I(

Ma

b

c

a

ARRA

KCIMLM

I

daMsedpa

tntcc

cDf

0d

Acta Histochemica 114 (2012) 87– 93

Contents lists available at ScienceDirect

Acta Histochemica

jou rna l h o mepage: www.elsev ier .de /ac th is

mmunohistochemical studies on the bovine lactating mammary glandBos taurus)

ohamed Alkafafya,∗, Reda Rashedb, Amr Helal c

Department of Cytology and Histology, Faculty of Veterinary Medicine, Minufiya University, Sadat City Branch, Sadat City 32897, Minufiya, EgyptDepartment of Anatomy and Embryology, Faculty of Veterinary Medicine, Minufiya University, Sadat City Branch, EgyptDepartment of Anatomy and Embryology, Faculty of Veterinary Medicine, Zagazig University, Egypt

r t i c l e i n f o

rticle history:eceived 12 December 2010eceived in revised form 22 February 2011ccepted 23 February 2011

eywords:ow

a b s t r a c t

The study aimed to evaluate the validity of immunohistochemistry in the differential labeling of thediverse components of the lactating mammary gland. Paraffin-embedded sections of lactating bovinemammary glands were stained by conventional and histochemical techniques. Primary antibodies againstS100, alpha smooth muscle actin (�-SMA), connexin-43 (Cx43), cytokeratin-14 (Ck14), galactosyltrans-ferase (GalTase), angiotensin converting enzyme (ACE) and vascular endothelial growth factor (VEGF)were applied on paraffin sections. Strong cytoplasmic and nuclear S100 immunoreactivity was mainlyexpressed by alveolar epithelium and to a lesser variable extent by ductal epithelium. The Golgi zone

mmunohistochemistryammary gland

actationyoepithelial cells

of the epithelial cells expressed strong GalTase immunostaining. Myoepithelial cells displayed a strongimmunostaining for �-SMA, Cx43 and Ck14, but not for S100. Vascular endothelium showed a moder-ate (for VEGF) to strong (for ACE) immunostaining. The presence of VEGF-immunoreactive mast cellswithin the interstitium may reflect their functional significance in angiogenesis, vascular permeabilityand migration of mononuclear leukocytes, suggesting their regulatory role in the secretory and immuno-logical functions of the mammary glands.

ntroduction

The bovine mammary gland consists of an extensively brancheductal network enclosed within a distinctive basement membranend embedded in a stromal compartment (Monterio-Riviere, 1998).ammary epithelium is organized as luminal secretory units and

urrounding myoepithelial cells (Monterio-Riviere, 1998; Welscht al., 1998; Adriance et al., 2005; Faraldo et al., 2006). The mainevelopment of mammary glands occurs postnatally, with distincteriods of intensive morphogenesis taking place during pregnancynd lactation (Deugnier et al., 2002).

During lactation, milk production depends on the action of thewo cell types of the secretory portions of the glands (alveolar lumi-al epithelial cells and myoepithelial cells). Luminal cells secrete

he milk components into the ductal lumina and myoepithelial cellsontract to aid in the milk ejection. The function of myoepithelialells in the mammary gland is more than just contractility (Adriance

Abbreviations: ACE, angiotensin converting enzyme; �-SMA, alpha smooth mus-le actin; BSA, bovine serum albumin; Ck14, cytokeratin-14; Cx43, connexin-43;AB, diaminobenzidine; IHC, immunohistochemistry; GalTase, galactosyl trans-

erase; PBS, phosphate buffered saline; VEGF, vascular endothelial growth factor.∗ Corresponding author.

E-mail address: dr alkafafy@yahoo.com (M. Alkafafy).

065-1281/$ – see front matter © 2011 Elsevier GmbH. All rights reserved.oi:10.1016/j.acthis.2011.02.012

© 2011 Elsevier GmbH. All rights reserved.

et al., 2005) with increasing evidence that they influence the pro-liferation, survival and differentiation of luminal cells, modulatestromal–epithelial interactions and actively participate in mam-mary morphogenesis (Faraldo et al., 2006). Moreover, the loss orchange of myoepithelial cell function may play a possible role inthe development of cancer (Adriance et al., 2005).

Histochemistry is a biological approach that permits study-ing the molecular characterization of tissues in relation to theirstructural organization in situ (Danguy et al., 1998). Thus, theaim of this study was to use immunohistochemical labeling ofthe various cellular components of the lactating bovine mammarygland and to determine structural–functional relationships. Forthat reason, the proteins studied were chosen according to theirfunctional relevance; mainly angiogenesis, intercellular communi-cation, secretion and contractility. The proteins studied included:S100, ACE, GalTase, Ck14, �-SMA, Cx43 and VEGF.

S100 belongs to a multifunctional subfamily of Ca2+-bindingproteins that have a broad range of functions including motility,chemotaxis, and secretion (Heizmann et al., 2002; Cruzana et al.,2003). ACE is a membrane-bound glycoprotein, which is detectablein all tissues and body fluids of mammals (Soffer, 1976). GalTase is

a member of a functional family of enzymes involved in the biosyn-thesis of carbohydrate moieties of glycoconjugates (Charron et al.,1998; Ramakrishnan et al., 2001). Cytokeratins are intermediatefilament proteins found in most epithelial cells including mam-

8 istochemica 114 (2012) 87– 93

mifdclcii2jevhd2p

M

A

cdfi

C

ousTtmeATsG

C

tde1t

I

vp0(bawtTfb3

ces,

and

wor

kin

g

dil

uti

ons

of

pri

mar

y

and

seco

nd

ary

anti

bod

ies.

tibo

die

sSe

con

dar

y

anti

bod

ies

Ori

gin

Sou

rce

Dil

uti

on

Incu

bati

on

tim

e

Typ

e

Sou

rce

Dil

uti

on

Ch

icke

nIn

stit

ute

of

Vet

erin

ary

An

atom

y,

LMU

-Mu

nic

h1:

500

Ove

rnig

ht

at

4◦ C

Bio

tin

ylat

ed

rabb

it

anti

-ch

icke

n

IgG

Roc

klan

d, U

SA

1:40

0C

hic

ken

Inst

itu

te

of

Vet

erin

ary

An

atom

y,

LMU

-Mu

nic

h1:

500

Ove

rnig

ht

at

4◦ C

Bio

tin

ylat

ed

rabb

it

anti

-ch

icke

n

IgG

Roc

klan

d, U

SA1:

400

Rab

bit

Dak

o,

Ham

burg

1:40

0 ½

h

at

room

tem

per

atu

re

Bio

tin

ylat

ed

pig

anti

-rab

bit

IgG

Dak

o,

Ham

burg

1:30

0M

ouse

Dak

o,

Ham

burg

1:20

0 1

h

at

room

tem

per

atu

re

Bio

tin

ylat

ed

rabb

it

anti

-mou

se

IgG

Dak

o,

Ham

burg

1:30

0M

ouse

BD

Bio

scie

nce

, Hei

del

berg

1:20

0

Ove

rnig

ht

at

4◦ C

Bio

tin

ylat

ed

rabb

it

anti

-mou

se

IgG

Dak

o,

Ham

burg

1:30

0G

uin

ea

pig

Acr

is, H

erfo

rd, G

erm

any

1:50

1

hr

at

room

tem

per

atu

re

Bio

tin

ylat

ed

rabb

it

anti

-

pig

IgG

Acr

is, H

erfo

rd, G

erm

any

1:50

0R

abbi

t

Dak

o,

Ham

burg

1:80

0

Ove

rnig

ht

at

4◦ C

Bio

tin

ylat

ed

pig

anti

-rab

bit

IgG

Dak

o,

Ham

burg

1:30

0

8 M. Alkafafy et al. / Acta H

ary epithelium (Mikaelian et al., 2006; Sun et al., 2010). ˛-SMAs a contractile protein mainly found in cells having contractileunctions and has been shown to be a useful marker for studyingifferentiation of smooth muscle cells in normal and pathologicalonditions (Skalli et al., 1989). Both �-SMA and Ck14 have beenong used as phenotypic markers for mammary gland myoepithelialells (Hellmén and Isaksson, 1999; Deugnier et al., 2002). Connex-ns constitute a large family of trans-membrane proteins that allowntercellular communication (El-Sabban et al., 2003; Dbouk et al.,009). Connexins are also considered tumor suppressors and gap

unctions are often down-regulated in breast cancer (McLachlant al., 2006). VEGF is a heparin-binding growth factor specific forascular endothelial cells, with a potent angiogenic capacity thatas been implicated both in physiological and pathological con-itions (Armesilla et al., 1999; Pepper et al., 2000; Hetian et al.,002). This may be attributed to its ability to increase microvascularermeability (Ekerbicer et al., 2008).

aterials and methods

nimals and tissues

Mammary gland tissues were collected from seven lactatingows (Bos taurus) from a slaughterhouse in Cairo, Egypt. Imme-iately after slaughter the tissues were immersed in differentxatives.

hemicals and methods

Specimens were fixed in Bouin’s fluid and also in a mixturef methanol/glacial acetic acid (2:1). Bouin-fixed specimens weresed for routine histological staining and immunohistochemicaltaining (ACE, S-100, �-SMA, Ck14 and Cx43). Some proteins (Gal-ase and VEGF) could not be resolved in Bouin-fixed sections, andhese specimens were fixed in the methanol/glacial acetic acid

ixture. Tissue specimens were dehydrated in a graded series ofthanol, cleared in xylene, embedded in Paraplast wax (Sigma-ldrich, St. Louis, MO, USA) and sectioned at 5 �m thickness.issue sections were mounted on positively charged and coatedlides (Thermo Scientific, Menzel-Gläser GmbH, Braunschweig,ermany).

onventional histological staining

Several conventional staining techniques were used to inves-igate the general histological structure according to protocolsescribed in Bancroft et al. (1996). These included hematoxylin andosin, Masson and Goldner’s trichrome stains, Alcian blue 8GX (pH.0 and 2.5), periodic acid-Schiff (PAS) reaction after McManus andoluidine blue.

mmunohistochemistry

Dewaxed and rehydrated sections were subjected to inacti-ation of endogenous peroxidases by incubation in 1% hydrogeneroxide (H2O2) for 15 min. Then the sections were placed in.01 mol/L citrate buffer (pH 6) and heated in a microwave oven700 W) for 10 min for antigen retrieval. The sections were blockedy phosphate buffered saline (PBS) containing 5% bovine serumlbumin (BSA) for an hour, and then each section was incubatedith its corresponding primary antibody (types, sources and dilu-

ions of antibodies, and the duration of incubation are shown in

able 1) in humidified chamber. The sections were washed by PBSor 5 min 3 times and incubated with biotinylated secondary anti-odies (types, sources and dilutions are shown in Table 1), for0 min at room temperature. The sections were washed by PBS for Ta

ble

1Id

enti

ty, s

our

Prim

ary

an

Aga

inst

AC

EG

alTa

seS1

00�

-SM

A

Cx4

3C

k14

VEG

F

M. Alkafafy et al. / Acta Histoch

Table 2Immunolocalization of different proteins in the lactating bovine mammary gland.

Proteins Luminal epithelium Myo Blood vessels CT MC

A D VE SMCs

S100 +++ −/++ − − − − −GalTase +++GZ −/+++GZ − − − − −ACE − − − +++ − − −VEGF − − − +/++ − − +++SMA − − +++ − +++ − −Ck14 − − +++ − − − −Cx43 − − +++ − − − −

Alveolus (A); duct (D); Golgi zone (GZ); myoepithelium (Myo); vascular endothe-lN(

1tePPrttpaL

P

onibs

R

H

asci

I

T

ium (VE); smooth muscle cells (SMCs); connective tissue (CT) and mast cell (MC).egative (−); negative to strong (−/+++); weak (+); moderate (++); weak to moderate

+/++) and strong (+++) reaction.

0 min. Then the secondary antibody was detected with Vectas-ain ABC kit (Vector Laboratories Inc., Burlingame, CA, USA) firstlyach section is covered with 100× dilution of A and B reagent inBS (1 �l reagent A + 1 �l reagent B + 98 �l PBS), then washed byBS for 10 min 3 times and the color was developed using DABeagent (Sigma-Aldrich, St. Louis, MO, USA). Sections were coun-erstained with hematoxylin for 30 s, washed in water, dehydratedhrough graded ethanol, cleared in xylene and mounted with DPXermanent mounting media (Sigma-Aldrich, St. Louis, MO, USA)nd photographed (Leica EC3 digital camera with LAS EZ software,eica Microsystems Ltd., Heerbrugg, Switzerland).

ositive and negative controls

Immunohistochemical negative controls, where each primaryr secondary antiserum or the ABC reagent was omitted, gaveo positive staining. Positive controls were used according to the

nstructions provided by the manufacturers of the primary anti-odies. For assessment of the immunolabelling a semi-quantitativeubjective scoring was performed by three independent observers.

esults

istological findings

The structural units of the lactating gland consisted of vari-bly shaped lobules. The lobules were composed of tubulo-alveolarecretory units that drain into small intralobular ducts (simpleuboidal epithelium), which leave the lobule and open into a largenterlobular duct (bistratified cuboidal epithelium) (Fig. 1).

mmunohistochemical findings

The main immunohistochemical findings are summarized inable 2.

Ck14: The myoepithelial cells were the sole component of theglandular tissue which displayed a distinct Ck14 immunostaining(Fig. 2A).˛-SMA: Apart from the myoepithelial cells and the vascular SMCsthat expressed a strong immunostaining for �-SMA, all otherparenchymal as well as stromal components exhibited no �-SMAbinding sites. The spatial distribution of myoepithelial cells wasvariable in the different segments of the alveolar–ductal system.The ductal myoepithelial cells were spindle-shaped and orientedparallel to the long axis of ducts as a continuous layer. The alve-

olar myoepithelial cells were discontinuous, stellate-shaped, andformed a basket-like network (Fig. 2B).ACE: Although the luminal and myoepithelial cells failed to displayACE-binding sites, a strong ACE immunostaining was found in the

emica 114 (2012) 87– 93 89

vascular endothelium, especially in the subepithelial blood vessels(Fig. 2C).VEGF: The VEGF immunostaining was restricted to the vascularendothelium (weak to moderate staining) and mast cells (strongstaining) within the interstitium (Fig. 2D).S100: A distinct S100 immunostaining existed in the luminalepithelial cells in alveoli and ducts. However, the immunostain-ing included all the alveolar cells, but only some of the ductal cells(Fig. 3A). Moreover, the intensity of reaction was higher in thealveolar cells. The positive cells expressed both cytoplasmic andnuclear immunostaining. Neither the myoepithelial cells nor theintraepithelial lymphocytes (Fig. 3B) displayed binding sites forS100. The interstitial stromal compartments, including the vascu-lar system, failed to express S100-IR.GalTase: The distribution of binding sites was variable in the differ-ent structures of the gland. A strong GalTase immunostaining wasexpressed by Golgi structures in the peri-nuclear cytoplasm of theepithelial cells lining the secretory alveoli and the small intralob-ular ducts (Fig. 3C and D), while the larger intralobular and theinterlobular ducts displayed no reactivity (Fig. 3D).

Discussion

The light microscopic structure of the lactating bovine mam-mary gland is in agreement with previous studies on differentspecies (Monterio-Riviere, 1998; Welsch et al., 1998). Similar tothe case in the African elephant (Welsch et al., 1998), mast cellswere consistently found in the interstitial connective tissue andstained with Alcian blue and metachromatically with toluidineblue. Moreover, mast cells expressed strong VEGF immunostaining.The functional relevance of mast cells will be discussed later.

In accord with previous immunohistochemical studies on mam-mary, salivary and eccrine sweat glands (Haimoto et al., 1987;Turusov, 1990), S100 was localized at high levels in exocrine cellsof lactating bovine mammary glands. As a Ca2+-binding protein,S100 has a broad range of functions (Heizmann et al., 2002) includ-ing secretion (Cruzana et al., 2003). Therefore, it may play rolein the calcium metabolism in the lactating mammary gland. Itis worth noting that human psoriasin has been described as amember of the family of S100 proteins, the bovine homolog ofhuman psoriasin exhibits antibacterial activity especially againstEscherichia coli. Psoriasin expression was induced in bovine mam-mary tissue by experimental intramammary infection with E. coli.Thus, S100 appears to be a part of the local host defense mecha-nisms in the udder (Regenhard et al., 2010).

Though S100 protein was described as a specific marker formyoepithelial cells, it was immunolocalized in both the myoepithe-lial and epithelial cells of the canine mammary gland. Consequentlyit was concluded that S100 is not a specific marker for myoep-ithelial cells (Möller and Hellmén, 1994). Since the myoepithelialcells of the lactating bovine mammary gland failed to express S100immunoreactivity, the current findings lend support to this conclu-sion.

The secretory cells possess an abundant Golgi complex activelyinvolved in synthesis and secretion of extracellular matrix. Further-more, the short isoform of GalTase is mainly confined to the Golgicomplex, serving a purely biosynthetic function (Shur et al., 1998).GalTase is specifically upregulated in mammary epithelium dur-ing lactation to participate in lactose biosynthesis (Charron et al.,1998; Shur et al., 1998; Hennet, 2002). In the present work, a strongimmunostaining was confined to Golgi areas of the epithelial cells

lining the secretory alveoli and the small intralobular ducts; butnot of the larger intralobular or the interlobular ducts. The variabledistribution pattern of GalTase-binding sites in the lactating bovinemammary gland may be attributed to different functional activities.

90 M. Alkafafy et al. / Acta Histochemica 114 (2012) 87– 93

Fig. 1. Photomicrographs of lactating bovine mammary gland: (A) H and E-stained section showing alveoli (asterisks) and ducts (concave arrowheads); (B) trichrome-stainedsection displaying alveolar epithelium (AE); blue-colored collagen fibers (arrows) and blood vessel (concave arrowhead) within an interlobular septum; (C) Alcian blue-stainedsection showing a positively-reacting mast cell (arrow) and basement membrane (concave arrowhead); and (D) toluidine blue-stained section showing a positively-reactingmast cell (concave arrowhead). Scale bars: 50 �m (A and B) and 20 �m (C and D). (For interpretation of the references to color in this figure legend, the reader is referred tothe web version of the article.)

Fig. 2. Photomicrographs of immunostained lactating bovine mammary gland. (A) Distinct Ck14-binding sites in the myoepithelial cells (concave arrowhead) surroundingthe secretory units (L); (B) distinct �-SMA-binding sites in the stellate shaped myoepithelial cells (concave arrowhead) and vascular SMCs (arrows); (C) the endothelia ofinteralveolar blood vessels (notched arrowheads) expressed a strong ACE immunostaining and the luminal (arrows) and myoepithelial (arrowheads) cells were negative;and (D) the vascular endothelium (arrow) and mast cells (arrowheads) in the interstitium surrounding an alveolus (A) expressed a weak and strong VEGF-IR, respectively.Scale bars: 10 �m (A), 50 �m (B and C) and 20 �m (D).

M. Alkafafy et al. / Acta Histochemica 114 (2012) 87– 93 91

Fig. 3. Photomicrographs of immunostained lactating bovine mammary gland. (A) Variable S100-immunostaining ranged from partially negative (concave arrowhead) tostrong binding (arrowhead) in the ducts and in the alveolar epithelium (arrows); (B) the alveolar epithelium (arrowheads) showed a strong S100 immunostaining, while bothintraepithelial lymphocyte (notched arrowhead), myoepithelial cell (concave arrowhead) and interstitial blood vessels (arrows) were negative; (C) GalTase-IR was strong int ells aa (notc5

Tl

1wmiaHehats

de2ilmsApsmbbt

mm

he Golgi complex (arrowheads) in the perinuclear cytoplasm of alveolar epithelial crrowheads); and (D) GalTase-binding sites were distinct in the intralobular ducts0 �m (A and D) and 20 �m (B and C).

hus, some secretory activity may be provided by the epitheliumining the intralobular ducts.

As phenotypic markers, both �-SMA (Hellmén and Isaksson,999; Deugnier et al., 2002) and Ck14 (Hellmén and Isaksson, 1999)ere used to characterize the differentiation process of mammaryyoepithelial cells. The present study showed that the myoep-

thelial cells expressed a strong immunostaining for �-SMA, Ck14nd Cx43. This agrees with previous studies (Welsch et al., 1998;ellmén and Isaksson, 1999; El-Sabban et al., 2003; Kanczuga-Kodat al., 2003; Talhouk et al., 2005; Li et al., 2006). Similarly, immuno-istochemical studies on the bovine (Hellmén and Isaksson, 1999)nd caprine (Li et al., 2006) mammary glands revealed that the spa-ial distribution of myoepithelial cells is variable in the differentegments of the alveolar–ductal system.

Gap junctions play a critical role in tissue development andifferentiation and probably in carcinogenesis (Kanczuga-Kodat al., 2003). In agreement with previous studies (El-Sabban et al.,003; Kanczuga-Koda et al., 2003; Talhouk et al., 2005), Cx43

mmunoreactivity was displayed by myoepithelial cells in theactating bovine mammary glands. This may indicate that the

yoepithelial cells may respond to stimuli, simultaneously, like ayncytium facilitating the milk letdown, at least at the lobular level.dditionally, the myoepithelial cells are assumed to influence theroliferation, survival and differentiation of luminal cells, modulatetromal–epithelial interactions and actively participate in mam-ary morphogenesis (Faraldo et al., 2006). These assumptions may

e attributed to the intermediary location of the myoepitheliumetween the parenchymal and stromal compartments; along with

he strong expression of Cx43 immunoreactivity.

The vascular tone in the mammary blood vessels is usuallyaintained, in part, by sympathetic innervation. However, theammary vasculature is probably also regulated through a variety

nd negative in myoepithelial cell (arrow) and intraepithelial lymphocytes (notchedhed arrowheads) and absent from the interlobular ducts (arrowheads). Scale bars:

of locally produced, vasoactive agents (Gorewit et al., 1993; Prosseret al., 1996). The present work showed strong ACE immunostainingin the vascular endothelium, especially in the subepithelial bloodvessels. This finding agrees with previous studies demonstratingthat the mammary tissue contains ACE, which converts circulatingangiotensin, into a potent vasoactive hormone. The latter is pro-duced by epithelial cells themselves, providing a mechanism for thefunctioning epithelium to control its own blood supply and, hence,the nutrient flow for milk synthesis (Prosser et al., 1996). Fur-thermore, the mammary arteries of lactating cows are responsiveto the angiotensin family and contain ACE and specific receptorsfor angiotensin II. This system may be important in the normalphysiological regulation of mammary blood flow of lactating cows(Gorewit et al., 1993). Also, a previous study on the ovine mammarygland showed that the ACE activity in lactating mammary gland wassix times higher than in non-lactating gland, suggesting a hormonaldependent expression of ACE in female mammals (Rao et al., 2007).Additionally, since it stimulates angiogenesis in vivo (Fernandezet al., 1985) and acts as a growth factor (Naftilan et al., 1989) incell culture systems, ACE may play a role in the development anddifferentiation of mammary tissue.

VEGF immunoreactivity in the bovine mammary gland wasrestricted to the vascular endothelium and MC in the intersti-tium. This agrees with the findings reported by Alkafafy (2005)who found VEGF-immunoreactive mast cells in the bovine epididy-mal interstitium. Mast cells release a variety of angiogenic factors(Crivellato et al., 2004), and therefore, the biological role of VEGF inthe mammary gland may be associated with regulation of vascu-

lar permeability (Lissbrant et al., 2003; Ekerbicer et al., 2008) andpromotion of monocyte chemotaxis (Clauss et al., 1990).

In conclusion, the histochemical and spatial distribution ofdifferent proteins in the diverse compartments of lactating mam-

9 istoch

merlCtbbi

R

A

A

A

B

C

C

C

C

D

D

D

E

E

F

F

G

H

2 M. Alkafafy et al. / Acta H

ary tissue displayed a variable immunoreactivity. The luminalpithelial cells expressed binding sites only for S100 and GalTase,eflecting their synthetic and secretory activities. The myoepithe-ial cells showed a distinct immunoreactivity for Ck14, �-SMA andx43, highlighting their ectodermal origin and synchronized con-ractile function. In addition to the VEGF-binding sites expressedy the interstitial mast cells, the vascular endothelium displayedinding sites for VEGF and ACE. This may underline their key role

n regulation of mammary gland circulation and immunity.

eferences

driance MC, Inman JL, Petersen OW. Myoepithelial cells: goodfences make good neighbors. Breast Cancer Res 2005;7:190–7.

lkafafy M. Glycohistochemical immunohistochemical and ultra-structural studies of the bovine epididymis. Munich, Germany:Ph.D. dissertation, Faculty of Vet. Med., LMU; 2005.

rmesilla AL, Lorenzo E, Gomezdel Arco P, et al. Vascular endothe-lial growth factor activates nuclear factor of activated T cells inhuman endothelial cells: a role for tissue factor gene expression.Mol Cell Biol 1999;19:2032–43.

ancroft JD, Stevens A, Turner DR. Theory and practice of histolog-ical techniques. 4th ed. London, Toronto: Churchill Livingstone;1996.

harron M, Shaper JH, Shaper NL. The increased level of beta-1,4-galactosyltransferase required for lactose biosynthesis isachieved in part by translational control. Proc Natl Acad Sci USA1998;95:14805–10.

lauss M, Gerlach M, Gerlach H. Vascular permeability factor:a tumor-derived polypeptide that induces endothelial celland monocyte procoagulant activity, and promotes monocytemigration. J Exp Med 1990;172:1535–45.

rivellato E, Beltrami CA, Mallardi F. The mast cell: an activeparticipant or an innocent bystander? Histol Histopathology2004;19:259–70.

ruzana MB, Budipitojo T, De Ocampo G. Immunohistochemicaldistribution of S-100 protein and subunits (S100-alpha andS100-beta) in the swamp-type water buffalo (Bubalus bubalis)testis. Andrologia 2003;35:142–5.

anguy A, Decaestecker C, Genten F. Applications of lectins andneoglycoconjugates in histology and pathology. Acta Anat1998;161:206–18.

bouk HA, Mroue RM, El-Sabban ME. Connexins: a myr-iad of functions extending beyond assembly of gapjunction channels. Cell Commun Signal 2009;7:4.http://www.biosignaling.com/content/7/1/4.

eugnier MA, Teulière J, Faraldo MM. The importance of being amyoepithelial cell. Breast Cancer Res 2002;4:224–30.

kerbicer N, Tarakci F, Barut T. Immunolocalization of VEGF VEGFR-1 and VEGFR-2 in lung tissues after acute hemorrhage in rats.Acta Histochem 2008;110:285–93.

l-Sabban ME, Abi-Mosleh LF, Talhouk RS. Developmental reg-ulation of gap junctions and their role in mammary epithe-lial cell differentiation. J Mammary Gland Biol Neoplasia2003;8:463–73.

araldo MM, Taddei-De La Hosseraye I, Teulière J. Mammarygland development: role of basal myoepithelial cells. J Soc Biol2006;200:193–8 [Abstract].

ernandez LA, Twickler J, Mead A. Neovascularization produced byangiotensin II. J Lab Clin Med 1985;105:141–5.

orewit RC, Jiang J, Aneshansleyi DJ. Responses of the bovine mam-

mary artery to angiotensins. J Dairy Sci 1993;76:1278–84.

aimoto H, Hosoda S, Kato K. Differential distribution ofimmunoreactive S100-alpha and S100-beta proteins in normalnon-nervous human tissues. Lab Invest 1987;57:489–98.

emica 114 (2012) 87– 93

Heizmann CW, Fritz G, Schafer BW. S100 proteins: struc-ture, functions, and pathology. Front Biosci 2002;7:d1356–68.

Hellmén E, Isaksson A. Immunohistochemical investigation into thedistribution pattern of myoepithelial cells in the bovine mam-mary gland. Histochem J 1999;31:379–93.

Hennet T. The galactosyltransferase family. Cell Mol Life Sci2002;59:1081–95.

Hetian L, Ping A, Shumei S. A novel peptide isolated from aphage display library inhibits tumour growth and metasta-sis by blocking the binding of vascular endothelial growthfactor to its kinase domain receptor. J Biol Chem 2002;277:43137–42.

Kanczuga-Koda L, Sulkowska M, Koda M. Expression of connexin43 in breast cancer in comparison with mammary dyspla-sia and the normal mammary gland. Folia Morphol (Warsz)2003;62:439–42.

Li P, Wilde CJ, Finch LM. Identification of cell types in the developinggoat mammary gland. J Soc Biol 2006;200:193–8.

Lissbrant IF, Lissbrant E, Persson A. Endothelial cell prolif-eration in male reproductive organs of adult rat is highand regulated by testicular factors. Biol Reprod 2003;68:1107–11.

McLachlan E, Shao Q, Wang HL, Langlois S, Laird DW. Connexinsact as tumor suppressors in three-dimensional mammary cellorganoids by regulating differentiation and angiogenesis. Can-cer Res 2006;66:9886–94.

Mikaelian I, Hovick M, Silva KA. Expression of terminal differ-entiation proteins defines stages of mouse mammary glanddevelopment. Vet Pathol 2006;43:36–49.

Möller AC, Hellmén E. S100 protein is not specific for myoep-ithelial cells in the canine mammary gland. J Comp Pathol1994;110:49–55.

Monterio-Riviere N. Integument. In: Dellmann HD, Eurell J, editors.Textbook of veterinary histology. 5th ed. Williams and Wilkins;1998. p. 303–32.

Naftilan AJ, Pratt RE, Dazu VJ. Induction of platelet-derived growthfactor A-chain and c-myc gene expressions by angiotensin IIin cultured rat vascular smooth muscle cells. J Clin Invest1989;83:1419–24.

Pepper MS, Baetens D, Mandriota SJ. Regulation of VEGF andVEGF receptor expression in the rodent mammary gland dur-ing pregnancy, lactation, and involution. Dev Dyn 2000;218:507–24.

Prosser CG, Davis SR, Farr VC. Regulation of blood flowin the mammary microvasculature. J Dairy Sci 1996;79:1184–97.

Ramakrishnan B, Shah PS, Qsaba PK. Alpha-Lactalbumin (LA) stim-ulates milk beta-1,4-galactosyltransferase I (beta 4Gal-T1) totransfer glucose from UDP-glucose to N-acetylglucosamine.Crystal structure of beta 4Gal-T1 x LA complex with UDP-Glc.J Biol Chem 2001;276:37665–71.

Rao NM, Udupa EG, Angiotensin converting enzyme from sheepmammary. lingual and other tissues. Indian J Exp Biol2007;45:1003–6.

Regenhard P, Petzl W, Zerbe H. The antibacterial psoriasin isinduced by E. coli infection in the bovine udder. Vet Microbiol2010;143:293–8.

Shur BD, Evans S, Lu Q. Cell surface galactosyltransferase: currentissues. Glycoconj J 1998;15:537–48.

Skalli O, Pelte MF, Peclet MC. Alpha-smooth muscle actin, adifferentiation marker of smooth muscle cells, is present in

microfilamentous bundles of pericytes. J Histochem Cytochem1989;37:315–21.

Soffer R. Angiotensin converting enzyme and the regulation ofvasoactive peptides. Annu Rev Biochem 1976;45:73–94.

istoch

S

TWelsch U, Feuerhake F, van Aarde R. Histo- and cytophys-

M. Alkafafy et al. / Acta H

un P, Yuan Y, Li A. Cytokeratin expression during mouse embryonicand early postnatal mammary gland development. HistochemCell Biol 2010;133:213–21.

alhouk RS, Elble RC, Bassam R. Developmental expression pat-terns and regulation of connexins in the mouse mammarygland: expression of connexin30 in lactogenesis. Cell Tissue Res2005;319:49–59.

emica 114 (2012) 87– 93 93

Turusov VS. Protein S-100 in the histological diagnosis of tumors.Arkh Patol 1990;52:71–8 [Abstract].

iology of the lactating mammary gland of the Africanelephant (Loxodonta africana). Cell Tissue Res 1998;294:485–501.