by a variety of
stimuli at any stage, but the most significant feature is a
gradual loss of secretory cells from peak lactation onwards,
which closely parallels the decline in milk yield. This loss most
probably occurs by apoptosis, which can be detected through-
out lactation, although it is clearly most prevalent during post-
lactational involution (Wilde et al., 1997). Between parturition
and peak lactation, the activity of individual secretory cells
increases markedly and remains high throughout declining
lactation. The local effects of milking frequency on cell activity
and the number of cells have been determined in goats
(Wilde et al., 1987b, 1989b) and cows (Hillerton et al., 1990). In
the short term (days to weeks), the change in yield results from
altered cell activity, but in the longer term, cell number is
affected. There is evidence in goats that a long period of milk-
ing three times a day increases mammary cell proliferation
(Wilde et al., 1987b), but it is also apparent that milk stasis
 Intramammary local control
Immediate
Milk
Medium term yield Short term
Udder
Cell
anatomy
FIL activity
FIL
Physical
stretch Milking
frequency
FIL? FIL
Cell Endocrine
number sensitivity
Long term Short term
Fig. 4. Schematic representation of the various local effects of milk-
ing frequency on the mammary gland.
stimulates apoptosis in both goats (Quarrie et al., 1994) and
mice (Quarrie et al., 1996). Long-term changes in cell number
due to milking three times a day most probably result, in part,
from increased proliferation and, in part, from decreased apop-
tosis. Both effects can occur in one gland independently of
other glands in the same animal and, thus, are clearly subject to
local regulation; the same is true of the short-term differ-
entiative changes.
Are changes in cell differentiation a consequence of FIL
action? Addition of FIL to primary cell cultures from mid-
pregnant mouse mammary gland inhibited hormone-induced
cell differentiation (Wilde et al., 1991), possibly through a
mechanism related to its main effect on the protein secretory
pathway. Some cellular proteins, including hormone receptors,
are synthesized and trafficked within the cell in a manner an-
alogous to the secreted milk proteins, and this trafficking
appears to be responsive to FIL. We have demonstrated that
milk accumulation reduces cell-surface receptors for prolactin
(and probably IGF-I) (Bennett et al., 1992), an effect that is re-
produced by intraductal FIL injection (Bennett et al., 1990).
Therefore, it appears likely that the effects of FIL on cell dif-
ferentiation are mediated by altered sensitivity of the secretory
cell to circulating lactogenic hormones.
Does FIL affect the number of cells? The same endocrine-
sensitivity mechanism may come into play here; both prolactin
and IGF-I are recognized mammary mitogens in a variety of
species (Forsyth, 1989) and both appear to be involved in mam-
mary cell survival through a complex interaction with the bind-
ing protein, IGFBP5 (Flint and Knight, 1997). In short, there
appear to be multiple effects of FIL (Fig. 4), which can all be re-
lated to a primary action at the level of intracellular protein
trafficking. The immediate effect on milk secretion is a direct
consequence, and the subsequent effect on cell activity and the
eventual effect on the number of cells are indirect consequences
mediated through altered hormone sensitivity.
111
Conclusions
We have presented evidence for local control of the onset and
maintenance of milk secretion, the latter by a small molecular
weight glycoprotein (FIL) that has immediate and direct effects
on protein and lactose secretion and long-term, probably in-
direct, effects on mammary development and involution. This
local control co-exists very successfully with systemic endo-
crine and metabolic control. It fulfills the vital function of
matching the rather different demands of the lactating mother
to those of her offspring and, at its most extreme, it enables the
lactating marsupial to nourish two young with very different
energetic requirements at one time.
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