Journal of Dairy Research (2002) 69 37–52.

# Proprietors of Journal of Dairy Research 2002 37

DOI : 10.1017\S0022029901005246 Printed in the United Kingdom

Mammary apoptosis and lactation persistency in dairy animals

B BRUNO STEFANON"*, MONICA COLITTI", GIANFRANCO GABAI#,

CHRISTOPHER H. KNIGHT$  COLIN J. WILDE$
" Department of Scienze della Produzione Animale, Faculty of Veterinary Medicine,
University of Udine, 33100 Udine, Italy
# Department of Scienze Zootecniche, Faculty of Veterinary Medicine,
University of Padova, Agripolis, 1-35020 Legnaro, Italy
$ Hannah Research Institute, Ayr KA6 5HL, UK

(Received 16 November 2000 and accepted for publication 5 September 2001)

S. The decline in milk yield after peak lactation in dairy animals has long
been a biological conundrum for the mammary biologist, as well as a cause of
considerable lost income for the dairy farmer. Recent advances in understanding the
control of the mammary cell population now offer new insights on the former, and
a potential means of alleviating the latter. The weight of evidence now indicates that
a change in mammary cell number, the result of an imbalance between cell
proliferation and cell removal, is a principal cause of declining production. Further,
it suggests that the persistency of lactation, the rate of decline in milk yield with
stage of lactation, is strongly influenced by the rate of cell death by apoptosis in the
lactating gland. Mammary apoptosis was first demonstrated during tissue involution
after lactation, but has now been detected during lactation, in mammary tissue of
lactating mice, goats and cattle. Those factors that determine the rate of cell death
by apoptosis are as yet poorly characterized, but include the frequency of milking in
lactating goats. Initial evidence suggests that nutrition also is likely to influence cell
survival after peak lactation, an important factor being the degree of oxidative stress
imposed by feed and the tissue’s ability to deal with, and prevent damage by,
reactive oxygen species. Comparison of cows in calf or not pregnant during declining
lactation also indicates a likely influence of reproductive hormones, with oestradiol
and progesterone acting to preserve mammary ductal and alveolar integrity during
the dry period, while allowing a degree of apoptosis and cell replacement. In each
case, the molecular mechanisms controlling mammary cell survival (or otherwise) are
as yet poorly defined. On the other hand, more persistent lactations are likely to
benefit animal welfare through fewer calvings and by placing less emphasis on
maximal production at peak lactation, and modelling of persistent lactation with
longer calving intervals indicates their likely economic benefits. In these cir-
cumstances, there is considerable incentive to elucidate the determinants of
mammary apoptosis, and the factors controlling the dynamic balance between cell
proliferation and cell death in the lactating mammary gland.

K : Lactation, apoptosis, mammary development, milk production

* For correspondence ; e-mail : bruno.stefanon!dspa.uniud.it

38 B. S  
Milk production systems have traditionally been designed for maximization of
daily yield in the belief that this would increase economic efficiency. Research was,
accordingly, concentrated on those factors in animal physiology and nutrition that
would increase milk production at the peak of lactation, rather than on the
maintenance of milk secretion during the declining phase of lactation. A variety of
considerations have now challenged that strategy, and re-focused research effort on
extending lactation rather than on increasing peak production levels. One
consideration is the fact that high-yielding animals managed conventionally and
calving at yearly intervals are increasingly being dried off while still producing
significant quantities of milk. Yields of 25–30 kg\d at drying off are not uncommon.
Another consideration is the perception that the high-yielding dairy cow is already
under metabolic stress, and any further increase in milk yield would be at the
expense of animal welfare. Whilst this perception is not wholly supported by
metabolic analysis (Knight, 1998), it is undoubtedly true that disease incidence is
greatest (by far) in early lactation (Erb et al. 1984). This association between high
output and disease implies that uptake of manipulative strategies that primarily
increase the maximum rate of milk secretion are likely to have limited application
within Europe. Further, the overproduction of male calves associated with yearly
calving intervals is now seen in many countries as unacceptable and unsustainable
in the long term. Finally, and most importantly, economic modelling of both totally
persistent lactation (Knight & Mainland, 1995) and of extended lactation (Galligan
& Lormore, 2001) suggests that there may be a significant financial advantage in
adopting such a system. For the biologist, the challenge is now to understand the
mechanisms that control loss of milk production after peak lactation, that is, to
identify the physiological determinants of declining milk yield after peak lactation,
and to find ways of reducing this decline, so as to increase the persistency of
lactation. In this article, we review evidence that declining production characteristic
of the lactating dairy animal is due to loss of cells from the secretory epithelium in
which milk is synthesized. We then consider the role of physiological factors such as
animal nutrition and management factors, such as frequency and efficiency of
milking, that influence these processes and which may be manipulated to improve
lactation persistency and animal welfare.

    

The cellular determinants of declining lactation have, despite their impact on
productivity, received relatively little attention. There is, however, persuasive
evidence from studies in lactating goats that falling milk yield is primarily due to a
progressive decrease in cell number in the lactating udder (Knight & Peaker, 1984).
This cell loss occurs in the parenchymal tissue of the gland where milk secretion takes
place. Milk secretion declines, therefore, because the tissue fails to maintain its
population of secretory epithelial cells (Wilde & Knight, 1989). Indeed, in goats, a
fall in milk yield of approximately 40 % between peak lactation and drying off is
accompanied by a similar percentage fall in mammary DNA content, indicating a net
decrease in tissue cell number (Knight & Peaker, 1984). On the other hand, the
surviving cells retain their capacity for milk synthesis. This is indicated by the
activities of a number of key enzyme markers of epithelial cell differentiation (Wilde
et al. 1989) and by the abundance of messenger RNA for the principal milk proteins
(J. M. Bryson and C. J. Wilde, unpublished observations). In other words, a sizeable
 Mammary apoptosis in dairy animals 39
fraction of milk-producing cells is lost during the course of lactation but, providing
husbandry and other factors do not change, the surviving mammary secretory cells
retain the intracellular machinery necessary for milk synthesis and secretion.
The most significant advance in understanding the cell biology underpinning
persistency of lactation has come from the demonstration that cell loss during
lactation occurs by apoptosis. Physiological cell death by this mechanism was
hitherto thought to be characteristic of involuting mammary tissue, and in
particular the involuting mammary tissue of non-ruminants (Walker et al. 1989 ;
Strange et al. 1992 ; Quarrie et al. 1996). It is now clear, however, that apoptosis is a
feature of ruminant mammary tissue remodelling after lactation under at least some
circumstances (see below). More importantly, evidence from studies in both rodents
and ruminants provides persuasive evidence that apoptosis is also part of the
repertoire of lactating mammary tissue. This conclusion is based primarily on the
detection of internucleosomal fragmentation in lactating mammary DNA, demon-
strated by the appearance of ‘ ladders ’ of oligonucleosomal fragments, of 180
basepairs and multiples thereof, in DNA extracts resolved by electrophoresis
(Quarrie et al. 1995 ; Wilde et al. 1997). DNA laddering is the classic indicator of cell
death (Wyllie et al. 1980). Insofar as it can be quantified (Quarrie et al. 1995), the
intensity of laddering in lactating mammary tissue indicates that the proportion of
mammary cells dying by apoptosis is a small fraction of that which can be lost when
lactation is terminated (Quarrie et al. 1995 ; Wilde et al. 1997). A similar conclusion
comes from identification of apoptotic cells by terminal deoxynucleotide end-
labelling of fragmented DNA in situ in tissue sections (TUNEL). Whereas mammary
tissue of non-pregnant cows contained up to 5 % of TUNEL-positive cells 1 week
after drying off in late lactation, the proportion of cells detected by this method in
lactating tissue was approximately 2 % (Wilde et al. 1997). A similar study in
lactating goats indicated that, whereas 5 % of cells in involuting tissue were again
apoptotic, less than 1 % of cells in lactating tissue were dying (Li et al. 1999). It
should, however, be noted that the DNA fragmentation detected by TUNEL
represents just one stage of apoptosis, and each apoptotic death is a transient event
compared with the duration of lactation (Pullan et al. 1996). Therefore, even a low
index of TUNEL-positive cells represents a quantitatively significant process, and
identifies apoptotic cell death as an important determinant of declining productivity
in lactating ruminants. Cell death may be counterbalanced by cell proliferation, since
even in declining lactation DNA synthesis indicative of cell proliferation is detected
(Wilde et al. 1987) and the importance of understanding relationships between the
two processes has been stressed recently (Knight, 2000). Nevertheless, the net fall in
cell number during this period suggests that in normal circumstances cell death is the
major contributor to mammary cell turnover, and it is on this that we concentrate
here.
If declining production in dairy cows is indeed the result of cell loss by apoptosis,
it follows that increased persistency of lactation may be achieved by increasing cell
survival. Some of the factors likely to influence the survival of luminal, i.e. secretory
epithelial cells in lactating tissue are predictable from experiments that have
progressively characterized the time course and control of mammary tissue
involution on cessation of milking. The following sections, summarized in Figs 1 and
2, discuss emerging evidence that nutrition, lactation management, i.e. milking
frequency and efficiency, and reproductive status all affect the incidence of apoptosis
in the lactating mammary gland, and are therefore key targets for strategies to
extend lactation by prolonging cell survival.
40 B. S  

Phase
Early Lactation Late Lactation Post-lactation
Involution

Key processes
Cell proliferation + Cell proliferation – Cell proliferation –/+

Apoptosis – – Apoptosis + Apoptosis + + +

MMPs – – – MMPs – MMPs + + +

Cell number  Cell number 

Outcome

Cell number 
Alveolar size  Alveolar regression
Milk yield 
Milk yield  ECM remodelling 

Fig. 1. Key events in mammary tissue development and remodelling during lactation and involution.
Arrows indicate relative activity of processes across phases : kkk jjj. MMP, matrix
metalloproteinase. ECM, extracellular matrix.

lactation
involution Galactopoietic
hormones

Basement + Chemical
+ +
membrane + – – feedback
degeneration + + +
–FIL, IGFBP5?
– +
–
+ + + Apoptosis
+

Metalloproteinase Alveolar
+ distension?
activation +/– + + +
+

Dietary factors,
reactive oxygen
species?
Fig. 2. Factors influencing mammary apoptosis during lactation and involution. Arrows indicate
relative activity of processes across phases : kkk jjj. FIL, feedback inhibition of lactation.
IGFBP5, insulin-like growth factor binding protein 5.

   

Nutrition
Mammary development during pregnancy and in lactation is influenced by
nutrition in both rodents and ruminants. Feeding either a restricted amount of a
nutritionally appropriate diet (Knight & Peaker, 1982) or adequate amounts of
protein-deficient diets (Pyska & Styczynski, 1979) to pregnant rodents impairs
mammary development. Cell proliferation was measured in the later work and was
reduced, but whether these treatments also increased apoptosis is not known. A stair-
step compensatory nutrition regime, whereby whole-body growth is first restricted
by energy deficiency and then allowed to catch up, has been shown to have
significant (but small) positive effects on mammary development in rats (Park et al.
 Mammary apoptosis in dairy animals 41
1994 ; Kim et al. 1998) and ruminants (Park et al. 1998). It was suggested that
reduced apoptosis may be part of the explanation (Kim et al. 1995 ; Moon & Park,
1999). In rat experiments, cell proliferation was increased during early lactation and
apoptosis was reduced during late lactation by the dietary regime, but since
treatment had ended some time earlier it is difficult to establish a cause and effect
relationship (Kim et al. 1995 ; Moon & Park, 1999). There are other indirect reasons
for hypothesizing an effect of energy status, at least on mammary cell division.
During early lactation, proliferation is proportionately greater in species that exhibit
less negative energy balance (compare rodents and cows). Similarly, mitogenic effects
of growth hormone have been observed in pre-partum goats (Knight et al. 1994) and
cows (Collier et al. 1993) but are absent during early lactation, only to return in late
lactation when the animal’s energy balance is restored (Capuco & Byatt, 1998). It is
not known whether similar but reciprocal effects would be observed on apoptosis,
although a recent study in cows in early lactation suggests that the relationship may
not be entirely simple. Heifers were fed diets containing 75 % or 25 % concentrate
(high and low energy, respectively) during the first 8 weeks of lactation. At the end
of this period, the chances of detecting proliferating cells in histological sections of
mammary biopsies were increased 11-fold by high nutrition, but whilst apoptosis
was not significantly affected it tended to be increased, rather than reduced
(Sorensen et al. 2000). Eight weeks later the differences had disappeared, suggesting
a more direct effect of the energy difference than was seen in the rat work. It is worth
pointing out that the 11-fold difference sounds very large but actually arises from
proliferation rates of approximately 0.2 and 0.02 %, emphasizing the difficulty of
interpreting numerically small differences in what are very low rates of turnover.
What is still needed is an evaluation of dietary effects on apoptosis during the
declining phase of lactation. Indirect evidence suggests that there may be an effect,
since increased intake can improve lactation persistency in dairy cows (Sorensen &
Knight, 2000) but the appropriate measurements have not yet been made.
Nutritional control of mammary apoptosis might be predicted from numerous
studies in other tissues that demonstrate induction of apoptosis by oxidative stress.
Generation of reactive oxygen species, or a reduction in the activity of cellular
antioxidant mechanisms increase susceptibility to apoptosis (Hockenbery et al.
1993). Mitochondrial DNA damaged by oxidative state was found to be higher in
apoptotic cells, and an increase of oxidized glutathione was found in mitochondria of
apoptotic cells in mammary tissue (Esteve et al. 1999). These effects may be mediated
by the actions of bcl-2 gene family members (Hockenbery et al. 1990 ; Kane et al.
1993), either by their ability to scavenge harmful free radicals (Hockenbery et al.
1993 ; Hochman et al. 1998), or possibly through bcl-2 protein interaction with
mitochondrial superoxide dismutase (Buttke & Sandstrom, 1994).
Oxidative stress in dairy cattle, and its possible consequences for mammary
apoptosis, is likely to be influenced by feed composition, and in particular its content
of antioxidants or thiol-containing compounds. For example, forage grown in the
presence of high levels of nitrogen fertilizer contain high concentrations of nitrates
and nitrites, which can be converted into peroxinitrite, a reactive species known to
modify proteins and stimulate lipid peroxidation (Radi et al. 1991 ; Beckman et al.
1994). There is direct evidence that dietary antioxidants influence the levels of
mammary bcl-2 and bax, two proteins implicated in the control of mammary
apoptosis (Metcalfe et al. 1999 ; Motyl et al. 2000). In lactating sheep fed a diet
deficient in vitamin E, a known antioxidant (Muller & Goss-Sampson, 1990), the
bcl-2 to bax protein ratio was increased during subsequent tissue involution after
42 B. S  
termination of suckling (Colitti et al. 2000). Interpretation of this response requires
further investigation, since inter-animal variation in the incidence of apoptotic cells
prevented a clear picture of its relation to apoptotic mechanisms in the tissue.
Moreover, it is likely that the roles of other members of the bcl-2 gene family, and
their possible nutritional modulation, must also be considered in defining the
relationship between oxidative stress and apoptosis (Metcalfe et al. 1999).
Nevertheless, this study demonstrates in vivo the likely involvement of dietary
factors in intracellular signalling mechanisms that contribute to the control of
mammary development.

Milking frequency and efficiency

It has been known for some time that mammary cell number in lactating
ruminants is controlled locally, within each mammary gland, by frequency of milk
removal (Wilde et al. 1987). Glands of lactating goats milked three times daily
contained, at the end of lactation, more luminal epithelial cells than the contralateral
glands in the same animals milked twice daily. The greater cell number in thrice-
milked glands was initially thought to be due to local stimulation of cell proliferation,
since DNA synthesis, measured ex vivo in fresh tissue explants, was higher than in
those prepared from tissue of twice-milked glands (Wilde et al. 1987). However, it was
also evident that the local response to frequent milking occurred against a
background of declining cell number after peak lactation (Knight & Peaker, 1984).
Thus, cell number had fallen in both glands during the period of differential milking
(three times milking of one gland, twice-daily milking of the other), but to a lesser
extent in the thrice-milked gland. In these circumstances, it seemed likely that cell
proliferation was not solely responsible for the greater cell number observed in glands
milked frequently. Once programmed cell death by apoptosis was demonstrated as
the means whereby luminal epithelial cells were removed after lactation (Wilde et al.
1997), attention turned to the possible role of apoptosis in the developmental
responses seen during lactation.
Local control of apoptosis by milk accumulation was first demonstrated by
inducing milk stasis in one gland of lactating goats. The resultant ipsilateral
induction in DNA laddering observed in the treated gland, with little or no effect on
the contralateral gland that continued to be milked normally, indicated that
mammary cell death was stimulated by milk storage irrespective of the continuing
presence of galactopoietic hormones (Quarrie et al. 1994). A similar response was
subsequently observed in lactating mice, following sealing of the teats on one half of
the body of lactating animals and removal of half the litter. Again, milk stasis
stimulated apoptosis in those glands, while lactation and cell survival were
maintained in adjacent glands through suckling of the remaining pups (Quarrie et al.
1996). These observations were confirmed in similar experiments elsewhere (Li et al.
1997 ; Marti et al. 1997). Interestingly, milk stasis appeared as effective a stimulant
of apoptosis as litter removal, inasmuch as DNA laddering was apparent in teat-
sealed glands within 24–48 h, and was of similar intensity to that observed after litter
removal (Quarrie et al. 1996). Whether milk stasis is as potent a stimulant of
apoptosis in ruminant mammary tissue remains to be seen, since the time course of
apoptosis following unilateral or complete cessation of milking has not been
compared in a single species. Bovine mammary apoptosis was easily detected
1 week after complete drying off (Wilde et al. 1997), and a significant increase in the
proportion of apoptotic cells was observed in sheep mammary tissue within 4 days
of removing the suckled lamb (Colitti et al. 1999). By comparison, unilateral cessation
 Mammary apoptosis in dairy animals 43
of milking in one goat mammary gland had little effect on cell survival after 3
days, but apoptosis was evident after 1 week and maximal 2 weeks after
induction of milk stasis in that gland (Li et al. 1999). Ruminants may, on this
evidence, tolerate better the effects of prolonged milk stasis and, by delaying
commitment to apoptosis, retain the ability to re-initiate lactation if circumstances
alter. Accordingly, milk production was reinstated in dairy cow quarters unmilked
for 12 d, yield recovering almost to pre-treatment values (Hamann & Reichmuth,
1990). Longer interruption of milking, when applied to all quarters, resulted in only
partial recovery of milk yield (Noble & Hurley, 1997). By contrast, mice can rescue
milk synthesis after 48 h of milk stasis, but after 72 h, recovery is already very
incomplete (Sorensen & Knight, 1997). Extended milk stasis should eliminate
differentiated gene expression and induce epithelial cell apoptosis (Wilde et al. 1997).
Partial re-initiation of lactation indicates, therefore, a potential for re-induction of
gene expression in surviving cells, the number of which may determine the extent of
recovery of milk yield. From the fragmentary evidence in dairy cows it seems likely
that in ruminants, cell survival depends on whether cessation of milking is partial or
complete, the former probably allowing more complete recovery of milk production.
This again implies that mammary apoptosis in ruminants is less sensitive to local
control by milk storage than is apoptosis in the rodent mammary cell population
such that, in the presence of systemic hormones, bovine and caprine mammary cell
survival is not immediately compromised.
Relative insensitivity of mammary cell number to milk accumulation suggested
that control of apoptosis by milking frequency, were it to occur, would have
significant effects only in the long term. This prediction was supported by the
response to differential milking of goat mammary glands : cell number in glands
milked thrice daily did not differ from their twice-milked counterparts after 13
weeks, and only after 37 weeks was a unilateral increase in cell number evident
(Wilde et al. 1987). However, a recent study of goat mammary apoptosis during
lactation suggests otherwise. In this case, goats were milked thrice daily in one gland
and once daily in the other for 10 weeks. Infrequent milking elicited a unilateral
decrease in milk yield and, after 4 weeks, an induction of DNA laddering and an
increase in the proportion of apoptotic cells in the once-milked gland (Li et al. 1999).
This indicates that mammary apoptosis is indeed sensitive to milking frequency
during lactation. It also suggests that, like the secretory response to frequent or
infrequent milking (Blatchford & Peaker, 1982 ; Wilde & Knight, 1990) and the effect
of post-lactation milk stasis (Li et al. 1999), the response is elicited by a local intra-
mammary mechanism, enabling each gland to respond independently. The apoptotic
response in glands milked once rather than thrice daily illustrated other aspects of
the physiological context in which this process operates. First, it was apparent that
apoptosis removed a proportion of luminal epithelial cells, and was then suppressed :
after 10 weeks of milking glands at different frequencies, the intensity of DNA
laddering was similar in the two glands. Thus, after an initial accelerated cell loss in
the once-milked gland, the two glands may then have lost cells at similar rates, such
that a new dynamic balance between cell proliferation (if any) and cell loss was
established, with no further divergence of milk yield between the two glands. The
apoptotic response is, therefore, adaptive rather than degenerative in nature. It was
also apparent that cell death did not occur randomly in the glands milked once daily.
Apoptotic, i.e. TUNEL-positive cells were distributed heterogeneously, being
concentrated in alveoli that, by immunocytochemical staining, appeared to be
involuting. The mechanism underpinning this heterogeneity of alveolar function and
44 B. S  
development is not understood, but has parallels in other species. Asynchronous
alveolar development has been reported in ultrastructural studies of the mid-
pregnant rodent mammary gland (Mills & Topper, 1970) and in situ hybridization
demonstrated a heterogeneous pattern of milk protein gene expression in sheep and
bovine mammary tissue (Molenaar et al. 1992).
The mechanism by which milking frequency influences mammary apoptosis is the
subject of on-going study, but investigation of the acute control of milk secretion by
milking frequency and efficiency suggest a possible underpinning mechanism. Those
studies indicate that control of milk secretion by milking frequency is through
feedback inhibition by a milk constituent (Peaker & Wilde, 1996). Accordingly,
screening of milk constituents in tissue and cell culture bioassays identified a small
milk protein that inhibited milk constituent secretion in tissue and cell cultures
rapidly, in a reversibly and concentration-dependent manner (Wilde et al. 1995). The
protein, which has been named FIL (feedback inhibitor of lactation), also inhibited
milk secretion reversibly when added to milk in the goat’s udder (Wilde et al. 1995).
Active immunization against FIL in lactating goats produced a temporary reversal
of the decline in milk production that normally occurs in late lactation (Wilde et al.
1996). FIL is synthesized in the same mammary epithelial cells that synthesize other
milk constituents, and so feedback inhibition is an autocrine mechanism, that is, the
luminal epithelial cells secrete an inhibitor of their own secretory activity. The
molecular basis of autocrine inhibition is considered elsewhere (Peaker & Wilde,
1996). It is notable, however, that one of the consequences of FIL’s ability to inhibit
mammary membrane trafficking (Rennison et al. 1993) is a down-regulation of
mammary hormone receptors, including those for prolactin (Bennett et al. 1990).
Prolactin influences mammary gene expression in ruminants (L. M. B. Finch, M.
Wells, C. J. Wilde, unpublished observations) and is known to control mammary
gene expression and cell survival in rodents (Travers et al. 1996). An ability to
desensitize the luminal epithelial cell population to systemic galactopoietic hormones
may therefore provide a mechanism by which FIL would influence both the
differentiated state of the cell population and its size, the latter through stimulating
cell removal by apoptosis. If, then, FIL concentration in milk is controlled
independently in each gland according to the frequency or completeness of milk
removal, this offers a mechanism for the local control of apoptosis by milking
frequency. Preliminary experiments in primary culture support such a mechanism :
FIL stimulated apoptosis of goat mammary epithelial cells in a concentration-
dependent manner (C. J. Wilde, unpublished observations).
Whether control of apoptosis by frequency of milk removal is through chemical
feedback by a milk constituent (FIL) remains to be tested. Nevertheless, the relative
ease with which milking frequency can be manipulated, and the non-invasive nature
of the manipulation, offers an attractive route for increasing mammary cell survival,
and thereby assessing the predicted economic benefits of the resultant increase in
lactation persistency. Elucidation of the underpinning cellular mechanism may in
due course provide the means of applying the mechanism directly, that is, by means
other than frequent milking.

Galactopoietic hormones
The hormones primarily responsible for maintaining milk output are growth
hormone (GH) and prolactin (Prl). The older view was that GH was effective in
ruminants and Prl in all other species, but this has proved to be very much an
oversimplification (Flint & Knight, 1997). The intricate interaction between these
 Mammary apoptosis in dairy animals 45
two hormones has been reviewed recently, and appears to include mechanisms
involved in the control of apoptosis and cell survival (Flint et al. 2001). As presently
understood, the story does, however, include a number of conundrums, beginning
with the observation that GH acts locally to stimulate milk secretion, but secretory
cells do not possess GH receptors. Nor is the effect mediated by IGF1 secreted by
mammary stromal cells in response to GH, since local IGF1 administration does not
mimic the GH effect. On the other hand, IGF1 is both a known mammary mitogen
and anti-apoptotic factor, so perhaps the effect is actually one of enhanced cell
survival rather than direct stimulation of secretion. If so, one would anticipate
decreased local production of IGF1 during mammary involution but, on the
contrary, milk IGF1 actually increases at this time. IGF1 action is far from simple,
however, because of the existence of at least six IGF binding proteins, one of which
(IGFBP5) has been implicated as an apoptotic factor in a number of tissues. Is
IGFBP5 elevated during mammary involution? Yes it is, but only when that
involution is accompanied by a reduction in circulating Prl. That is, local induction
of involution by milk stasis in animals that are still being suckled is through a
different mechanism, as described above. In summary, GH and Prl interact to
prevent mammary apoptosis, GH by stimulating local production of IGF1 and Prl
by inhibiting local production of IGFBP5 which would otherwise abrogate the action
of IGF1 (Flint et al. 2001). This mechanism almost certainly explains the very
persistent milk yield seen in frequently milked, GH-treated goats (Knight et al.
1990), the frequent milking acting through local enhancement of Prl receptors (see
above). It is not known whether the GH–IGF axis is also involved in some of the
nutritional effects previously described, but the possibility must be considered that
it mediates the effects of energy intake in particular.

Reproductive status
In an earlier section we presented evidence that apoptosis should be considered
a normal part of the mammary cycle and an important determinant of bovine
mammary cell number during declining lactation, as it is in the goat (Li et al. 1999).
Nevertheless, the incidence of apoptosis during tissue remodelling between lactations
appears markedly lower in cows than in rodents or goats (Capuco & Akers, 1999),
which has led to debate concerning the significance of apoptosis in dairy cows. One
reason for this apparent species difference could be that dairy cows are, typically,
pregnant during most of the lactation cycle, and any mammary remodelling during
the dry period, promoted by milk stasis, usually takes place against a background of
elevated reproductive, mammogenic hormones. Accordingly, little tissue remodelling
was evident in pregnant cows when milking ceased in late lactation (Capuco et al.
1997). On the other hand, histological and ultrastructural comparison of bovine
mammary involution in pregnant and non-pregnant Holstein cows suggested that
mammary involution proceeded with little loss of epithelial cells in either condition,
and no disengagement of epithelial cells from basement membrane attachment
(Holst et al. 1987). Ultrastructurally, the involuting epithelium showed progressive
loss of tight junction integrity, with resultant alteration in the composition of
mammary secretion during the dry period (Athie et al. 1996). The organelles
intimately involved in milk protein synthesis and secretion (rough endoplasmic
reticulum and Golgi apparatus) degenerated during involution, this deterioration of
cellular ultrastructure beginning 2 days after drying off irrespective of re-
productive state (Holst et al. 1987).
On the basis of this histological evaluation of involuting tissue there appears,
46 B. S  
therefore, to be little support for an influence of reproductive hormones on the loss
of bovine mammary epithelial cells during lactation. Nevertheless, direct measure-
ment of apoptosis during drying off of non-pregnant cows indicates that, while in the
short term the tissue is spared the widespread alveolar degeneration characteristic of
rodent mammary involution, apoptosis is induced to a degree comparable to that in
involuting mouse mammary tissue (Wilde et al. 1997). By comparison, mammary cell
proliferation between lactations in dry, pregnant cows suggests a modest degree of
cell replacement, but not to the extent that tissue DNA content is altered (Capuco
et al. 1997). Therefore, whilst no direct comparison is available, it seems likely that
drying off after lactation promotes the death and removal of a proportion of the
epithelial cell population, the extent of the process being dependent on the
reproductive status of the animal. It is noteworthy, however, that mammary
apoptosis was observed in late-lactating concurrently-pregnant mice and, based on
intensity of DNA laddering, was as extensive as that observed in non-pregnant
animals (Quarrie et al. 1996). Another caveat in interpreting observations made in
ruminants is that the degree of mammary involution may vary with location in the
gland. Quantitative histology identified three distinct regions in involuting bovine
mammary glands (Capuco et al. 1997). One region adjacent to the gland cistern (zone
1) exhibited the highest rate of thymidine incorporation, i.e. cell proliferation and
appeared most sensitive to stimuli that would promote cell turnover. A second zone
midway between the gland cistern and the periphery of the gland and a third at the
margin of parenchyma adjacent to the ventral body wall showed progressively less
involution and lower rates of cell proliferation, with zone 3 exhibiting near-normal
epithelium. Conversely, whereas alveolar integrity and cell ultrastructure showed a
lactating phenotype in zones 2 and 3, cells in zone 1 appeared swollen, engorged with
lipid and disorganized, and alveoli contained areas denuded of cells and a high
proportion of cells with pyknotic nuclei.
In the cow, milk yield is maintained at high levels until month 5–6 of pregnancy
and decreases afterwards (Bertilsson et al. 1997). How pregnancy influences milk
yield is not known, but it seems likely that the sex steroids that maintain pregnancy
are involved. Oestrogens and progesterone have long been recognized as mammo-
trophic hormones. Oestrogen or anti-oestrogen implants in mouse mammary tissue
demonstrated a direct effect of oestrogen on ductal growth (Silberstein & Daniel,
1987 ; Silberstein et al. 1994). Similar effects have been reported in other species
(Cowie et al. 1980), and the presence of oestrogen receptors and oestradiol
involvement in mammary development have been reported in bovine mammary
tissue (Rotondi & Auricchio, 1978 ; Woodward et al. 1993). Progesterone receptors
are present in rat (Quirk et al. 1982), mouse (Haslam & Shyamala, 1979), goat
(Markland & Hutchens, 1977), dog (Rutteman et al. 1988) and human mammary
tissue (Verma et al. 1978), but there is as yet no direct evidence for progesterone
receptors in the bovine mammary gland. Study of prepubertal bovine mammary
tissue suggests that progesterone receptors may be present but do not, on the other
hand, promote cell proliferation. Woodward et al. (1993) studied the effect of
exogenous oestradiol and progesterone on the proliferative response of mammary
epithelial cells, fibroblasts, adipocytes and endothelial cells in prepubertal heifers
and found no effect of progesterone alone, and an inhibitory effect of progesterone on
oestradiol-stimulated cell proliferation. Interestingly, oestradiol stimulation of
bovine epithelial cell proliferation preceded a similar response in the stromal cell
population. In contrast, oestradiol stimulation of mouse mammary epithelial cell
proliferation required paracrine signalling between stromal and epithelial cells,
 Mammary apoptosis in dairy animals 47
including the oestradiol-mediated induction of epithelial progesterone receptors
(Haslam, 1988). Perhaps in consequence, progesterone stimulates ductal growth in
prepubertal mice (Skarda et al. 1989) and, in gestation, lobuloalveolar development
(Topper & Freeman, 1980). Progesterone receptor knockout experiments initially
suggested that both stromal and epithelial receptors are necessary for ductal and
lobuloalveolar development (Humphreys et al. 1997), but the predominant effect
may be exerted by epithelial receptors (Brisken et al. 1998). In addition, only a
proportion of progesterone receptor-positive epithelial cells need be engaged in the
hormone response (Brisken et al. 1998). Neighbouring cells may be recruited into
alveolar development through autocrine and paracrine signalling exerted possibly by
epidermal growth factor (EGF) (Ankrapp et al. 1998) or Wnt 5B (Humphreys et al.
1997). Interactions between EGF, oestrogens and progesterone depend upon the
developmental stage of the mammary gland, their effects being most pronounced in
the mature mammary gland (Haslam et al. 1993). Together, this suggests that rodent
and ruminant mammary tissue may differ with respect to their oestradiol and
progesterone responsiveness and, further, that effects observed in heifers are not
necessarily indicative of their actions in pregnant, lactating cattle.
The presence of systemic oestrogens and progesterone may, as discussed above,
serve to control the extent of tissue remodelling between lactations, but the cellular
effects of these hormones in declining lactation in pregnant, lactating cows, and their
consequent impact on lactation persistency, is not easily predicted. If progesterone
were to influence mammary development in pregnant late-lactating cows, one
possible effect could be to promote survival of the mammary epithelial cells.
Progesterone administered locally in hormone implants inhibited apoptosis of
mammary epithelial cells in the mouse, and appeared to maintain the lactating
phenotype of the tissue (Feng et al. 1995). Locally administered glucocorticoid had
a similar effect, and both appeared to act by preventing AP-1 induced transcription
of apoptosis-related genes such as spg-2, plasminogen activator, stromelysin-1 and
c-jun. It is also noteworthy that progesterone is a potent repressor of matrix
proteinases (MMP) in the uterus (Hulboy et al. 1997) and it may exert the same effect
on the mammary gland. Metalloproteinase inhibition would prevent degradation of
basement membrane, a key feature of mammary involution (Talhouk et al. 1992),
and a promoter of epithelial cell apoptosis (Boudreau et al. 1995 ; Pullan et al. 1996).
On the other hand, while oestrogens may enhance progesterone inhibition of MMP
expression, they stimulate AP-1 dependent transcription (Hulboy et al. 1997) and, in
breast cancer cell lines, induce tissue plasminogen activator (tPA) (Davies et al.
1995). Oestrogens, through tPA induction of extracellular proteinase activity, would
therefore be expected to promote tissue remodelling. Accordingly, oestradiol
injection for several days before drying off accelerated mammary involution (Athie
et al. 1996), possibly by promoting plasminogen activation (Athie et al. 1997).
Preservation of a functional mammary gland, while allowing a limited extent of
tissue remodelling in pregnant, lactating animals, may then depend on the relative
concentrations of oestrogen, progesterone and galactopoietic hormones prior to and
following drying off.
The circulating concentrations of the principal oestrogens and progesterone
hormones in cattle are well-documented. Circulating oestradiol is very low until the
fourth to sixth month of pregnancy, increases gradually until the last month of
gestation, and then rises rapidly to a peak value (Cowie et al. 1980). Plasma
progesterone concentration during pregnancy is similar to that found during the
luteal phase of the oestrous cycle and fluctuates around 4–13 ng\ml, although a
48 B. S  
modest decline between 160 and 230 d of pregnancy is sometimes reported (Cowie et
al. 1980). Other less well characterized but potentially important hormone variants
are the subject of on-going study. For example, 5-androstene-3 beta, 17 beta-diol, a
DHEA metabolite of feto-placental origin, is secreted in increasing concentrations
during pregnancy and may contribute significantly to the total oestrogenic activity
in the ruminant (Gabai et al. 1999). However, the relative influence of these hormones
on mammary development in declining lactation may be determined more by local
factors within mammary tissue. The bovine mammary gland is capable of
metabolizing circulating steroids. Progesterone is subject to metabolism and
inactivation by 5, alpha-reductase (Belvedere et al. 1996), the metabolite 5, alpha-
dihydroprogesterone having lower affinity for progesterone binding sites. Changes in
mammary 5, alpha-reductase activity during declining lactation in concurrently
pregnant cattle may, therefore, have a significant influence on tissue responsiveness
to progesterone. Similarly, bovine mammary steroid aromatase activity may enable
local oestradiol production in late pregnancy (Peaker, 1991). These local mechanisms
are, therefore, likely to modulate the endocrine control of mammary development
and function in declining lactation, and so influence significantly the persistency of
lactation.

Metabolic activity in the mammary epithelial cell population is subject to
modulation during lactation, acutely through translational or post-translational
control of synthetic and secretory mechanisms, with longer-term adaptations being
achieved by transcriptional control of key mammary genes, such as those intimately
concerned with milk constituent synthesis. Changes in intracellular synthetic
machinery are probably the means by which milk supply is adapted to acute (hours
to days) or medium-term (days to weeks) changes in production conditions and
nutrition (Wilde et al. 1987 ; Wilde & Knight, 1990 ; Park et al. 1994). We have,
however, argued here that while these metabolic adaptations may increase or
decrease the rate of milk secretion at any point, they are probably not the principal
determinant of the rate at which milk secretion declines after peak lactation. To
understand the cellular biology of lactation persistency, it is necessary to understand
the dynamics of the mammary cell population, and in particular the control of cell
turnover in established lactation. Clearly, the mammary cell population is in a
dynamic state during lactation. It continues to expand in early lactation in both
rodents and ruminants (Knight & Peaker, 1982, 1984) and decreases after peak
lactation (Wilde & Knight, 1989), prior to the more extensive remodelling
precipitated by drying off (Capuco et al. 1997 ; Wilde et al. 1997). A key factor in this
dynamic balance is the rate at which cells are removed by apoptosis. In the preceding
sections we have reviewed the evidence that apoptosis is indeed an integral part of
the mammary gland’s repertoire during lactation, and highlighted the, as yet,
relatively few instances where this process has been shown responsive to endocrine
status, nutrition and husbandry. Further research will undoubtedly provide a more
integrative picture of the cellular determinants of mammary apoptosis in the
ruminant, and should identify more clearly potential targets for manipulation, if the
goal is a longer, more persistent lactation. This may have far-reaching implications
for dairy production strategies, but is not necessarily dependent on radical
intervention such as endocrine manipulation. Significant benefits may be achievable
through careful choice of nutrition to minimize oxidative stress, through targeted
application of frequent milking, perhaps assisted by robotic milking, and by careful
 Mammary apoptosis in dairy animals 49
planning of re-breeding. These approaches are attractive in terms of public
acceptability, will benefit animal welfare and should contribute to the achievement
of sustainable production.

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