www.elsevier.com/locate/livprodsci

Livestock Production Scie

Milk accumulation decreases expression of genes involved in

cell–extracellular matrix communication and is associated

with induction of apoptosis in the bovine mammary gland

K. Singh a,*, J. Dobson a, C.V.C. Phyn a, S.R. Davis b, V.C. Farr a,

A.J. Molenaar a, K. Stelwagen a

a AgResearch, Ruakura Agricultural Research Centre, P.B. 3123, Hamilton, New Zealandb Present address: ViaLactia Biosciences (NZ) Ltd., Newmarket, Auckland, New Zealand

Abstract

Forty-eight primiparous, non-pregnant, Friesian cows in mid-lactation, were used to investigate the cell–extracellular

matrix (ECM) communication in mammary epithelial cell (MEC) survival during induced mammary involution and to

examine cell survival and apoptotic signalling events. Cows were sacrificed and alveolar mammary tissue was obtained at 0,

6, 12, 18, 24, 36, 72 and 192 h (n =6 per group) after termination of milking. Tissue mRNA concentrations, measured

by quantitative real-time RT–PCR, of different integrins (h1, a6 and a5), down-stream signal transduction factors FAK and

14-3-3, and cell survival members BAG-1 and Bcl-xlong of the Bcl-2 family were decreased by 24 h compared to 6 h, with

no further decrease to 8 days (192 h). The pro-apoptotic member of the Bcl-2 family, aBax mRNA level was increased by

8 days. Apoptosis within the epithelial cell layer surrounding the alveolar lumen, measured by in situ end-labelling (ISEL),

was evident. There were a low number of ISEL nuclei, in lactating mammary tissue (6 h), which was increased by 72 h of

involution. By 8 days of induced involution, there was a dramatic increase in apoptotic products within the lumen,

accounting for the majority of total apoptotic cells, which was accompanied by neutrophil infiltration. In conclusion, cell–

ECM communication becomes compromised in the bovine mammary gland 18–24 h after termination of milking, as

indicated by a decrease in the expression of integrins and cell survival factors. The different down-regulated integrins

implicate crosstalk between integrins and growth factor receptors may occur during involution of the bovine mammary

gland. Pro-apoptotic factors are up-regulated by 8 days of involution.

D 2005 Elsevier B.V. All rights reserved.

Keywords: Involution; Bovine; Mammary; Apoptosis; Integrins

0301-6226/$ - see front matter D 2005 Elsevier B.V. All rights reserved.

doi:10.1016/j.livprodsci.2005.10.016

* Corresponding author. Tel.: +64 7 838 5196; fax: +64 7 838

5628.

E-mail address: kuljeet.singh@agresearch.co.nz (K. Singh).

1. Introduction

In dairy animals, the decline in milk yield,

following either peak lactation or termination of

nce 98 (2005) 67–78

K. Singh et al. / Livestock Production Science 98 (2005) 67–7868

milking, is associated with MEC loss by apoptosis

(Knight and Peaker, 1984; Molenaar et al., 1996;

Wilde et al., 1997; Li et al., 1999; Capuco et al.,

2001). The molecular mechanisms regulating bovine

mammary involution and apoptosis are not well

understood and further studies may provide insights

that lead to extended or increased persistency of

lactation. At cessation of milking, the udder becomes

milk-engorged (Holst et al., 1987; Hurley, 1989) and

we have recently shown that this is accompanied by

wide spread changes in gene expression in the

involuting mammary gland of cows compared to

lactating glands at mid-lactation (Davis et al., 2003;

Singh et al., 2004). Microarray analysis showed that

many of the genes that were differentially expressed

at 36 h post-milking were involved in cellular

processes such as cell death (apoptosis) and growth

and/or maintenance (Davis et al., 2003; Singh et al.,

2004). The present study has focussed on molecular

mechanisms involved in these processes, in particular

MEC maintenance via cell survival mechanisms. In

the bovine, there is evidence that MEC apoptosis

occurs during involution in late lactation (Molenaar et

al., 1996; Wilde et al., 1997; Capuco et al., 2001). In

rodents, it is well established that weaning or teat

sealing causes engorgement of the alveoli within a

few hours. This is associated with changes in gene

expression and removal of MECs via apoptosis, as

the glands return to their pre-lactation state (Strange

et al., 1992; Walker et al., 1989; Feng et al., 1995;

Marti et al., 1997, 1999; Clarkson et al., 2003; Stein

et al., 2003). Involution is triggered by local,

mammary-derived signals and several mechanisms

have been postulated (Li et al., 1997; Green and

Streuli, 2004).

Maintaining the three-dimensional structure of

epithelia is crucial to biological function and one

possible trigger of involution is the change in cell

shape as the gland becomes engorged with milk and

stretching of the MECs occurs. Two systems may be

affected by the altered cell shape. Firstly, tight

junctions become leaky (Stelwagen et al., 1994,

1995, 1997) and pro-apoptotic factors may diffuse

from the apical to the basolateral side of the MECs

to induce involution and apoptosis. The second

system possibly influenced by cell shape, which we

have focussed on in this study, is the interaction of

the cell with ECM, through focal adhesion com-

plexes. In rodents, adhesion of luminal MECs to the

ECM is essential for cell survival (Boudreau et al.,

1995; Pullan et al., 1996; Farrelly et al., 1999;

Streuli and Gilmore, 1999). Research in vitro

demonstrates that MECs require a basement mem-

brane rich in laminin for survival and these survival

signals are mediated via integrins, a family of

heterodimeric transmembrane glycoproteins (Hynes,

1992; Boudreau et al., 1995; Pullan et al., 1996).

The integrins are composed of at least 16 a- and 8 h-integrin subtypes, which form heterodimers to

produce more than 20 different receptors, and are

linked to the cytoskeleton and mediate signals

through unique cytoplasmic domains (Hynes, 1992;

Clark and Brugge, 1995; Giancotti and Ruoslahti,

1999). In the mammary gland, the most abundant

integrin receptors which are specific for laminin and

which have been implicated as having a role in MEC

survival are a6h4 and those associated with the h1subunit such as a6h1 (Wewer et al., 1997; Faraldo et

al., 1998; Farrelly et al., 1999; Muschler et al., 1999;

Weaver et al., 2002). The a6h1 receptor interacts

only with fibronectin (Ruoslahti, 1991) and earlier in

vivo studies in rodents demonstrate that this integrin

receptor may play a role in involution (Huang and

Ip, 2001).

The mechanism of survival signalling via the

integrins is unclear. Recently we have demonstrated

in vivo a critical role of h1 integrin via focal adhesion

kinase (FAK), which interacts directly with integrins,

and the down-stream signal transduction pathway via

protein kinase B/Akt, during involution in the rodent

mammary gland (McMahon et al., 2004). The

molecular mechanisms linking MEC adhesion and

the survival signals with the intrinsic apoptotic

machinery are still unclear. This may result from

activation of caspases via regulation of anti- and pro-

apoptotic Bcl-2 family members (Marti et al., 1999;

Schorr et al., 1999a,b; Gilmore et al., 2000).

We have previously demonstrated the importance

of the junctional complex in maintenance of caprine

and bovine mammary function (Stelwagen et al.,

1995, 1997); in the present study, we have focussed

on cell–ECM communication. The aim of this study

was to investigate the molecular mechanisms that may

be involved in cell–matrix interactions during involu-

tion of the bovine mammary gland and to examine

resultant apoptotic signalling events.

K. Singh et al. / Livestock Production Science 98 (2005) 67–78 69

2. Materials and methods

2.1. Animals

Involution of the bovine mammary gland was

induced by abrupt termination of milking in 48 non-

pregnant Friesian dairy cows at mid-lactation (average

days in milk, 92F3.0). The cows were in their first

lactation and exclusively pasture-fed. Average daily

milk yield was 14.3F0.3 kg/cow. Somatic cell count

at the start of the experiment was 159F20�103 cells/

ml. Alveolar mammary tissue was obtained following

slaughter at 0, 6, 12, 18, 24, 36, 72 and 192 h (n =6

per group) after the last milking. Animals were killed

by electrical stunning and exsanguination. Samples

(approximately 30 g) of secretory alveolar mammary

tissue were obtained from the middle of the upper

one-third of the gland of a rear quarter and snap-

frozen in liquid nitrogen for subsequent RNA

extraction. Approximately 1.5 cm thick samples of

alveolar tissue (10 g) were also obtained for histolog-

ical analysis and in situ end-labelling (ISEL). Animal

experimentation was conducted in compliance with

the rules and guidelines of the local animal ethics

committee.

2.2. Quantitative real-time reverse transcription–

polymerase chain reaction analysis (RT–PCR)

Total RNA was extracted from ground tissue using

TRIzol (Invitrogen, Carlsbad, CA, USA) and 1 Agwas treated with 1 U DNase I (Invitrogen). Samples

were further purified through RNeasy gel columns

Table 1

Primers, primer concentrations, product size and annealing temperatures u

bovine mammary by quantitative real-time RT–PCR analysis using Syber

Gene name Forward Reverse

5VY3V 5VY3V

h1 integrin cag atg agg tga aca gcg aa atg cag gaa gtg gta ccc

a6 integrin gtt gtc gtc tcc aca tcc ct cac tct gga ggc tga aaa

a5 integrin aag gct cag atc ttg ctg ga gca gac gac tct ggt tca

Fak ctg ggg cca tgg agc gag ta tct ggt ggg tgg gca agt

14-3-3 agt taa ggg cca gac cca gt aga cgg aag gtg ctg aga

BAG-1 atg gtt gcc ggg tta tgt ta gga agg cct gaa tcc ttt t

Bcl-xlong gcg tag aca agg aga tgc ag gtt cca caa aag tgt ccc a

aBax cga gtg gcg gct gaa atg tt gca gcc gct ctc gaa gga

hactin cgc acc act ggc att gtc at ttc tcc ttg atg tca cgc ac

(Qiagen Sciences, MD, USA) and converted to

cDNA using a SuperScript II First-Strand Synthesis

system as described by the manufacturer (Invitrogen).

The cDNA products were diluted 10-fold and

samples (1 Al) were assayed in duplicate, by real-

time quantitative RT–PCR using an ABI PRISM

7700 or 7900 Sequence Detection System (PE

Applied Biosystems, Foster City, CA, USA). Detec-

tion of the product was by SYBER Green I (Morrison

et al., 1998), using the Universal PCR Master Mix

(PE Applied Biosystems). For each assay, two control

reactions were included; a reverse transcriptase-

negative control, and omission of the template (no

template control). Any amplification occurring in

these control reactions would indicate the presence of

non-RNA template. PCR primer sequences for

detection of Bcl-xlong were designed using GCG

Wisconsin Package, version 10.3 (Accelys Inc., San

Diego, CA, USA) or for FAK and aBax using

windows 32 PrimerSelect version 3.10, DNASTAR

Inc. For h1, a6 and a5 integrins, BAG-1 and 14-3-3,

primer sequences were from Coussens and Nobis

(2002), which were generated for bovine genes and

ortholog-selected bovine EST sequences. All primer

sequences and conditions for amplification are listed

in Table 1. The thermal cycling programs were 95 8Cfor 10 min followed by 40 cycles of 95 8C for 15 sec,

56–60 8C for 30 sec and 72 8C for 30 sec. hactin(primer sequences were kindly donated by Dr. R.

Lee, AgResearch, Hamilton, New Zealand) was used

as an internal control. Dissociation curve analysis

confirmed a single product. Products were amplified

by PCR and verified by sequencing (Waikato DNA

sed for investigation of gene expression in lactating and involuting

Green chemistry

Product

size (bp)

Annealing

temperature (8C)Primer

concentration (nM)

ag 289 56 100

gg 331 58 100

ca 274 56 300

tca ta 265 60 100

aa 318 56 100

c 324 56 100

g 119 60 100

agt 165 60 300

207 56–60 100

K. Singh et al. / Livestock Production Science 98 (2005) 67–7870

Sequencing Facility, Hamilton, New Zealand). The

threshold cycle (CT) number for each gene generated

by real-time RT–PCR was used to quantify the

relative abundance of each gene using the relative

standard curve method (PE Applied Biosystems,

Sequence Detection System, Chemistry Guide,

2003). Values for each gene were normalised to

hactin and then log10-transformed. Results were

expressed as the back-transformed mean fold-change

relative to the 6 h mean. The 6 h time point was used

in preference to the 0 h time point because 0

h represents tissue taken after a 12-h milking interval

(i.e. the regular milking interval immediately prior to

the start of the experiment was 12 h), whereas the 6-

h sample was the sample taken after the shortest

milking interval.

2.3. In situ end-labelling (ISEL) and histological

analysis

Tissue samples for ISEL were fixed in 4%

phosphate-buffered paraformaldehyde for 1 day.

Slices (~25�30�2 mm3) were then cut from the

fixed material and processed automatically through

an increasing series of graded alcohols (70%, 80%,

95%), then toluene, and finally embedded in paramat

wax (BDH laboratory supplies, Dorset, England).

Serial sections (8 Am) of each sample were cut and

mounted onto polysine glass slides (BioLab Scien-

tific, New Zealand). Every sample was examined

histologically by staining with haematoxylin and

eosin; and for subsequent ISEL for each of the early

time points, 0, 6, 18, and 24 h engorgement, 3 cows

were examined and for the later time points, 36, 72

and 192 h, 6 cows were examined. ISEL was

performed using a modified method of Ansari et

al. (1993). The sections were de-waxed, treated with

10 Ag/ml proteinase K (Invitrogen) and dried as

previously described (Molenaar et al., 1991). ISEL

was performed directly on the slide in a 50 Al volume

per section using the Klenow fragment of a DNA

polymerase to incorporate digoxigenin-11-2V-deoxy-uridine-5V-triphosphate (alkali stable) (DIG-11-

dUTP) (Roche Applied Science, Mannheim, Ger-

many), into fragmented or damaged DNA, a

characteristic of apoptosis. For each reaction 0.2

mM dGTP, dCTP and dATP (PCR grade, Roche

Applied Science), 20 Ag/ml bovine serum albumin

(Roche Applied Science), 1 mM DTT (Invitrogen)

and 1 AM DIG-11-dUTP in a final concentration of

1� React 2 Buffer comprising 100 mM Tris–HCl

pH 7.6, 10 mM MgCl2, 150 mM NaCl (Invitrogen).

One unit of Klenow enzyme (Roche Applied

Science) was added to each reaction and omitted

from the control. The reactions were allowed to

proceed for 2 h at 37 8C in a humidified chamber

then washed in several changes of water, before

blocking with block buffer (buffer 1; 100 mM Tris–

HCl pH 7.5 and 150 mM NaCl, containing 2%

blocking solution (Roche Applied Science)), for to 1

h at 37 8C. The slides were washed several times

with buffer 1 then incubated with anti-digoxigenin-

AP-Fab fragments (Roche Applied Science) diluted

500-fold in block buffer for 1 h at 37 8C. The slides

were washed with several changes of buffer 1, then

buffer 3 (100 mM Tris–HCl pH 9.5, 100 mM NaCl,

50 mM MgCl2) before the addition of the substrates:

450 Ag/ml 4-nitroblue tetrazolium chloride (NBT)

(Roche Applied Science) and 175 Ag/ml 5-bromo-4-

chloro-3-indolyl-phosphate, 4-toluidine salt (BCIP)

(Roche Applied Science) in buffer 4 (100 mM Tris–

HCl pH 9.5, 100 mM NaCl containing 1 mM

levamisole (Sigma, St. Louis, MO, USA)). The

alkaline phosphatase reaction was allowed to pro-

ceed for 1.5 h and then stopped by rinsing the

sections with water followed by dehydration in 70%

ethanol (2 min), 95% ethanol (30 min), 70% ethanol

(2 min), water (2 min) to darken the ISEL signal

from brown to blue/black. Sections were counter-

stained with nuclear fast red for 2–4 min, dehydrated

and coverslipped using DPX (BDH Laboratory

Supplies, Poole, England).

Quantitative analysis of cells with fragmented

DNA in the histological sections was carried out

using light microscopy (Olympus BH-2). Ten ran-

domly selected fields (100� magnifications) were

photographed per sample and the number of ISEL

nuclei and alveoli counted using the mark and count

analysis tool in ImageJ (US National Institute of

Health, http://rsb.info.nih.gov/nih-image). DIG–ISEL

nuclei were identified as either located within the

secretory epithelial layer or within the lumen of

mammary alveoli. The value 1 was added to each

count of ISEL nuclei per field followed by a

correction for the number of alveoli per field. Data

were log10-transformed.

0.1

1.0

10.0

-20 0 20 40 60 80 100 120 140 160 180 200

β1α6

α5

** **

0.1

1.0

10.0

-20 0 20 40 60 80 100 120 140 160 180 200

hours post milking

Bag-1

Bclxlong

α BA X

****

**

**

*

*

0.1

1.0

10.0

-20 0 20 40 60 80 100 120 140 160 180 200

gen

e ex

pre

ssio

n le

vels

rel

ativ

e to

6 h

po

st m

ilkin

g

(fo

ld d

iffe

ren

ce)

FAK

14.3.3

*

*

**

*

*

*

*

A

B

C

Fig. 1. Changes in mRNA levels of (A) integrins (h1, a6 and a5), (B) FAK and 14-3-3, and (C) Bag-1, Bcl-xlong and pro-apoptotic aBax with

time in mammary alveolar tissue of lactating cows at mid-lactation following the last milking (n =6 per time point). Data are expressed as

meanFS.E.M. and P values (*P b0.05, **P b0.01) are relative to 6 h time point for respective genes. For (A) P values represent significance at

the different time points for all genes, for (B) and (C) P values are shown for each gene.

K. Singh et al. / Livestock Production Science 98 (2005) 67–78 71

K. Singh et al. / Livestock Production Science 98 (2005) 67–7872

2.4. Data and statistical analyses

For real-time RT–PCR analysis, the differences

between means were analysed by ANOVA in GenStat

7.0 (Lawes Agricultural Trust, 2003) and data were

expressed as meanF the standard error of the mean

(S.E.M.). For in situ end-labelling analysis, the log10-

transformed data was analysed by ANOVA and data

were expressed as the back transformed mean

(1+ISEL nuclei) per alveolus with the standard error

of the difference (S.E.D.). The least significant

differences identify the means significantly different

from each other (*P b0.05, **P b0.01, ***P b0.001).

3. Results

3.1. Gene expression

The mRNA levels of the integrins h1, a6 and a5

were decreased by 24 h after the last milking relative

to 6 h by 1.8-, 3.7- and 2.1-fold, respectively (Fig. 1).

There was no further decrease after 24 h. The mRNA

levels of FAK, 14-3-3, BAG-1 and Bcl-xlong were also

decreased by 24 h after last milking relative to 6 h by

1.6-, 2.2-, 2.2- and 2-fold, respectively, with no further

decrease after 24 h (Fig. 1). By 8 days, the level of pro-

apoptotic aBax mRNA was increased by 2.5 fold

compared to 6 h following the last milking (Fig. 1).

3.2. Histological analysis

Fig. 2 shows representative samples of the mor-

phology of the lactating and involuting gland at

different times following ISEL and staining with

nuclear fast red. Initially, every cow (n =6) at each

time point (0, 6, 12, 18, 24, 36, 72, 192 h post-milking)

was examined by haematoxylin and eosin staining of

the paraffin-embedded tissue (data not shown). There

was very little difference in the morphology of the

tissue between cows at the early time points, but

considerable variation in the degree of involution was

Fig. 2. Qualitative analysis of in situ end-labelling and morphological chang

0, 6, 18, 24, 36, 72 and 192 h after the last milking. DIG–ISEL nuclei are lab

of epithelial ISEL nuclei, representative examples of luminal ISEL

leucocytes (neutrophils).Magnification=200�, scale bar=100 AM. (For inte

referred to the web version of this article.)

evident between cows by 36 h and at 72 h and 8 days.

Therefore, in this study, we report qualitative and

quantitative analysis of apoptosis, by ISEL for n =3

cows at the early time points, 0, 6, 18 and 24 h post-

milking excluding 12 h, and n =6 for the later time

points, 36, 72 and 192 h. The 6 h time point was the

most representative of actively lactating mammary

tissue, characterised by cuboidal shaped MECs at-

tached to the basement membrane of the ECM

surrounding the alveolar lumen, large areas of uniform

and moderate sized alveoli, minimal stromal areas and

minimal vesicles and fat droplets within MECs (Fig.

2). At the early time points, the accumulation of milk

occurring in the alveolar lumen over time resulted in

larger and engorged alveolar lumen and the MECs

being stretched and flattened in shape (Fig. 2). By 24

h, although there were a few localised areas of

involution, characterised by collapsed or smaller

alveoli with a ruffled luminal surface and occasional

small vesicles, most alveoli were large, open and

engorged (Fig. 2). By 36 h, there were clear differ-

ences in the degree of involution between the animals

(Fig. 2). There was also heterogeneity in tissue

structure within samples. In some areas, whole lobules

of alveoli were involuted, while in other areas alveoli

retained the appearance of lactation. By 72 h and

8 days, the highly involuted samples were consistent

with a non-lactating phenotype, characterised by

smaller collapsed alveoli, moderate to high amounts

of large vesicles in the cells, more thickened areas of

stromal tissue between alveoli and large broad bands

of supportive connective tissue. Large ducts were

more evident and a low number of leukocytes were

present in areas with large vesicles within the cells

(Fig. 2). There were also samples that showed a

moderate degree of involution and some where very

little involution had occurred (data not shown).

3.3. In situ end-labelling analysis

The samples from 0 to 36 h had very low levels of

positive ISEL nuclei, and hence, low apoptosis (Figs.

es in paraffin-embedded tissue sections of bovine mammary gland, at

elled blue/black and indicate apoptosis. representative examples

nuclei (single or regions), Y representative examples of regions of

rpretation of the references to colour in this figure legend, the reader is

0 h 6 h

18 h 24 h

36 h 72 h

192 h 192 h negative control

K. Singh et al. / Livestock Production Science 98 (2005) 67–78 73

hours post milking

0 20 40 60 80 100 120 140 160 180 200

(1 +

ISE

L n

ucl

ei)

per

alv

eolu

s (l

og

-sca

le)

0.01

0.1

TotalEpithelial Luminal

SED withinTotal

SED withinType

**

**

******

**

min rep. SED between Type

max rep. SED between Type

***

****

Fig. 3. Quantitative analysis of in situ end-labelled nuclei in bovine mammary glands, at 0, 6, 18, 24 h (n =3 per time point) and at 36, 72 and

192 h (n =6 per time point) following the last milking. Data are expressed as the back transformed mean number of total, epithelial and luminal

1+ISEL nuclei per alveolus, with the max–min replicates (rep.) (n =3 vs. n =6) S.E.D. within total and S.E.D. within type (epithelial or luminal)

for comparing different time points to the 6 h time point. The between-type S.E.D.s for comparing type at each time point (i.e. epithelial vs.

luminal) are shown for time points with either the min rep. (n =3) or the max rep. (n =6). (*P b0.05, **P b0.01, ***P b0.001).

K. Singh et al. / Livestock Production Science 98 (2005) 67–7874

2 and 3). By 72 h, the total number of ISEL nuclei

was increased per 100� magnification field (data not

shown) and per alveolus (Figs. 2 and 3), compared

with 6 h post-milking, and also at 8 days per 100�magnification field (data not shown) and per alveolus

(Figs. 2 and 3). The number of ISEL nuclei located

within the secretory epithelial cell layer surrounding

the alveoli followed a similar pattern with significant-

ly more per alveolus at 72 h and at 8 days, compared

with 6 h post-milking (Figs. 2 and 3).

There were more ISEL nuclei per alveolus within

the epithelial cell layer than the alveolar lumens at 36

h (P b0.05) and 72 h (P b0.001). However, by 8 days

post-milking, a dramatically increased number of

ISEL nuclei located within the lumen accounted for

the majority of ISEl nuclei detected at that time point

(Figs. 2 and 3). Apoptotic nuclei were not labelled in

the negative control (Fig. 2).

4. Discussion

This study examined changes in gene expression

and apoptosis in the bovine mammary gland over

8 days of involution. In rodents, it is well established

that induced involution results in wide spread changes

in gene expression and loss of MECs via apoptosis

(Strange et al., 1992; Walker et al., 1989; Feng et al.,

1995; Marti et al., 1997, 1999; Clarkson et al., 2003;

Stein et al., 2003). There is very information about the

mechanisms that may occur in dairy cows when

involution is induced by termination of milking. The

present study is an in vivo time-course of mammary

involution and apoptosis in cows, to identify molec-

ular mechanisms that may be critical in MEC

maintenance or survival. By 18 h, following the

termination of milking, the alveoli became milk-

engorged and the expression of different integrin

genes (h1, a6 and a5) had significantly declined by

24 h. MEC apoptosis was increased by 72 h following

the termination of milking. The results suggest that

changes in expression of integrin and integrin signal

transduction factors are an early event in MEC

survival and suggest a role for integrins in apoptosis

during involution. Together, the down-regulation of

integrins and the significantly increased apoptosis of

milk-secreting cells support the hypothesis that there

is a rapid decrease in communication between MECs

and the ECM during involution (McMahon et al.,

2004). The time-frame in which the interaction

K. Singh et al. / Livestock Production Science 98 (2005) 67–78 75

between MECs and the ECM decreases is very similar

to that for the loss of cell–cell contact between

adjacent MECs, which has been reported to occur

also following 18 h of milk accumulation (Stelwagen

et al., 1994, 1995, 1997).

Studies in vitro show that survival of MECs require

that they adhere to ECM proteins of the basement

membrane. Perturbation of integrin function in vitro

using anti-h1 or anti-a6 integrin antibodies results in

enhanced apoptosis in MECs (Boudreau et al., 1995;

Pullan et al., 1996; Farrelly et al., 1999). In

accordance, recent in vivo studies in our laboratory

in rodents suggest a role for h1 integrin in the cell

survival pathway (McMahon et al., 2004). Earlier in

vivo studies in mice expressing a dominant-negative

form of h1 integrin in their mammary glands show

that apoptosis is increased (Faraldo et al., 1998) and

a6 integrin has been shown to be associated with anti-

apoptotic signals, promoting the survival of metastatic

human breast carcinoma cells (Wewer et al., 1997). In

agreement, results from the present study suggest that

h1 and a6 integrins changed in vivo in secretory

MECs of lactating cows. In addition, a5 integrin has

been implicated in bovine mammary cell survival.

Most integrin heterodimers recognise several ECM

proteins; however, the a5h1 receptor interacts only

with fibronectin (Ruoslahti, 1991). Earlier in vivo

studies in rodents demonstrate a5h1 integrin may

play a role in involution (Huang and Ip, 2001),

although primary cultures of MECs and mammary

cell lines require laminin-rich basement membrane for

survival and undergo apoptosis on other types of

ECM such as collagen I or fibronectin (Boudreau et

al., 1995; Pullan et al., 1996). The a6h1 integrin

mediated cell survival in MECs occurs via the

survival PI-3-kinase/Akt pathway (Farrelly et al.,

1999). The a5h1 integrin has also been shown to

mediate survival via the PI-3-kinase pathway, in

intestinal epithelial cells, possibly via activation of

EGFR signalling to the PI-3-K/PKB survival pathway

(Lee and Juliano, 2000, 2002). This suggests that

crosstalk between integrins and growth factor recep-

tors may be occurring during involution of bovine

mammary tissue.

Previous in vitro studies suggest that FAK, which

is a nonreceptor protein tyrosine kinase and is

phosphorylated in response to adhesion to the ECM,

is involved in integrin-mediated signalling (Schaller et

al., 1992; Hanks et al., 1992). Inhibition of FAK

function by anti-FAK antibodies or by expression of a

dominant negative FAK results in apoptosis in serum-

deprived fibroblasts and cancer cell lines (Hungerford

et al., 1996; Ilic et al., 1998), whereas expression of

constitutively activated FAK leads to anchorage-

independent survival of MDCK cells (Frisch et al.,

1996). Our study demonstrated that FAK may play a

role in integrin signalling in vivo during bovine

mammary involution, and it may be linked to the

intrinsic intracellular apoptotic machinery involving

anti- and pro-apoptotic members of the Bcl-2 family.

The levels of gene expression of FAK, 14-3-3 and

anti-apoptotic members of the Bcl-2 family, Bcl-xlongand BAG-1 were all decreased, by 24 h. Although

pro-apoptotic aBax gene expression was increased by

8 days, the increased apoptosis of MEC observed by

72 h, was not accompanied by an increase in aBax

gene expression. Studies in vitro suggest that Bax

localisation, rather than transcriptional regulation,

plays a role in apoptosis (Gilmore et al., 2000).

Integrin signalling through FAK is essential for

retaining Bax in the cytoplasm, however, detachment

of MECs from the ECM induces a rapid translocation

of Bax to the mitochondria, which was reversible and

occurs before caspase activation and apoptosis (Gil-

more et al., 2000), thus explaining the lack of change

in expression. FAK may mediate survival via the PI-3-

K/PKB survival pathway. A downstream target for

PKB/Akt is pro-apoptotic Bad, a member of the Bcl-2

family of apoptosis regulators. Phosphorylation of

Bad via PI 3-K and PKB axis, allows 14-3-3 protein

to bind and sequester it within the cytoplasm (Zha et

al., 1996; Parrizas et al., 1997; Songyang et al., 1997).

BAG-1 binds to anti-apoptotic Bcl-2 (Takayama et al.,

1995) and may inhibit apoptosis either alone, or in co-

operation with Bcl-2, as a heterodimer. Previous

studies have shown that mice with a conditional

deletion of the Bcl-x gene have accelerated apoptosis

during involution (Walton et al., 2001). In agreement

with the present study, studies in vivo in mice have

shown pro-apoptotic members of the Bcl-2 family

increase and anti-apoptotic members decrease during

involution, which is accompanied by an induction of

apoptosis (Heermeier et al., 1996; Li et al., 1997;

Merlo et al., 1997). Previously in goats, Bax protein

expression is increased in late lactation (Wareski et al.,

2001). While we have demonstrated changes in gene

K. Singh et al. / Livestock Production Science 98 (2005) 67–7876

expression are associated with involution we need to

establish the extent to which the invasion of leuco-

cytes during the first few days of mammary involution

impacts upon the expression levels observed.

The results from the present study support our

earlier research in rats, identifying a potential role for

signal transduction via FAK in MEC survival

(McMahon et al., 2004). Induced involution was

associated with decreased h1 integrin and cytochrome

C protein levels from mitochondria as early as 6 h in

rats (McMahon et al., 2004). In the present study, our

results suggest that factors influencing apoptosis and

mammary cell survival during involution also change

rapidly in lactating cows. However, in comparison to

rodents, the number of apoptotic products in involut-

ing tissues was only slightly increased, and the

massive cell death that occurs in rodents at involution

did not occur, supporting studies showing that

involution in ruminants occurs to a lesser extent, only

replacing senescent or damaged cells (Capuco et al.,

2001). This may help to explain that milk production

in cows can be reinitiated in quarters unmilked for 12

days, and with the yield recovering almost to pre-

treatment values (Hamann and Reichmuth, 1990).

Longer interruption of milking, when applied to all

quarters, results in only partial recovery of milk yield

(Noble and Hurley, 1997). By contrast, mice can

rescue milk synthesis up to 48 h of milk stasis, but

after 72 h, recovery is limited (Sorensen and Knight,

1997).

The present study describes an in vivo examination

of the time-course of apoptosis during involution.

Previously, increased apoptosis has been demonstrat-

ed during gradual involution occurring during the

latter part of lactation in goats and cows (Knight and

Peaker, 1984; Wilde et al., 1997; Li et al., 1999;

Capuco et al., 2001). When cells die they are

phagocytosed by leucocytes or neighbouring cells

(Martin et al., 1994). In the present study, there was a

low level of MEC apoptosis in the lactating mammary

tissue of cows (6 h post-milking) at mid-lactation,

which increased by 72 h in the epithelial layer

surrounding the alveolar lumen, following the termi-

nation of milking. However, by 8 days of involution,

the majority of apoptotic products were detected in the

alveolar lumen, which was associated with an increase

in leucocytes (neutrophils) in alveolar lumen. We

were unable to distinguish if the positively labelled

nuclei (apoptotic products) were from the sloughed

MECs from the layer surrounding the alveolar lumen

or from the invading leucocytes. Similar results have

been demonstrated previously in preliminary studies

in sheep and cows (Molenaar et al., 1996).

This study showed that 24 h after milking, a

decrease in expression of genes that contribute to

ECM communication occurs in bovine mammary

glands that are involuting undergoing apoptosis.

Apoptosis was shown to be much more gradual and

late occurring when compared with that of the rodent

mammary gland.

Acknowledgements

The authors gratefully acknowledge the contribu-

tions of Harold Henderson for statistical support.

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