acta histochemica 113 (2011) 137–149

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acta histochemica

0065-12

doi:10.1

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E-m

journal homepage: www.elsevier.de/acthis

Cellular localization of aquaporins along the secretory pathway of thelactating bovine mammary gland: An immunohistochemical study

Ali Mobasheri a,�, Bryony Heather Kendall b, Judith Elizabeth Joan Maxwell b, Ami Veronica Sawran b,Alexander James German b, David Marples c, Martin Richard Luck d, Melissa Dawn Royalb

a Division of Veterinary Medicine, School of Veterinary Medicine and Science, Faculty of Medicine and Health Sciences, University of Nottingham, Sutton Bonington Campus,

Leicestershire LE12 5RD, United Kingdomb School of Veterinary Science, Faculty of Health and Life Sciences, University of Liverpool, Liverpool L69 3BX, United Kingdomc Institute of Membrane and Systems Biology, University of Leeds, Leeds LS2 9JT, United Kingdomd Division of Animal Sciences, School of Biosciences, University of Nottingham, Sutton Bonington Campus, Leicestershire LE12 5RD, United Kingdom

a r t i c l e i n f o

Article history:

Received 16 April 2009

Received in revised form

14 September 2009

Accepted 16 September 2009

Keywords:

Mammary gland

Bovine

Immunohistochemistry

Aquaporin

Water channel

Milk production

81/$ - see front matter & 2009 Elsevier Gmb

016/j.acthis.2009.09.005

esponding author.

ail address: ali.mobasheri@nottingham.ac.uk

a b s t r a c t

In this study we examined the cellular localization of aquaporins (AQPs) along the secretory pathway of

actively lactating bovine mammary glands using immunohistochemistry. Mammary tissues examined

included secretory ducts and acini, gland cisterns, teats, stromal and adipose tissues. Aquaporin 1

(AQP1) was localized in capillary endothelia throughout the mammary gland in addition to

myoepithelial cells underlying teat duct epithelia. AQP2 and AQP6 were not detected and AQP9 was

found only in leukocytes. AQP3 and AQP4 were observed in selected epithelial cells in the teat, cistern

and secretory tubuloalveoli. AQP5 immunopositivity was prominent in the cistern. AQP3 and AQP7 were

found in smooth muscle bundles in the teat, secretory epithelial cells and duct epithelial cells. These

immunohistochemical findings support a functional role for aquaporins in the transport of water and

small solutes across endothelial and epithelial barriers in the mammary gland and in the production

and secretion of milk.

& 2009 Elsevier GmbH. All rights reserved.

Introduction

Aquaporins are a family of membrane bound proteins that areextensively distributed in microorganisms (Calamita, 2000),animals (Agre et al., 2002; Agre and Kozono, 2003; King et al.,2004) and plants (Chrispeels and Maurel, 1994; Johansson et al.,2000; Schaffner, 1998). Aquaporins play fundamental roles inwater and small solute transport across epithelial and endothelialbarriers (Verkman, 2002; Verkman and Mitra, 2000). To date, 13members of the aquaporin gene family have been identified inhumans: AQP0–AQP12 (Castle, 2005). Animal genome projectshave also confirmed the presence of multiple aquaporin genesencoding distinct protein isoforms. The proteins encoded byaquaporin genes have been classified into two major groups basedon their substrate permeabilities: (1) some aquaporins arepermeated by water and include AQP1, AQP2, AQP4, AQP5 andAQP8 (Agre et al., 2002); (2) others, the aquaglyceroporins exhibitpermeability to water and small neutral solutes such as glyceroland urea and include AQP3, AQP7, AQP9 and AQP10 (Agre et al.,2002; Hibuse et al., 2006). In mammals, aquaporins are located atstrategic membrane sites in endothelia and a variety of epithelia,

H. All rights reserved.

(A. Mobasheri).

most of which have well-defined physiological functions in fluidabsorption or secretion (Brown et al., 1995).

The mammalian mammary gland is a specialized, enlargedsudoriferous or sweat (apocrine) gland that produces and secretesmilk. Milk consists of simple sugars, lipids, proteins, vitamins andminerals dissolved in water (Shennan and Peaker, 2000). Wateraccounts for up to 88% per unit volume of milk (although thispercentage will vary depending on the species under investigationand the physiological state of the lactating animal (McManamanand Neville, 2003). Current understanding is that water is secretedacross the mammary epithelium in a transcellular manner, inresponse to an osmotic gradient produced largely by the lactosecontent of the milk (Shennan and Peaker, 2000; McManaman andNeville, 2003).

The yield and quality (in terms of constituent yield i.e. proteinand butterfat) of milk are important criteria for the dairy industryand exact requirements will vary depending on the milk buyerand the end product, and obviously the water content has a largerole to play here. However, despite the economic importance ofmilk yield and its constituent quality, very little is known aboutthe physiological mechanisms responsible for water transport inthe bovine mammary gland. Nothing is known about theexpression of aquaporins in the mammary glands of economicallyimportant milk producing animals. Recent immunohistochemicalstudies of aquaporins in the rat and mouse mammary gland have

A. Mobasheri et al. / acta histochemica 113 (2011) 137–149138

confirmed the presence of AQP1 and AQP3 proteins in bothspecies (Matsuzaki et al., 2005). Our own work using humantissues has provided immunohistochemical evidence for AQP1and AQP3 in human mammary glands (Mobasheri and Marples,2004; Mobasheri et al., 2005b). AQP1 was located in both theapical and basolateral membranes of capillary endothelia in therodent mammary gland (Matsuzaki et al., 2005). AQP3 waspresent in basolateral membranes of secretory epithelial cellsand intralobular and interlobular duct epithelial cells in rat andmouse mammary tissue (Matsuzaki et al., 2005). Although theexpression of mRNA transcripts encoding AQP4, AQP5, AQP7 andAQP9 were demonstrated in rodent mammary by RT-PCR, thepresence of the corresponding proteins was not thoroughlyinvestigated (Matsuzaki et al., 2005).

The objectives of this study were as follows: (1) to determine ifAQP1 and AQP3 are present in the lactating bovine mammary gland;(2) to study the cellular localization of other aquaporin isoforms inthe lactating bovine mammary gland. Accordingly, we employedimmunohistochemistry to examine the cellular distribution ofaquaporins 1–7 and 9 in lactating bovine mammary glands usingisoform-specific antibodies raised against rat aquaporins.

Materials and methods

Chemicals: Unless otherwise stated, all chemicals were mole-cular biology grade and were purchased from Sigma/Aldrich(Poole, Dorset, UK).

Table 1Summary of the peptide sequences used for developing polyclonal antibodies to AQP5

AQP antibody Peptide sequence N

AQP5 NH2-(C)WEDHREERKKTIEL–COOH

AQP6A NH2-(C)LEPQKKESQTNSED–COOH

AQP6B NH2-(C)EPQKKESQTNSEDTE–COOH

AQP7A NH2-(C)MVQASGHRRSTRGS–COOH

AQP7B NH2-(C)AYEDHGITVLPKMG–COOH

AQP7C NH2-(C)APPLHESMALEHF–COOH

AQP9 NH2-(C)KAEPSENNLEKHEL–COOH

The amino terminus of the peptide begins with an N-terminal cysteine, which is essentia

used two peptides for AQP6 and three peptides for AQP7 to maximize the chances of pro

the NCBI accession number of corresponding protein sequences producing 100% identi

Antibodies: Affinity purified rabbit polyclonal antibodies werepreviously developed against rat AQP1 and AQP2 (Mobasheriet al., 2004b, 2005b). These polyclonal antibodies have beensuccessfully used in a variety of different applications on human(Mobasheri et al., 2005a), equine and rodent tissues (Floyd et al.,2007). The species cross-reactivity of some of these antibodies hasalso been demonstrated using tissues derived from severaldomestic animal species including pig, horse (Floyd et al., 2007;Mobasheri, 2006; Mobasheri et al., 2004b) and cow. Polyclonalantibodies were developed against rat AQP3–AQP7 and AQP9 inpartnership with Sigma-Genosys (Poole, Dorset, UK). Detailsrelating to the characterisation of our rabbit polyclonal antibodiesagainst rat AQP3 and AQP4 and their cross-reactivity to otherspecies have recently been published (Floyd et al., 2007). In thispaper, we describe our approach to developing polyclonalantibodies against AQP5–AQP7 and AQP9 using peptide sequencesderived from rat and human AQP proteins. The peptide sequencesused are provided in Table 1. All the sequences used were carefullyselected to ensure isoform specificity and high antigenicity.Furthermore, all peptide sequences were checked on the proteinblast database to ensure that they corresponded to thecorrect protein (http://www.ncbi.nlm.nih.gov/BLAST/). A 77-dayimmunization protocol was chosen which consisted of pre-immune serum collection, injection with 200mg peptideconjugated to keyhole limpet hemocyanin in complete Freund’sadjuvant on day one. This was followed by 5�100mg boosterinjections in incomplete Freund’s adjuvant on days 14, 28, 42, 56and 70. A monoclonal antibody raised against the a1 subunit of

, AQP6, AQP7 and AQP9 with corresponding NCBI accession numbers.

CBI accession number

NP_036911 – Rat AQP5 [Rattus norvegicus]

NP_033831 – Mouse AQP5 [Mus musculus]

NP_001642 – Human AQP5 [Homo sapiens]

CAD56689 – Equine AQP5 [Equus caballus]

XP_001110608 – PREDICTED: rhesus monkey AQP5 [Macaca mulatta]

XP_001157125 – PREDICTED: similar to AQP5 [Pan troglodytes].

NP_071517 – Rat AQP6 [Rattus norvegicus]

NP_780296 – Mouse AQP6 [Mus musculus]

XP_606158 – PREDICTED: similar to bovine AQP6 [Bos taurus]

NP_071517 – Rat AQP6 [Rattus norvegicus]

NP_780296 – Mouse AQP6 [Mus musculus]

XP_606158 – PREDICTED: similar to bovine AQP6 [Bos taurus]

NP_001161 – Human AQP7 [Homo sapiens]

XP_001100020 PREDICTED: similar to rhesus monkey AQP7 [Macaca mulatta]

Q4R691nn PREDICTED: similar to crab-eating macaque [Macaca fascicularis]

NP_001161 – Human AQP7 [Homo sapiens]

XP_001100020 – PREDICTED: similar to rhesus monkey AQP7 [Macaca mulatta]

NP_001069846 – Hypothetical protein LOC615498 [Bos Taurus]

XP_001498227 – PREDICTED: hypothetical protein [Equus caballus]

NP_001161 – Human AQP7 [Homo sapiens]

XP_001100020 – PREDICTED: similar to rhesus monkey AQP7 [Macaca mulatta]

XP_605822 – PREDICTED: hypothetical protein [Bos taurus]

NP_075249 – Rat AQP9 [Rattus norvegicus]

NP_071309 – Mouse AQP9 [Mus musculus]

NP_066190 – Human AQP9 [Homo sapiens]

l for conjugation to the carrier protein, in this case keyhole limpet hemocyanin. We

ducing good antibodies to these aquaporin isoforms. The right-hand column shows

ty or significant alignments with proteins of other mammalian species.

A. Mobasheri et al. / acta histochemica 113 (2011) 137–149 139

Na, K-ATPase (designated a6F), which exhibits a very broadcapacity for cross-reactivity to other species (i.e. avian,amphibian, mammalian) was used as a positive control in thebovine kidney, liver and mammary gland. This was done to makesure that the structural integrity of the mammary epithelium inour tissues was intact and that the Na, K-ATPase maintained itspolarized basolateral distribution in mammary epithelial cells.The a6F monoclonal (originally developed by D. Fambrough, JohnsHopkins University) was obtained as a hybridoma supernatantfrom the Developmental Studies Hybridoma Bank, under theauspices of the National Institute of Child Health andDevelopment and maintained by The University of Iowa,Department of Biological Sciences, Iowa City. This hybridomasupernatant was used undiluted for immunohistochemistry.

Animals and Tissue Processing: Bovine kidneys, livers andmammary glands (n=3 for each organ) were obtained fresh fromlactating animals (immediately after slaughter) from a localabattoir. Samples of kidney and liver were included as thesetissues are positive controls for several aquaporins. Positivecontrol tissues were fixed and processed as previously described

Fig. 1. Histology of the lactating bovine mammary gland. Panel A: section of deep mamm

Panel B: section of mammary cistern showing larger ducts and stromal connective tissue

running through the adipose tissue. Panel D: section of the teat showing the teat duct

cortical glomeruli (G) and proximal tubules in the renal cortex and peritubular capillari

panels: 200� . Bars represent 100mm.

(Floyd et al., 2007). Mammary samples included tissue from theteat, cistern and secretory portions of mammary glands of activelylactating cows. The tissues were transported to the laboratory onice within 1 h of harvesting and were carefully dissected beforefixing in 10% neutral buffered formalin for 24 h for immunohis-tochemical studies. Formalin fixation was not allowed to continuebeyond 24 h to ensure adequate tissue fixation and to preventdrastic reduction of antigen recognition which may occur forcertain proteins exposed to formalin. Sections were then cut (6mmthickness) using a microtome, and mounted on positively chargedglass microscope slides and stained with Papanicolaou’s haema-toxylin and eosin or immunolabelled for the binding of polyclonalAQP antibodies as described below.

Immunohistochemistry: Immunohistochemical labelling wasperformed on bovine tissues using a DakoCytomation EnVision+Dual Link System, Peroxidase (DAB+) kit, essentially as describedin several recent papers (Floyd et al., 2007; Mobasheri et al.,2004a, 2004b; Simpson et al., 2006; Trujillo et al., 2004). The kitused takes advantage of the superior sensitivity of a horseradishperoxidase-labeled polymer which is conjugated with secondary

ary gland showing active acini (tubuloalveoli) and ducts filled with milk droplets.

. Panel C: section of mammary adipose tissue showing adipocytes and microvessels

and surrounding stromal tissue. Panels E and F: sections of bovine kidney showing

es and collecting tubules in the renal medulla. Original magnifications of the main

Table 2Table summarizing the immunolocalisation of AQP proteins in the lactating bovine

mammary gland.

AQPProtein

Comments on immunolocalisation

AQP1 Present in capillary endothelia (microvessels) in the deep

mammary gland, cistern, teat, and adipose tissue

AQP1 was also present in myoepithelial cells underlying cistern

and teat duct epithelia

AQP2 Not detected in any portion of the bovine mammary gland

AQP3 Present in selected epithelial cells in teat, cistern and acini

(tubuloalveoli)

Also detected in teat smooth muscle bundles (data are not shown)

AQP4 Diffuse immunopositivity in selected epithelial cells in teat, cistern

and acini

Apically localized in selected cells in cistern and teat duct epithelia

Low immunopositivity in teat smooth muscle bundles

AQP5 Weak but prominent immunopositivity in acini. Prominent

immunopositivity in small cistern ducts

AQP6 No immunopositivity in any part of the bovine mammary gland

AQP7 Weak immunopositivity in epithelial cells in the teat and secretory

acini

Also present in adipocytes, teat ducts and smooth muscle bundles

within the mammary gland and the teat

AQP9 No immunopositivity in any epithelial, stromal or endothelial

structure in the bovine mammary gland. Only detected in

leukocytes within the mammary gland (data are not shown)

A. Mobasheri et al. / acta histochemica 113 (2011) 137–149140

antibodies. The labelled polymer does not contain avidin or biotinand, consequently, non-specific labelling resulting from endogen-ous avidin–biotin activity in tissues such as liver and kidney iseliminated. Briefly, slides were deparaffinised in xylene for 20 minto remove embedding media and washed in absolute ethanol for3 min. The slides were gradually rehydrated in a series of alcoholbaths (96%, 85% and 50%) and placed in distilled water for 5 min.Endogenous peroxidase activity was blocked for 1 h in a 97%methanol solution containing 3% hydrogen peroxide and 0.01%sodium azide. The slides were then incubated for 30 min at roomtemperature (RT) with 2% protease-free bovine serum albumin(Sigma–Aldrich) in phosphate buffered saline (PBS) supplementedwith 0.5% Tween-20 (PBS–Tween) to block non-specific antibodybinding. Slides were incubated overnight at 4 1C with primaryanti-AQP antibodies diluted 1:100–1:1000 (depending on prioroptimisation experiments) in PBS–Tween. The slides were thenwashed 3 times for 5 min each in PBS–Tween before incubationwith horseradish peroxidase-labeled polymer conjugated toaffinity purified goat anti-rabbit and goat anti-mouse immuno-globulins, prepared according to manufacturer’s instructions,for 30 min at RT. The sections were washed 3 times for 5 min inPBS–Tween before applying liquid DAB+ chromogen (DAKO; 3,30-diaminobenzidine solution, prepared according to manufacturer’sinstructions) for up to 10 min. The development of the brown-coloured reaction was stopped by rinsing in distilled water. Thelabelled slides were immersed for 5 min in a bath of Papanico-laou’s haematoxylin to counterstain cell nuclei. Finally the slideswere washed for 5 min in running water and dehydrated in aseries of graded ethanol baths before rinsing in 3 xylene baths andmounting in 1,3-diethyl-8-phenylxanthine (DPX, BDH Labora-tories, UK). Control experiments were performed by omittingprimary antibodies to aquaporins and Na, K-ATPase and byusing appropriate positive control tissues (i.e. bovine kidney andliver).

Data acquisition and analysis: The labelled slides were exam-ined using a Nikon Eclipse 80i microscope and images capturedusing a Nikon Digital Sight DS-5M camera connected to a PCrunning Eclipsenet imaging software (v. 1.20, Laboratory Imagingfor Nikon Instruments, Kingston-upon-Thames, UK).

Results

Histology of the actively lactating bovine mammary gland and kidney

Light microscopical evaluation of sections of actively lactatingbovine mammary glands revealed an abundance of activetubuloalveoli in the deep part of the mammary gland (Fig. 1,Panel A). The morphological appearance of mammary cistern andducts is shown in Fig. 1, Panel B. Mammary ducts in the teat areshown in Fig. 1, Panel D. The remainder of the mammary tissuespace consists of stromal (connective) tissue and adipose tissue(mammary adipocytes and microvessels are shown in Fig. 1,Panel C). The histological appearance of bovine kidney (used as apositive control for the immunolocalisation of aquaporins 1, 2and 7) is shown in Fig. 1, Panels E and F (renal cortex and medulla,respectively).

The immunolocalisation of selected AQPs in bovine mammarytissue is summarized in Table 2.

Aquaporin 1

The AQP1 results are summarized in Fig. 2. In the lactatingbovine mammary gland, there was prominent labelling for AQP1in endothelial cells throughout (indicated by the boxed areas andinsets in Fig. 2A, B and D). There was no AQP1 immunopositivityin epithelial cells, secretory tubuloalveoli (acini), cistern and teatducts or in mammary adipose cells (Fig. 2C). However, AQP1 wasdensely labelled in the myoepithelial cells underlying teat ductepithelia (as indicated by the arrows in Fig. 2B and the magnifiedinset in Fig. 2B) and microvessels in mammary adipose tissue(Fig. 2C and magnified inset). In the bovine kidney AQP1immunopositivity was present in apical (labelled A) andbasolateral (labelled B) membranes of proximal tubules in thecortex (Fig. 2E) and loops of Henle in the medulla (Fig. 2F) andpapilla (Fig. 2G).

Aquaporin 2

AQP2 was not detected in any part of the lactating bovinemammary gland (Fig. 3, Panels A–D). Immunohistochemicalexamination of the bovine kidney confirmed the presence ofAQP2 in cortical collecting ducts (in apical membranes ofprincipal cells). AQP2 immunopositivity in the apicalmembranes of collecting duct principal cells of the bovinekidney is indicated by arrows in Fig. 3, Panel E and again in themagnified inset.

Aquaporins 3 and 4

AQP3 immunopositivity was found in basolateral selectedepithelial cells in the tubuloalveoli, cistern ducts, teat ducts andteat smooth muscle bundles (data are not shown). Immunola-belling of AQP3 in the basolateral membranes of medullaryrenal collecting duct epithelial cells in the bovine kidney isshown in Fig. 4, Panels E and F (basolateral localizationindicated by arrows in the magnified inset in Panel E). Insome epithelial cells lining cistern and teat ducts AQP3 waslocalized in apical membranes (Fig. 4, Panels B and D; apicallocalization indicated by arrows in the magnified inset in bothpanels).

AQP4 immunopositivity was weakly detected in epithelialcells in tubuloalveoli in the deep mammary gland (Fig. 5,Panel A). The precise membrane localization of AQP4 was notclear although the immunopositivity observed was closer tothe apical membrane. AQP4 immunopositivity in the cistern

Fig. 2. Immunohistochemical localization of AQP1 in different regions of the lactating bovine mammary gland and bovine kidney. Panel A: Distribution of AQP1 in

microvessels in tubuloalveoli in the deep mammary gland. The area in the square with dotted lines is shown in higher magnification in the inset. Panel B:

Immunolocalisation of AQP1 in myoepithelial cells lining ducts in the cistern (arrows) and microvessels (shown in squares with solid lines). The area in the square with

dotted lines is shown in higher magnification in the inset. Panel C: Immunolocalisation of AQP1 in microvessels in mammary adipose tissue microvessels. The area in the

square with dotted lines is shown in higher magnification in the inset. Panel D: AQP1 immunolocalisation in myoepithelial cells lining ducts in the teat. The area in

the square with dotted lines is shown in higher magnification in the inset. Panel E: AQP1 immunolocalisation in apical and basolateral membranes of proximal tubules in

the renal cortex. Apical (A) and basolateral (B) immunolabelling is highlighted using arrows in Panel E. Panel F: AQP1 immunolocalisation in loops of Henle (arrows) in the

renal medulla. Panel G: AQP1 immunolocalisation in loops of Henle (arrows) in the renal papilla. Original magnifications of the main panels: 200� . Bars represent 100mm.

A. Mobasheri et al. / acta histochemica 113 (2011) 137–149 141

was also weak but more specific than the tubuloalveoli (Fig. 5,Panel B). AQP4 was not detected in adipose tissue (Fig. 5, PanelC). In teat ducts AQP4 immunopositivity was clearly apical(Fig. 5, Panel D). Detection of AQP4 in the basolateralmembranes of cortical collecting duct epithelial cells in thebovine kidney is shown in Fig. 5, Panel E (basolateral

localization indicated by arrows in the magnified inset inPanel E).

AQP3 and AQP4 were generally weakly detected in the acini(mammary tubuloalveoli). The AQP3 and AQP4 results aresummarized in Figs. 4 and 5. The labelling seen waspredominantly basolateral for AQP3 in the tubuloalveoli of

Fig. 3. Lack of AQP2 immunodetection in various regions of the lactating bovine mammary gland. Panel A: deep mammary gland showing active tubuloalveoli. Panel B:

section of mammary cistern. Panel C: Mammary adipose tissue. Panel D: teat end. Positive labelling of AQP2 was only observed in apical membranes epithelial cells lining

cortical collecting ducts in the bovine renal cortex (arrows in Panel E). The area in the square with dotted lines highlights the apical localization of AQP2 and is magnified in

the inset. Original magnifications of the main panels: 200� . Bars represent 100mm.

A. Mobasheri et al. / acta histochemica 113 (2011) 137–149142

the deep mammary gland (Fig. 4) and apical for AQP4 in teatducts (Fig. 5).

Aquaporins 5 and 6

AQP5 was found to be very weakly detected in tubuloalveoli ofthe lactating cow (arrows in Panel A, Fig. 6). However, AQP5 wasdensely labelled in smaller ducts in the cistern (squares with dottedlines in Fig. 6B). There was no AQP5 labelling in mammary adiposetissue (data are not shown). AQP5 was only present is selected cellsin the teat (squares with solid lines in Fig. 6C). AQP6 which is achloride channel rather than a water channel was not detected inany of the mammary tissues studied (data are not shown).

Aquaporin 7

AQP7 was found to be relatively diffusely labelled in the apicalmembranes of selected epithelial cells in the tubuloalveoli in thedeep mammary gland and absent from many ducts in the cistern

(arrows in Fig. 7, Panel B). AQP7 was present in cell bodiesof adipocytes in mammary adipose tissue (Fig. 7, Panel C). In theteat AQP7 labelling was prominently apical in ducts (Fig. 7, PanelD). AQP7 was present in smooth muscle bundles in the mammarygland and the teat (Fig. 7, Panels E and F, respectively). AQP7immunopositivity in the bovine kidney was observed in apicalmembranes of proximal tubule cells (Fig. 7, Panel E).

Aquaporin 9

AQP9 was not detected in any of the mammary tissuesexamined; however immunopositivity was evident in leukocyteswithin the glands and blood vessels (data are not shown).

Na, K-ATPase

Detection of the a1 subunit of Na, K-ATPase was used as apositive control and basolateral marker of epithelial cells. Wedecided to use this protein as a marker of epithelial transport in

Fig. 4. Immunohistochemical localization of AQP3 in different regions of the lactating bovine mammary gland. Panel A: distribution of AQP3 in epithelial cells lining active

tubuloalveoli in the deep mammary gland. The area in the square with dotted lines is shown in higher magnification in the inset. Panel B: apical immunolocalisation of

AQP3 in selected epithelial cells lining ducts in the cistern. The area in the square with dotted lines is shown in higher magnification in the inset. Panel C: lack of AQP3

immunopositivity in mammary adipose cells. Panel D: immunolocalisation of AQP3 in selected cells lining ducts in the teat. The area in the square with dotted lines is

shown in higher magnification in the inset. Panel E: AQP3 immunolocalisation in basolateral membranes of collecting ducts in the renal medulla. The area in the square

with dotted lines highlights the basolateral localization of AQP3 (arrows) and is magnified in the inset. Panel F: AQP3 immunolocalisation in collecting ducts (arrows) in the

renal papilla. Original magnifications of the main panels: 200� . Bars represent 100mm.

A. Mobasheri et al. / acta histochemica 113 (2011) 137–149 143

the mammary gland. The abundant labelling and preferentiallocalization of Na, K-ATPase in basolateral membranes ofepithelial cells confirmed the expected polarity and structuralintegrity of the bovine mammary and the renal tissues used ascontrols in this study. The Na, K-ATPase was highly immunopo-sitive in basolateral membranes of epithelial cells lining tubu-loalveoli in the deep mammary gland (arrows in Panel A, Fig. 8).Na, K-ATPase labelling was significantly lower in the basolateralmembranes of epithelial cells lining interlobular and intralobularducts in the cistern (arrows in Panels B and D, Fig. 8) andwas virtually undetectable in mammary adipocytes (exceptfor capillary endothelial cells which labelled positive forNa, K-ATPase) (square with dotted lines in Panel C, Fig. 8). Na,K-ATPase was localized abundantly in basolateral membranesof cells lining the bovine renal cortex (proximal and distaltubules) and medulla (collecting ducts) (arrows in Fig. 8, PanelsE and F, respectively). The Na, K-ATPase results are summarizedin Fig. 8.

Images of negative control sections with the primary anti-bodies omitted are shown in Fig. 9 for each region of the bovinemammary gland (Panels A–D) and kidney (Panels E and F).

Discussion

The mammary gland is a source of nourishment for theneonate and growing mammal from birth to weaning byproducing milk containing vital nutrients and colostrum whichprovides passive mucosal immunity (Salmon, 1999; Shennanand Peaker, 2000). In terms of anatomical architecture,mammary glands are essentially modified and enlarged sweatglands. Mammary lobes are comprised of secretory acini, whichare formed from cuboidal epithelial cells, responsible for thesynthesis and secretion of milk, surrounded by myoepithelialcells. The latter contract during the milk ejection reflex,increase the pressure in acini and affect the expulsion of milk.

Fig. 5. Immunohistochemical localization of AQP4 in different regions of the lactating bovine mammary gland. Panel A: weak and diffuse immunopositivity of AQP4 in

epithelial cells lining active tubuloalveoli in the deep mammary gland. The magnified area in the inset highlights the diffuse labelling. Panel B: weak but prominent labelling

of AQP4 in a selected number of epithelial cells lining ducts in the cistern (arrows). The magnified area in the inset highlights the apical immunolocalisation. Panel C: lack of

AQP4 labelling in mammary adipose cells. Panel D: prominent labelling of AQP4 in cells lining ducts in the teat (arrows). The magnified area in the inset highlights the

prominent immunopositivity. Labelling of AQP4 was positive but generally characterized by weak immunoreactivity in most regions of the bovine mammary gland except

the teat ducts. Panel E: AQP4 labelling in basolateral membranes of collecting ducts in the renal cortex. The magnified area shown in the inset highlights the basolateral

localization of AQP3 (arrows). Panel F: AQP3 labelling in collecting ducts (arrows) in the renal medulla. Original magnifications of the main panels: 200� . Bars represent

100mm.

A. Mobasheri et al. / acta histochemica 113 (2011) 137–149144

Interstitial spaces between acini, ducts and lobes are filled withconnective tissue and for the most part, adipose tissue. Inlactating animals, active acini secrete milk, which drains intosmall intralobular excretory ducts. Ducts are also lined withepithelial cells and may have an outer layer of myoepithelialcells derived from the same lineage (Gudjonsson et al., 2005;Pechoux et al., 1999). Intralobular ducts join to form a networkof interlobular ducts leading into a complex system oflactiferous ducts. In the cow, lactiferous ducts converge into asingle lactiferous cistern within each quarter where milkcollects before being ‘‘let down’’ through the teat canal duringthe milking process or suckling.

Aquaporins are a family of small integral membrane proteinsthat are expressed in a variety of epithelial tissues where they areresponsible for regulating rapid water movement across epithelialbarriers driven by osmotic gradients. Milk consists of water pluslipids, electrolytes, vitamins, sugars and specific milk proteins.

The delivery of water to the mammary gland by the circulatorysystem and the movement of water across endothelial andepithelial barriers are critical for milk synthesis and secretion inlactating animals. There is some published information on sodiumand chloride transport in mammary epithelia (Blaug et al., 2001).However, the functional anatomy of the mammary gland has notbeen studied in the context of water transport across endothelialand epithelial barriers and bulk fluid movement in lactatinganimals. Consequently, little is known about the process of watertransport across the mammary epithelium and very littlepublished information is available about aquaporin expression,distribution and function in mammary tissues. The only availabledata relate to the identification of AQP1 and AQP3 proteins incapillary endothelia and epithelial cells of mouse mammaryglands, respectively (Matsuzaki et al., 2005). Matsuzaki and co-workers also used RT-PCR to study the expression of AQP1–AQP7in the rat mammary gland. In addition to AQP1 and AQP3 they

Fig. 6. Immunohistochemical localization of AQP5 in different regions of the

lactating bovine mammary gland. Panel A: weak detection of AQP5 in epithelial

cells lining active tubuloalveoli in the deep mammary gland (arrows). Panel B:

strong labelling of AQP5 in apical membranes of selected epithelial cells lining

smaller ducts in the cistern (squares with dotted lines). AQP5 immunolabelling

was much weaker or absent in larger ducts. Panel C: weak labelling of AQP5 in

selected cells lining ducts in the teat end (squares with solid lines). Original

magnifications: 200� . Bars represent 100mm.

A. Mobasheri et al. / acta histochemica 113 (2011) 137–149 145

found evidence for the presence of AQP4, AQP5, AQP7 and AQP9transcripts in the rat mammary gland. However, they did not useantibodies to localize these proteins in the rodent mammarygland.

Milk yield is of major economic importance to the dairyindustry. Therefore, it is necessary to understand the under-

lying molecular physiology involved in fluid movement inlactation. The aim of this study was to determine the distribu-tion of aquaporins within the lactating bovine mammary gland.This study confirms the presence of at least six AQP proteins indifferent anatomical locations within the lactating bovinemammary gland.

The data we have presented in this paper confirm some ofthe recent observations made by Matsuzaki et al. (2005) in rodentmammary glands and our own observations in human mammaryglands (Mobasheri and Marples, 2004; Mobasheri et al., 2005b).In addition, we have made a number of novel observationsin support of the RT-PCR findings made by Matsuzaki et al. (2005)in rodent mammary glands; these authors were unable toconfirm the presence of AQPs 4, 5 and 7 in the rodent mammarygland by immunohistochemical methods. However, our dataconfirm the presence of these proteins in the bovine mammarygland, although in distinct cellular locations. AQP9 is probablynot a feature of the bovine mammary gland, rather an indicatorof tissue resident leukocytes. We also observed a strikingand previously unreported abundance of AQP1 in myoepithelialcells underlying teat duct epithelia (Fig. 1, Panel B). Thephysiological reason for this is at present unknown, but highAQP1 levels in this location may contribute to increased perme-ability of teat duct epithelia in the lactating bovine mammarygland. The relative abundance of the protein also appearsto represent a distinct aspect of differentiation of myoepith-elial cells in this location, compared with those surrounding theacini.

Goats’ milk is known to contain numerous cell fragmentsknown as ‘‘christiesomes’’ which originate from secretory epithe-lial cells of the mammary gland (Clegg, 1978; Wooding et al.,1977). These cell fragments are known to contain intact andwell-preserved endoplasmic reticulum, mitochondria and lipiddroplets. Furthermore, they are capable of triglyceride synthesis.Although cows’ milk has been shown to contain very few cellularfragments, it does contain different and denser particles contain-ing fewer vesicles and numerous microvillus-like protrusions onone side – known as ‘‘sunbursts’’ (Wooding et al., 1977). Althoughit has been suggested that these particles are residues of deadcells, it is possible that these fragmented cytoplasm-containingentities contain membrane proteins which are targeted to theapical membranes of mammary secretory epithelial cells. Recentstudies have provided evidence for urinary excretion of AQP2water channel proteins during pregnancy (Buemi et al., 2001;Schrier et al., 1998). Biochemical analysis of ‘‘christiesomes’’ and‘‘sunbursts’’ should reveal if these cellular fragments containmembrane proteins of the apical membranes of alveolar epithelialcells.

In summary, we have demonstrated the presence of AQP1,AQP3–AQP5 and AQP7 in the bovine mammary gland. Compar-ing our data with published data from rodent studies clearlyindicates that some of the AQPs expressed in the lactatingmammary tissues were found in expected anatomical locations;immunohistochemical labelling in the rat mammary glandsuggests that AQP1 is localized to the capillaries and AQP3 islocalized to the basolateral membranes of the alveolar secretorycells. These results suggest that aquaporins are present inlactating mammary glands and may be participants in thecontrol of milk water content by diluting the sugar, protein andlipid contents of milk to an isotonic solution as it descendsthrough the teat duct system.

The data presented here have provided interesting newinformation about the possible regulation of water homeostasisin the bovine mammary gland. Economically important produc-tion traits within the dairy industry such as milk yield traits havelarge phenotypic variation and high heritability (Brotherstone

Fig. 7. Immunohistochemical localization of AQP7 in different regions of the lactating bovine mammary gland and in the bovine kidney. Panel A: diffuse immunodetection

of AQP7 in epithelial cells lining tubuloalveoli in the deep mammary gland. Panel B: weak labelling of AQP7 in epithelial cells lining ducts in the cistern and microvessels

(arrows). More prominent immunopositivity of AQP7 can be seen in structures resembling microvessels in the mammary adipose tissue microvessels (shown in small

squares with solid lines). Panel C: detection of AQP7 in adipocyte cell bodies and membranes (arrows). Panel D: prominent AQP7 labelling in apical membranes of selected

cells lining large ducts in the teat and weaker labelling in smaller ducts (shown in small squares with solid lines). The magnified area in the inset highlights the prominent

apical immunolocalisation. Panels E and F: AQP7 detection in smooth muscle bundles within the mammary gland and the teat, respectively. Panel G: diffuse labelling of

AQP7 in the apical (brush border) region of proximal tubules in the renal cortex. The magnified area highlights the prominent brush borderlocalization. Panel H: diffuse

AQP7 labelling in ducts in the renal medulla. Original magnifications of the main panels: 200� . Bars represent 100mm.

A. Mobasheri et al. / acta histochemica 113 (2011) 137–149146

et al., 1997; Jamrozik and Schaeffer, 1997; Royal et al., 2002; Wallet al., 2003) and thus are largely influenced by the genotype of thecow. However, the underlying physiological mechanisms respon-sible for the genetic variation between animals and indeed withina lactation period have not been clearly identified. Gaining further

information about the underlying genetic regulation of aquapor-ins and their production and activity may have the potential tohelp improve the accuracy of breeding value prediction for yieldtraits allowing producers to make more informed selectiondecisions.

Fig. 8. Immunohistochemical localization of a1 subunit of Na, K-ATPase in different regions of the lactating bovine mammary gland and in the bovine kidney. Panel A:

immunolabelling of the a1 subunit of Na, K-ATPase in basolateral membranes of epithelial cells lining tubuloalveoli in the deep mammary gland (the magnified area and

arrows in the inset highlight the basolateral localization). Panel B: weak labelling of Na, K-ATPase in the basolateral membranes of epithelial cells lining interlobular and

intralobular ducts in the cistern (arrows). Panel C: absence of Na, K-ATPase immunolabelling in mammary adipocytes and positive labelling in capillary endothelial cells

(shown in the square with dotted lines). Panel D: weak immunopositivity of Na, K-ATPase in the basolateral membranes of teat duct epithelial cells (arrows). Panels E and F:

abundant Na, K-ATPase immunolabelling in basolateral membranes of cells lining the bovine renal cortex (proximal and distal tubules) and medulla (collecting ducts)

(arrows in Panels E and F, respectively). The magnified area in the inset of Panel F highlights the specific basolateral localization. Panel G: weaker detection of Na, K-ATPase

in the basolateral membranes of selected cells in the renal papilla. Original magnifications of the main panels: 200� . Bars represent 100mm.

A. Mobasheri et al. / acta histochemica 113 (2011) 137–149 147

In conclusion, this study provides the first report of theaquaporins present within the bovine mammary gland. This datawill be highly relevant to the livestock and animal nutritionindustries. A better understanding of the molecular mechanisminvolved in milk production will have significant benefits for

animal breeding programmes. Further work is required to developa larger bank of mammary tissue samples for more comprehen-sive immunohistochemical analysis using custom designed tissuemicro-arrays. It will also be useful to obtain further informationabout aquaporin expression in mammary glands of non-lactating

Fig. 9. Negative controls. Tissue sections from all the same regions of the bovine mammary gland and kidney were treated in exactly the same way during the

immunohistochemical procedure except that the primary antibody was omitted. Original magnifications of the main panels: 200� . Bars represent 100mm.

A. Mobasheri et al. / acta histochemica 113 (2011) 137–149148

animals, those undergoing mammary development during firstpregnancy, and those recovering from mastitis.

Acknowledgements

We are grateful to BSAS/Genesis Faraday (http://www.genesis-faraday.org/) for a Vacation Scholarship awarded to M.D. Royaland A. Mobasheri. A.J.G holds a Royal Canin Senior Lectureship inSmall Animal Medicine. The authors thank Mr. A.F. Brandwoodand Mr. S. Williams for histology support. The authors are alsograteful to Dr. Kieron Salmon for useful discussions and to LeeMoore and Nigel White for procuring abattoir tissues.

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