Pw

MD

a

ARRA

KACLPTP

I

u3igmct

acfhomPl

U

0d

Acta Histochemica 113 (2011) 570–577

Contents lists available at ScienceDirect

Acta Histochemica

journa l homepage: www.e lsev ier .de /ac th is

regnancy-associated glycoprotein (PAG) family localized in chorionic cellsithin the epitheliochorial/diffuse placenta of the alpaca (Lama pacos)

arta Majewska1, Grzegorz Panasiewicz, Bozena Szafranska ∗

epartment of Animal Physiology, Faculty of Biology, University of Warmia and Mazury in Olsztyn, Oczapowskiego Str 1A, 10-719 Olsztyn-Kortowo, Poland

r t i c l e i n f o

rticle history:eceived 25 March 2010eceived in revised form 9 June 2010ccepted 13 June 2010

eywords:lpacahorionama pacos

a b s t r a c t

Pregnancy-associated glycoproteins (PAGs) are abundant embryo-originated products expressed in thepre-placental trophoblast and later in the post-implantational chorionic epithelium of some ungulatespecies. This paper describes the cellular immunolocalization of the chorionic PAG family in the epithe-liochorial placenta type of the alpaca (Lama pacos—Lp), in which the PAGs were named ‘LpPAGs’. PlacentalLp sections (5 �m) of different females near mid-pregnancy (150 days post coitum; dpc), advanced preg-nancy (244–263 dpc) and late pregnancy (347 dpc) were used for cross-species (heterologous—ht) doublefluorescent immunohistochemistry (htdF-IHC). The htdF-IHC was performed with primary rabbit polyva-lent anti-porcine PAG polyclonals. The LpPAG immuno-complexes were visualized with secondary goatanti-rabbit immunoglobulins-conjugated with Alexa 488 fluorophore (green), among all nuclei of pla-

regnancy-associated glycoproteinsrophectodermlacenta

cental cells stained with propidium iodide (red). This is the first study reporting the immunolocalizationof the LpPAG family identified by htdF-IHC at the feto/maternal interface during different pregnancystages of the alpaca. The most dominant and strongest immune-positive LpPAG signals were found in thewell-developed chorionic cell layer. Our htdF-IHC indicated relatively high epitope resemblance to thatof the PAGs in camelids and pigs. These data increase our general knowledge of chorionic PAG localiza-tion during pregnancy-stage dependent development of the epitheliochorial diffuse placenta type in thealpaca.

ntroduction

The PAG family is a large group of secretory chorionic prod-cts belonging to placental aspartic proteinase superfamily (EC.4.23). Structural exon-intron identification of the PAG gene fam-

ly (Szafranska et al., 2001b) permitted an initial search of useful

enetic marker (Szafranska et al., 2001a), and chromosomal assign-ent of the PAG family (Majewska et al., 2010). Distinct cloned

omplementary DNA (cDNA) or numerous purified native placen-al proteins permitted us to identify diversity and multiplicity of

Abbreviations: PAGs, pregnancy-associated glycoproteins; A488, Alexa 488 dye;nti-pPAG-P, anti-porcine PAG polyvalent polyclonals; BNC, binuclear cells; cDNA,omplementary DNA; dpc, days post coitum; END, endometrium; fV, fetal vein; fA,etal artery; GTC, giant trophectoderm cells; H&E, hematoxylin and eosin staining;tdF-IHC, heterologous double fluorescent immunohistochemistry; ht-ISH, heterol-gous in situ hybridization; Lp, Lama pacos; LpPAG, PAGs of the alpaca; MNC,ononuclear cells; NRS, normal rabbit serum; RT, room temperature; pPAG, porcine

AG; PI, propidium iodide; TRF, trophoblast; TRD, trophectoderm-chorionic epithe-ium.∗ Corresponding author.

E-mail address: szafran@uwm.edu.pl (B. Szafranska).1 Present address: Department of Physiology, Faculty of Medical Sciences,niversity of Warmia and Mazury in Olsztyn, Poland.

065-1281/$ – see front matter © 2010 Elsevier GmbH. All rights reserved.oi:10.1016/j.acthis.2010.06.002

© 2010 Elsevier GmbH. All rights reserved.

the PAG family in some domestic and wild species belonging tothe Artiodactyla, Perissodactyla, Carnivora and Rodentia orders (see:Szafranska et al., 2006a).

Diversified species-multiplicity and pregnancy-stage depen-dent chorionic expression of the PAG family starts in pre-placentaltrophoblast (TRF) before implantation, and then is continuedin the post-placental trophectoderm (TRD—chorionic epithelium)until term in some examined eutherians (see: Szafranska et al.,2006a). So far, high heterogeneity of PAG transcripts activated ina pregnancy-stage dependent manner (mRNA expressed withinTRF or/and developed TRD cells) has been identified in cattle andsheep (Green et al., 2000; Xie et al., 1997b), pig (Panasiewicz et al.,2004a; Szafranska and Panasiewicz, 2002; Szafranska et al., 1995),cat (Green et al., 1998), horse and zebra (Green et al., 1999), goat(Garbayo et al., 2000, 2008) and white-tail deer (Brandt et al., 2007).Cellular localization of placental PAG-like mRNA (by ht-ISH) hasalso been identified in the European bison (Szafranska et al., 2005)and camelid species (Majewska et al., 2009).

Although a role(s) for the chorionic PAG family is still not clear,measurements of the PAG concentrations in maternal blood ormilk of domestic and wild ruminants is used as a prenatal markerfor pregnancy diagnosis by various homologous or heterologousradioimmunologic and immunoenzymatic tests (see: Szafranska

istoche

e6f(dsL(gfbosbh(poa

ugac2r(eu2ee22(fPid1(22todcnf

M

P

ooPV2ttcps

M. Majewska et al. / Acta H

t al., 2006a). For such PAG-based pregnancy tests (U.S. Patent869770/2005), specific standards, tracers and antigens originatedrom purified native or recombinant PAG proteins are requiredGreen et al., 2005, 2009). Therefore, multiple isoforms and manyistinct N-terminal sequences of native highly-diversified glyco-ylated PAGs have been identified in domestic cattle (Klisch andeiser, 2003; Klisch et al., 2005b, 2008; Zoli et al., 1991), sheepAtkinson et al., 1993; El Amiri et al., 2003, 2004; Xie et al., 1997a),oat (Garbayo et al., 1998), zebu (Sousa et al., 2002), water buf-alo (Barbato et al., 2008) and in the American and Europeanison (Kiewisz et al., 2008, 2009). Moreover, immunodetectionf spatial and temporal specificity of the placental PAG expres-ion (detected by immunohistochemistry [IHC] and/or Westernlot) within chorionic mononuclear (MNC) or binuclear cells (BNC)as been identified, mainly in cattle (Wooding et al., 2005), pigMajewska et al., 2006), deer (Brandt et al., 2007) and the Euro-ean bison (Majewska et al., 2005, 2008). However, to the best ofur knowledge, no IHC data on the PAGs of the camelid placentare known.

Among camelids (Camelidae family belonging to even-toedngulates—Artiodactyla order) only a few taxa still exist: alpaca,uanaco and llama (Lama genus) or vicugna and camel genus (one-nd two-humped camels). Limited studies have dealt with thelassification of the placenta of alpaca (Allen et al., 2003; Carter,001; Klisch and Mess, 2007), in which a diffuse epitheliocho-ial placental type is developed during 342–348 days of gestationOlivera et al., 2003a,b). In the camelids, specific implantation andarly placentation (Skidmore et al., 1996), unusual immunoglob-lins (Su et al., 2002), detailed follicular waves (Vaughan et al.,004) and mitotic polyploidization of chorionic giant cells (Klischt al., 2005a) have been characterized. Others have indicatedstradiol secretion by pre-implantation blastocysts (Powell et al.,007), placental efficiency due to age of the dam (Bravo et al.,008), placental expression of miscellaneous regulatory factorsHombach-Klonisch et al., 2000; Wooding et al., 2003) and use-ul reproductive performances in camelids (Tibary et al., 2008).lacenta type- and species-specific glycosylation patterns of var-ous molecules expressed at feto-maternal interfaces have beenescribed in non- and inter-breeding related species (Jones et al.,997, 1999a,b, 2000), camelids (Jones et al., 2002), peccary speciesJones et al., 2004), pigs (Jones et al., 1995; Panasiewicz et al.,004b; Szafranska et al., 2003, 2004) and cattle (Klisch and Leiser,003; Klisch et al., 2006, 2008). However, cellular localization ofhe PAG family has not been identified in placental proteomesf many wild or domesticated Artiodactyla eutherians, includingifferent camelids. The objective of this study was to identifyellular localization of placental LpPAG family in the alpaca (aew-world camelid), as the first examined taxon among Camelidae

amily.

aterials and methods

regnant animals and placental tissue harvesting

Placental tissues were harvested from Peruvian alpacas (n = 5,ne pregnant female per stage), mainly during the second halff gestation (150, 244, 257, 263 and 347 days post coitum—dpc).regnancy stage was confirmed by ultrasonography (Scanner 480ET 03-93 E-R, Pie Medical) as described previously (Olivera et al.,003a,b), and then all alpaca placental tissues were collected with

he agreement of the local ethical authorities. In addition, endome-rial tissues of cyclic pigs slaughtered during luteal phase (negativeontrol) and placental tissues of pregnant pigs (31 dpc) harvestedreviously (Majewska et al., 2006) were used during this study aspecies-specific positive controls for the PAG immunodetection.

mica 113 (2011) 570–577 571

Placental section preparation and morphological staining

Alpaca placental tissues were fixed in 4% phosphate-bufferedparaformaldehyde, rinsed in 0.1 M phosphate-buffered saline (PBS;pH 7.4), dehydrated in a graded series of ethanol, embedded inParaplast Plus® paraffin (Sherwood Medical, St. Louis, MO, USA),then sectioned (5 �m) and prepared as previously (Majewska etal., 2009). In addition, porcine 5 �m placental sections (used asthe positive controls) were mounted on Superfrost®/Plus slides(Fisher Scientific, Pittsburgh, PA, USA) and stained as previouslydescribed (Majewska et al., 2006). Briefly, the placental sectionswere similarly fixed and stained with hematoxylin and eosin (H&E)or with fluorescent propidium iodide (PI; 535ext/617em nm)—usedfor standard morphological fluorescence of nuclei of all placentalcells.

Double fluorescent immunohistochemistry (dF-IHC) withanti-pPAG polyclonals

Alpaca placental sections (150–347 dpc) were subjected to het-erologous (ht; cross-species) F-IHC detection using the selectedrabbit polyvalent anti-porcine PAG (pPAG) polyclonals, previouslyproduced and characterized by immunoscreening of fusion pro-teins coded by cloned cDNAs (Panasiewicz et al., 2004b; Szafranskaet al., 2002, 2003). The ht-IHC was required because anti-PAG seraagainst alpaca antigens are not yet available. The diagnostic pri-mary anti-pPAG-polyvalent (anti-pPAG-P) polyclonals were raisedagainst nine native secretory glycosylated antigens produced invitro and against a recombinant non-glycosylated pPAG2 antigen.This mixture of the anti-pPAG-P polyclonals was very advantageousfor the identification of various PAG epitopes by numerous homo-and ht-Western blottings performed for porcine placental proteinsduring early pregnancy (Szafranska et al., 2002, 2003, 2004), aswell as for placental cotyledon proteins during advanced- and late-pregnancy of some other ungulates, including various domesticand wild Artiodactyla taxa (Majewska et al., 2005; Szafranska etal., 2005).

During this study on placental sections of the alpaca (Lamapacos—Lp), the cellular LpPAG-immunodetection was performedby the method described for the porcine sections (Majewskaet al., 2006) and modified ht/dF-IHC—for the bison placenta(Majewska et al., 2008). Briefly, the Lp placental sections wereblocked (5 h/37 ◦C) with 2.5% ovalbumin solution, rinsed in PBS(3×/10 min/RT) and incubated (24 h/4 ◦C) with the primary anti-pPAG-P (1:300), or with normal rabbit serum (NRS) similarlydiluted (1:300). In addition, the porcine luteal-phase endome-trial (negative control) and placental sections—used as positivecontrol for PAG immunodetection, were equally treated. Twoadditional negative control-types were prepared with similarlydiluted NRS and with PBS only, whereas the primary anti-pPAG-Ppolyclonals were omitted. After incubation, all placental sectionswere rinsed in PBS (3× 10 min), then incubated (1.5 h/37 ◦C)with specific secondary goat anti-rabbit immunoglobulins (IgG;1:1000)—conjugated with Alexa 488 dye (A488; 495ext/519em nm),as the visualizing fluorophore (Molecular Probes®, Invitrogen,Carlsbad, CA, USA). The commercial goat anti-rabbit IgG/A488 werehighly cross-adsorbed by the manufacturer against human, mouse,rat and bovine IgGs. After rinsing in PBS (3× 10 min), the pla-cental specimens were counterstained (1 min/RT) with propidiumiodide (PI; 0.6 �g/ml) for fluorescent nuclei staining. The speci-mens were rinsed again, and coverslipped with antifade solution

(90% glycerol and 2.3% Dabco [Sigma–Aldrich, St. Louis, MO, USA]in 20 mM Tris–HCl, pH 8.0) for extended multicolor fluorescenceprotection.

Our ht/dF-IHC (A488/PI) permitted the identification of thecellular localization of chorionic immunoreactive PAG-like sig-

572 M. Majewska et al. / Acta Histochemica 113 (2011) 570–577

Fig. 1. Histo-morphological development of the alpaca placenta during 150–347 days of pregnancy (dpc) monitored by hematoxylin/eosin (A–D) or fluorescent propidiumi arrow2 rrow)a unit.

nsiO(a

odide (E–M) staining. TRD—trophectoderm (chorionic epithelium) layer, in which.5× in C and D inserts); GTC—giant trophectoderm cell (arrowheads); Ar—areola (artery; END—endometrial tissue; and G—uterine END glands of maternal placental

als (A488—green), among all fluorescent placental cells with

tained nuclei (PI—red). The morphological and fluorescentmages (10×, 20× and 40× objectives) were examined (BX51,lympus, Japan), analyzed (ACDSee 7.0; USA) and archived

analySIS Software ver. 3.2, Olympus, Japan) before final figurerrangements.

indicates an example of very frequently dispersed TRD cells (digitally magnified; M—mesenchymal tissue of developed fetal placental unit; fV—fetal vein; fA—fetal

Results

Morphological alpaca placental changes as the pregnancyadvanced

Either histomorphological staining with hematoxylin and eosin(H&E) (Fig. 1A–D) or with PI (Fig. 1E–M) revealed many devel-

M. Majewska et al. / Acta Histochemica 113 (2011) 570–577 573

Fig. 2. Multicolour fluorescent localization of the PAG family (LpPAGs) within diffuse/epitheliochorial placenta of the alpaca during middle- (150 dpc), advanced- (257–263dpc) and late-pregnancy (347 dpc). Cellular localization of the LpPAGs within trophectoderm (TRD—chorionic epithelium) cells identified by single heterologous immun-odetection (Alexa 488 alone; A–C, L and M), and by double fluorescence (A488/propidium iodide—PI; D–K, N–S) with the use of polyvalent anti-porcine PAG polyclonals( by PIa escenG hyme;i to the

oa(p

anti-pPAG-P) visualized by A488 (green) among all fetal-maternal cells stainednti-pPAG-P (I). Porcine placental section—used as positive control for double fluorTC—giant trophectoderm cell (arrowheads); Ar—areola (arrow); M—fetal mesenc

nterpretation of the references to color in this figure legend, the reader is referred

pmental placenta structure changes with the progress of thelpaca gestation. During the examined stages: near mid-pregnancy150 dpc), then advanced pregnancy (244–263 dpc) and late-regnancy (347 dpc), the increased placental surface was parallel

(red). Alpaca placenta section—used as negative control with omitted polyvalentt PAG immunodetection (S); and digital reductions of negative control (insert in S).fV—fetal vein; END—endometrial tissue of maternal advanced placental unit. (Forweb version of the article.)

to its developmental folding, caused by very intensive cell prolifer-ation and consequential increased mass of the chorionic epitheliumcell layer (TRD). The shape of primary non-branched TRD foldsvaried, from initial flattened ‘balloon-like’ forms (Fig. 1A–E) to

5 istoch

‘wftai(cTwDstlgo

piagsH(

P

papPipwwsn

Lc

(tsaTtowaFcswcLoL

nvTsD

74 M. Majewska et al. / Acta H

grape-like’ structures during advanced placental development,hen specific forming of the secondary/auxiliary-branched TRD

olds were identified (Fig. 1F–M). During mid-pregnancy (150 dpc),he balloon-like shape of the TRD villi-folding dominated (Fig. 1End F), but occasionally, the structure of some TRD folds var-ed, and more slender and longer TRD villi-folds were observedFig. 1G–J). However, the TRD cell layer structure was markedlyhanged in some placental regions, in which many dispersedRD cells moved towards the residential mesenchymal tissue (M)ithin several developed fetal units (inserts in Fig. 1C and D).uring advanced pregnancy stages (244–263 dpc), the increased

econdary-branching of the TRD folds descended more deeply intohe maternal uterine endometrium (END) layer (Fig. 1H–J). Duringate-pregnancy (347 dpc), fully developed TRD folds (Fig. 1K–M)enerally resembled cotyledon-like structures of the late-placentaf ruminants.

Rare giant trophectoderm cells (GTC) were observed in somelacental regions (Fig. 1A, and 2M and P), although, some neighbor-

ng TRD folds also mimicked GTC (Fig. 2A). Also, areola (Ar) shapend structure varied (Figs. 1A and B, and 2B). Moreover, the ENDland (G) development was increased (Fig. 1F), nearby fetal ves-el (fV—vein; fA—artery) expansion locally augmented (Fig. 1F and), and the END epithelium flat cell layer was markedly slimmer

Fig. 1I and J).

lacental specificity of immune-PAG controls

The negative controls for the alpaca sections (with omittedrimary anti-PAG-P) show only fluorescent red PI that stronglyccumulated in all nuclei of the placental cells (Fig. 2I). In porcinelacental sections (used as two additional controls for specificAG-immunodetection) strong fluorescent positive PAG-signal wasdentified only in the TRD cell layer (Fig. 2S—in green by A488), com-aring to the negative controls, in which the primary antiserumas altered into pre-immune NRS, or the anti-pPAG-P polyclonalsere omitted (insert in Fig. 2S—in red by PI). In the endometrial

ections (negative control) specific immunostaining signals wereot detected (data not show).

ocalization of the LpPAG family expression in the alpacahorionic cells

Multicolor ht/F-IHC on the alpaca sections identified the LpPAGsAlexa 488—green; Fig. 2) in the trophectoderm cell layer ofhe placenta at all stages of pregnancy (150–347 dpc). Thetrongest immune-positive LpPAG-stains (Fig. 2D–H) were equiv-lently located in very similar regions, in which the dispersedRD cells (moved toward mesenchyma) were also identified byhe H&E staining (compare Fig. 1C and D). During the second halff gestation, the LpPAG-signal was straightforward recognizableith extremely stage-adequate folding of the TRD layer increased

s pregnancy advanced (150 dpc; Fig. 2A–C, 257 and 263 dpc;ig. 2D–L). During late-pregnancy (347 dpc), the LpPAG immune-omplexes were prolifically located in very condensed/compactedtructure of the fully developed alpaca TRD folds, which coincidedith the cotyledon-like morphology (Fig. 2M–R). Mononuclear TRD

olumnar cells were detected (Fig. 2J and K), in which the greenpPAG signal-intensity seemed to be reduced within the cytoplasmf the TRD cells (green and red resulted yellow), compared to thepPAG-staining with A488 alone (Fig. 2L).

In some placental regions, relatively intensive fluorescent sig-

al of the LpPAGs was identified within secretory materials ofery rare GTCs, which were mainly located at the apex of theRD folds—deeply interdigitating the maternal uterine END tis-ue (Fig. 2P) within the cotyledon-like TRD structures (Fig. 2M).ispersed areolas, occurring on the feto-maternal surface, were

emica 113 (2011) 570–577

characteristic for the diffuse and epitheliochorial alpaca placentadevelopment (Fig. 2B). Areolar cavities were frequently filled byimmunopositive TRD-originated secretions (A488), but the oppo-site maternal uterine END was unstained.

Discussion

This is the first study reporting the cellular localization of thePAG-like protein family within diffuse epitheliochorial placentatype of the alpaca, in which the PAG family was named ‘LpPAGs’.The cellular identification of the LpPAGs by the heterologous dF-IHC(Alexa 488/PI) revealed immunoreactive LpPAG signal distributionwithin placental surface of the alpaca. This ht/dF-IHC permittedthe LpPAG identification within the chorionic cells during var-ious advanced pregnancy stages (150–347 dpc). The dominantimmunoreactive LpPAG signals (Fig. 2) were mainly detected in themononuclear TRD cell layer (see also Fig. 1C and D); and in rare giantTRD cells (GTC) that were located at the apex as well as at the base ofdeveloped chorionic villi folds. A similar intensity of PAG-stainingwas detected in some columnar TRD cell layers and areolar cavities,with relatively intensive condensation of the identified fluorescentsecretory LpPAGs.

Our LpPAG results correspond very strongly to the cellularlocalization of the PAG family identified during transcript analy-ses (by ISH) of the alpaca placenta (Majewska et al., 2009). Theidentification of the LpPAGs by the ht/dF-IHC was possible due tothe PAG-epitope similarities in pigs and alpacas, both Artiodactylaspecies—developing related diffuse/epitheliochorial placenta type.Previously, moderately high epitope-homology of numerous het-erogeneous secretory PAG family members (35–90 kDa) andN-terminal sequences of many distinct PAGs (55–76 kDa), as well asthe identified polypeptide precursors (341–380 amino acids), haveonly been found in some Eutherian mammals (see: Szafranska etal., 2006a). During the middle and advanced alpaca pregnancy, thePAG epitope-similarities were expected on the basis of the high-est morphological resemblance of the diffuse placenta (microvillarinterdigitations) in the Camelidae and the Suidae species (Majewskaet al., 2006). During the late-pregnancy of the alpaca, our ht/dF-IHC was specific for the LpPAGs detected within cotyledon-likestructure, similarly to the PAG-immunodetection in synepithelio-chorial cotyledonary placenta of the European bison (Majewska etal., 2008).

Cellular localization of the LpPAG (Fig. 2) is difficult to com-pare with other results because similar data are very limited fordifferent species of the Camelidae family. Recently, a single study(Bella et al., 2007) demonstrated the presence of the PAG family (byht-detection with anti-bovine PAG2 polyclonals) in semi-purifiedprotein fractions extracted from alpaca and dromedary placen-tal tissues. However, our data concerning morphological changesduring epitheliochorial placenta development are consistent withsome other studies. Our histochemical data of placenta morphol-ogy are comparable to some results obtained in alpacas (Oliveraet al., 2003a,b) and pigs (Dantzer, 1985; Keys and King, 1990;Majewska et al., 2006). The observed changes in the alpaca pla-centas (Figs. 1–2) are fairly similar to the epitheliochorial placentastructure identified in one-humped camels (Abd-Elnaeim et al.,1999, 2003; Skidmore et al., 1996), and surprisingly, partially toplacentome-like structure (cotyledons/caruncles) of the synep-itheliochorial bovine placenta (Leiser et al., 1997; Majewska etal., 2008; Wooding et al., 2005). In the alpaca, such morpho-logical modification of the diffuse/epitheliochorial placental type

with accompanying development of the cotyledon-like structurepermits increased feto-maternal communication. It seems that spe-cific placental morphology may be a characteristic adaptation forincreased oxygen requirements of the fetus survival at high alti-tudes, especially in alpacas and llamas.

istoche

aCTvtva

(KrosMa2afidinLo

fddPpeasta2epsooffiShptbi

tfika

A

w(g(P(A

M. Majewska et al. / Acta H

Specific areolar dispersion on the chorionic surface is generallystructural attribute for the diffuse/epitheliochorial placenta of theamelidae and Suidae family. In the alpaca placental sections, giantRD cells were detected, in which fluorescent LpPAG signals wereery intensive (Fig. 2A and P). In part, the LpPAG localization withinhe areolar structure (Fig. 2B) can be confirmed by facilitated inter-illous transfer of feto-maternal substances in pigs (Dantzer, 1984),s well as in camels (Abd-Elnaeim et al., 1999, 2003).

Many placental molecules are strongly glycosylated in camelidsAbd-Elnaeim et al., 1999; Skidmore et al., 1996), pigs (Keys anding, 1990; Majewska et al., 2006; Szafranska et al., 2004) anduminant species (Majewska et al., 2005). On the placental surfacef different mammals, species-specific post-translational glyco-ylation is related to the diversity and carbohydrate contents.iscellaneous glycan classes were identified within epithelial TRD

nd END cells in the alpaca and the dromedary camel (Jones et al.,002), the pig (Jones et al., 1995), the horse and donkey (Jones etl., 1997, 1999a,b, 2000). The glycan content and glycosylation pro-le may play crucial role(s) in conceptus anchoring and adhesionuring peri-implantation, whereas suitable feto-maternal glycan

nteractions may control TRF invasiveness and determine preg-ancy maintenance (Jones et al., 2004). Our data indicate that thepPAG, as glycoprotein family, is involved as well in the mechanismf alpaca placental development.

The identified dominant immunoreactive LpPAG signals at theeto-maternal surface may suggest the presence of numerous andiversified glyco-forms of the LpPAGs within stage-dependentevelopment of the alpaca placenta. Miscellaneous glycosylatedAG forms may suggest potential function(s) of this multiple glyco-rotein family in many eutherians (Atkinson et al., 1993; Kiewiszt al., 2008, 2009; Klisch et al., 2005b, 2006, 2008; Szafranska etl., 2004, 2005; Xie et al., 1997a,b). In the alpaca, we can expectome glyco-forms of the LpPAGs that can interact with other pro-eins (targets or receptors), similar to those identified in the pignd cattle (Panasiewicz et al., 2007; Szafranska et al., 2006a,b,007). Presumably, the PAG family may clench the contact ofpithelial TRD and END layers and may be involved in peptide trans-ort or conceptus nourishing and important protection againsteveral proteinases within the uterine lumen, influencing embry-nic mortality in the alpaca. Hopefully, further studies on freshr immediately liquid nitrogen-frozen alpaca placental samplesor proteome or transcriptome investigations will provide puri-ed native LpPAGs or cDNA for recombinant LpPAGs, respectively.uch studies will allow examination for a network of compre-ensive interactions between the LpPAGs and other embryonicroducts, and additionally between them and maternal uterine fac-ors. Understanding the nature of possible signalling interactionsetween the LpPAGs and other placental factors require further

nvestigation.In conclusion, this is the first study reporting cellular identifica-

ion of the LpPAG family within chorionic cells of the alpaca, as therst selected Camelidae species. Our report increases our generalnowledge about the LpPAGs in placental proteome throughoutlpaca pregnancy.

cknowledgements

This study, as a part of Ph.D. thesis of M. Majewska,as supported by the State Committee for Scientific Research

UWM522-0206.0206 and UWM528-0206.0805 projects, Poland)

ranted to the corresponding author; and the first authorEFS/ZPORR 2004-06). We are very grateful to Dr. L. Olivera (Univ.eruana Cayetano Heredia, Lima, Peru) and Dr. V.J.M. PalominoNational Univ. of Altiplano, Casilla, Peru) for placental samples.lso, our appreciations are to Prof. M. Majewski (Division of Clin-

mica 113 (2011) 570–577 575

ical Physiology, Department of Functional Morphology, Faculty ofVeterinary Medicine, UWM in Olsztyn, Poland) and Dr. K. Borkowski(Olympus PL, Warsaw, Poland) for their suggestions regarding mul-ticolor imaging.

References

Abd-Elnaeim MM, Pfarrer C, Saber AS, Abou-Elmagd A, JonesCJ, Leiser R. Fetomaternal attachment and anchorage in theearly diffuse epitheliochorial placenta of the camel (Camelusdromedarius). Light, transmission, and scanning electron micro-scopic study. Cells Tissues Organs 1999;164:141–54.

Abd-Elnaeim MM, Saber A, Hassan A, Abou-Elmagd A, Klisch K,Jones CJ, et al. Development of the areola in the early placentaof the one-humped camel (Camelus dromedarius): a light, scan-ning and transmission electron microscopical study. Anat HistolEmbryol 2003;32:326–34.

Allen WR, Carter AM, Chavatte-Palmer P, Dantzer V, Enders AC,Freyer C, et al. Comparative placentation—a workshop report.Placenta 2003(Suppl. A):S100–3.

Atkinson YH, Gogolin-Ewens KJ, Hounsell EF, Davies MJ, BrandonMR. Seamark RF Characterization of placentation-specific binu-cleate cell glycoproteins possessing a novel carbohydrate. J BiolChem 1993;268:26679–85.

Barbato O, Sousa NM, Klisch K, Clerget E, Debenedetti A, Barile VL,et al. Isolation of new pregnancy-associated glycoproteins fromwater buffalo (Bubalus bubalis) placenta by Vicia villosa affinitychromatography. Res Vet Sci 2008;85:457–66.

Bella A, Sousa NM, Dehimi ML, Watts J, Beckers JF. Westernanalyses of pregnancy-associated glycoprotein family (PAG)in placental extracts of various mammals. Theriogenology2007;68:1055–66.

Brandt GA, Parks TE, Killian G, Ealy AD, Green JA. A cloningand expression analysis of pregnancy-associated glycoproteinsexpressed in trophoblasts of the white-tail deer placenta. MolReprod Dev 2007;74:1355–62.

Bravo PW, Garnica J, Puma G. Cria alpaca body weight and peri-natal survival in relation to age of the dam. Anim Reprod Sci2008;111:214–9.

Carter AM. Evolution of the placenta and fetal membranes seen inthe light of molecular phylogenetics. Placenta 2001;22:800–7.

Dantzer V. An extensive lysosomal system in the maternal epithe-lium of the porcine placenta. Placenta 1984;5:117–29.

Dantzer V. Electron microscopy of the initial stages of placentationin the pig. Anat Embryol 1985;172:281–93.

El Amiri B, Remy B, de Sousa NM, Joris B, Ottiers NG, Perenyi Z,et al. Isolation and partial characterization of three pregnancy-associated glycoproteins from the ewe placenta. Mol Reprod Dev2003;64:199–206.

El Amiri B, Remy B, de Sousa NM, Beckers JF. Isolation and charac-terization of eight pregnancy-associated glycoproteins presentat high levels in the ovine placenta between day 60 and day 100of gestation. Reprod Nutr Dev 2004;44:169–81.

Garbayo JM, Remy B, Alabart JL, Folch J, Wattiez R, Falmagne P,et al. Isolation and partial characterization of a pregnancy-associated glycoprotein family from the goat placenta. BiolReprod 1998;58:109–15.

Garbayo JM, Green J, Manikkam M, Beckers JF, Kiesling DO, Ealy AD,et al. Caprine pregnancy-associated glycoproteins (PAGs): theircloning, expression and evolutionary relationship to other PAG.

Mol Reprod Dev 2000;57:311–22.

Garbayo JM, Serrano B, Lopez-Gatius F. Identification of novelpregnancy-associated glycoproteins (PAG) expressed by theperi-implantation conceptus of domestic ruminants. AnimReprod Sci 2008;103:120–34.

5 istoch

G

G

G

G

G

H

J

J

J

J

J

J

J

K

K

K

K

76 M. Majewska et al. / Acta H

reen JA, Xie S, Roberts RM. Pepsin-related molecules secreted bytrophoblast. Rev Reprod 1998;3:62–9.

reen J, Xie S, Szafranska B, Newman A, Gan X, McDowell K, etal. Identification of a new aspartic proteinase expressed by theouter chorionic cell layer of the equine placenta. Biol Reprod1999;60:1069–77.

reen JA, Xie S, Quan X, Bao B, Gan X, Mathialagan N, et al.Pregnancy-associated bovine and ovine glycoproteins exhibitspatially and temporally distinct expression patterns duringpregnancy. Biol Reprod 2000;62:1624–31.

reen JA, Parks TE, Avalle MP, Telugu BP, McLain AL, Peterson AJ, etal. The establishment of an ELISA for the detection of pregnancy-associated glycoproteins (PAGs) in the serum of pregnant cowsand heifers. Theriogenology 2005;63:1481–503.

reen JC, Volkmann DH, Poock SE, McGrath MF, Ehrhardt M,Moseley AE, et al. Technical note: a rapid enzyme-linkedimmunosorbent assay blood test for pregnancy in dairy and beefcattle. J Dairy Sci 2009;92:3819–24.

ombach-Klonisch S, Abd-Elnaeim M, Skidmore JA, Leiser R,Fischer B, Klonisch T. Ruminant relaxin in the pregnant one-humped camel (Camelus dromedarius). Biol Reprod 2000;62:839–46.

ones CJP, Dantzer V, Stoddart RW. Changes in glycan distributionwithin the porcine interhaemal barrier during gestation. CellTissue Res 1995;279:551–64.

ones CJP, Dantzer V, Leiser R, Krebs C, Stoddart RW. Localisationof glycans in the placenta: a comparative study of epithelio-chorial, endotheliochorial and haemomonochorial placentation.Microsc Res Tech 1997;38:100–14.

ones CJ, Enders AC, Wooding FP, Dantzer V, Leiser R, StoddartRW. Equine placental cup cells show glycan expression dis-tinct from that of both chorionic girdle progenitor cells andearly allantochorionic trophoblast of the placenta. Placenta1999a;20:347–60.

ones CJ, Wooding FB, Dantzer V, Leiser R, Stoddart RW. A lectinbinding analysis of glycosylation patterns during developmentof the equine placenta. Placenta 1999b;20:45–57.

ones CJP, Wooding FBP, Abd-Elnaeim MM, Leiser R, Dantzer V,Stoddart RW. Glycosylation in the near-term epitheliochorialplacenta of the horse, donkey and camel: a comparative studyof interbreeding and non-interbreeding species. J Reprod Fert2000;118:397–405.

ones CJP, Abd-Elnaeim M, Bevilacqua E, Oliveira LV, LeiserR. Comparison of uteroplacental glycosylation in the camel(Camelus dromedarius) and alpaca (Lama pacos). Reproduction2002;123:115–26.

ones CJ, Santos TC, Abd-Elnaeim M, Dantzer V, Miglino MA. Pla-cental glycosylation in peccary species and its relation to that ofswine and dromedary. Placenta 2004;25:649–57.

eys JL, King GJ. Microscopic examination of porcine conceptus-maternal interface between days 10 and 19 of pregnancy. Am JAnat 1990;188:221–38.

iewisz J, Melo de Sousa N, Beckers JF, Vervaecke H, PanasiewiczG, Szafranska B. Isolation of pregnancy-associated glycopro-teins from placenta of the American bison (Bison bison)at first half of pregnancy. Gen Comp Endocrinol 2008;155:164–75.

iewisz J, Melo de Sousa N, Beckers JF, Panasiewicz G, GizejewskiZ, Szafranska B. Identification of multiple pregnancy-associatedglycoproteins (PAGs) purified from the European bison (Eb;Bison bonasus L.) placentas. Anim Reprod Sci 2009;112:229–50.

lisch K, Leiser R. In bovine binucleate trophoblast giantcells, pregnancy-associated glycoproteins and placentalprolactin-related protein-I are conjugated to asparagine-linked N-acetylgalactosaminyl glycans. Histochem Cell Biol2003;119:211–7.

emica 113 (2011) 570–577

Klisch K, Mess A. Evolutionary differentiation of Cetartiodactyl pla-centae in the light of the viviparity-driven conflict hypothesis.Placenta 2007;28:353–60.

Klisch K, Bevilacqua E, Olivera LV. Mitotic polyploidization introphoblast giant cells of the alpaca. Cells Tissues Organs2005a;181:103–8.

Klisch K, Sousa N, Beckers JF, Leiser R, Pich A. Pregnancy associatedglycoprotein-1, -6, -7, and -17 are major products of bovine bin-ucleate trophoblast giant cells at midpregnancy. Mol Reprod Dev2005b;71:453–60.

Klisch K, Boos A, Friedrich M, Herzog K, Feldmann M, SousaN, et al. The glycosylation of pregnancy-associated glyco-proteins and prolactin-related protein-I in bovine binucleatetrophoblast giant cells changes before parturition. Reproduction2006;132:791–8.

Klisch K, Jeanrond E, Pang PC, Pich A, Schuler G, Dantzer V, et al. Atetraantennary glycan with bisecting N-acetylglucosamineand the Sd(a) antigen is the predominant N-glycan onbovine pregnancy-associated glycoproteins. Glycobiology2008;18:42–52.

Leiser R, Krebs C, Klisch K, Ebert B, Dantzer V, Schuler G, et al. Fetalvillosity and microvasculature of the bovine placentome in thesecond half of gestation. J Anat 1997;191:517–27.

Majewska M, Panasiewicz G, Dabrowski M, Gizejewski Z, BeckersJF, Szafranska B. Multiple forms of pregnancy-associated gly-coproteins released in vitro by porcine chorion or placentomaland interplacentomal explants of wild and domestic ruminants.Reprod Biol 2005;5:185–203.

Majewska M, Panasiewicz G, Majewski M, Szafranska B. Localiza-tion of chorionic pregnancy-associated glycoprotein family inthe pig. Reprod Biol 2006;6:205–30.

Majewska M, Panasiewicz G, Szafranska B, Gizejewski Z, MajewskiM, Borkowski K. Cellular localisation of the pregnancy-associated glycoprotein family (PAGs) in the synepitheliochorialplacenta of the European bison. Gen Comp Endocrinol2008;155:422–31.

Majewska M, Panasiewicz G, Klisch K, Olivera LVM, Mamani JM,Abd-Elnaeim MM, et al. Pregnancy-associated glycoprotein fam-ily (PAG): transcripts and gene amplicons in Camelids. ReprodBiol 2009;9:127–50.

Majewska M, Panasiewicz G, Szafranska B. Chromosomal assign-ment of porcine pregnancy-associated glycoprotein gene family.Anim Reprod Sci 2010;117:127–34.

Olivera LV, Zago DA, Jones CJ, Bevilacqua E. Developmentalchanges at the materno-embryonic interface in early preg-nancy of the alpaca, Lamos pacos. Anat Embryol 2003a;207:317–31.

Olivera L, Zago D, Leiser R, Jones C, Bevilacqua E. Placentation in thealpaca Lama pacos. Anat Embryol 2003b;207:45–62.

Panasiewicz G, Majewska M, Szafranska B. Trophoblastic cDNAcloning of porcine pregnancy-associated glycoprotein genes(pPAG) and in silico analysis of coded polypeptide precursors.Reprod Biol 2004a;4:131–41.

Panasiewicz G, Majewska M, Szafranska B. The involvement ofluteinizing hormone (LH) and pregnancy-associated glycopro-tein family (PAG) in pregnancy maintenance in the pig. ReprodBiol 2004b;4:143–63.

Panasiewicz G, Majewska M, Romanowska A, Dajnowiec J, Szafran-ska B. Radiocompetition of secretory pregnancy-associatedglycoproteins as chorionic ligands with luteal and uterinegonadotrophin receptors of pregnant pigs. Anim Reprod Sci

2007;99:285–98.

Powell SA, Smith BB, Timm KI, Menino Jr AR. Estradiol productionby preimplantation blastocysts and increased serum proges-terone following estradiol treatment in llamas. Anim Reprod Sci2007;102:66–75.

istoche

S

S

S

S

S

S

S

S

S

S

M. Majewska et al. / Acta H

kidmore JA, Wooding FB, Allen WR. Implantation and early pla-centation in the one-humped camel (Camelus dromedarius).Placenta 1996;17:253–62.

ousa NM, Remy B, El Amiri B, De Figueiredo JR, Banga-MbokoH, Dias Goncalves PB, et al. Characterization of pregnancy-associated glycoproteins extracted from zebu (Bos indicus)placentas removed at different gestational periods. Reprod NutrDev 2002;42:227–41.

u C, Nguyen VK, Nei M. Adaptive evolution of variable region genesencoding an unusual type of immunoglobulin in camelids. MolBiol Evol 2002;19:205–15.

zafranska B, Xie S, Green J, Roberts RM. Porcine pregnancy-associated glycoproteins: new members of the asparticproteinase gene family expressed in the trophectoderm. BiolReprod 1995;53:21–8.

zafranska B, Panasiewicz G, Waclawik A. Length polymorphism ofPCR-amplified genomic fragments of the pregnancy-associatedglycoprotein (PAG) gene family in the pig and some otherdomestic or wild mammals. J Appl Genet 2001a;42:335–49.

zafranska B, Miura R, Ghosh D, Ezashi T, Xie S, Roberts RM,et al. Gene for porcine pregnancy-associated glycoprotein 2(poPAG2): its structural organization and analysis of its pro-moter. Mol Reprod Dev 2001b;60:137–46.

zafranska B, Panasiewicz G. The placental expression of theporcine pregnancy-associated glycoprotein (pPAG) gene familyexamined in situ and in vitro. Anim Reprod Sci 2002;72:95–113.

zafranska B, Ziecik A, Okrasa S. Primary antisera against selectedsteroids (E17(, E1, A4, T, B, F) or proteins (pLH, pPAG) and sec-ondary antisera against (-globulins—an available tool for studiesof reproductive processes. Reprod Biol 2002;2:187–204.

zafranska B, Panasiewicz G, Majewska M, Beckers JF. Chori-onic expression of heterogeneous products of the PAG(pregnancy-associated glycoprotein) gene family secreted invitro throughout embryonic and foetal development in the pig.Reprod Nutr Dev 2003;43:497–516.

zafranska B, Majewska M, Panasiewicz G. N-glycodiversity of thepregnancy-associated glycoprotein family (PAG) produced invitro by trophoblast and trophectoderm explants during implan-tation, placentation and advanced pregnancy in the pig. ReprodBiol 2004;4:67–89.

mica 113 (2011) 570–577 577

Szafranska B, Panasiewicz G, Dabrowski M, Majewska M,Gizejewski Z, Beckers JF. Chorionic mRNA expression andN-glycodiversity of pregnancy-associated glycoprotein family(PAG) of the European bison (Bison bonasus). Anim Reprod Sci2005;88:225–43.

Szafranska B, Panasiewicz G, Majewska M. Biodiversity of multiplepregnancy-associated glycoprotein (PAG) family: gene cloningand chorionic protein purification in domestic and wild eutheri-ans (Placentalia)—a review. Reprod Nutr Dev 2006a;5:481–502.

Szafranska B, Panasiewicz G, Majewska M. Porcine pregnancy-associated glycoprotein family (pPAGs)—as in vitro-producedchorionic ligands for luteal and uterine gonadotropin receptors.Reprod Biol 2006b;6:105–11.

Szafranska B, Panasiewicz G, Majewska M, Romanowska A,Dajnowiec J. Pregnancy-associated glycoprotein family(PAG)—as chorionic signaling ligands for gonadotropinreceptors of cyclic animals. Anim Reprod Sci 2007;99:269–84.

Tibary A, Rodriguez J, Sandoval S. Reproductive emergencies incamelids. Theriogenology 2008;70:515–34.

Vaughan JL, Macmillan KL, D’Occhio MJ. Ovarian follicular wavecharacteristics in alpacas. Anim Reprod Sci 2004;80:353–61.

Wooding FBP, Ozturk M, Skidmore JA, Allen WR. Developmentalchanges in localization of steroid synthesis enzymes in camelidplacenta. Reproduction 2003;126:239–47.

Wooding FB, Roberts RM, Green JA. Light and electron micro-scope immunocytochemical studies of the distribution ofpregnancy associated glycoproteins (PAGs) throughout preg-nancy in the cow: possible functional implications. Placenta2005;26:807–27.

Xie S, Green J, Bao B, Beckers JF, Valdez KE, Hakami L, et al. Multi-ple pregnancy-associated glycoproteins are secreted by day 100ovine placental tissue. Biol Reprod 1997a;57:1384–93.

Xie S, Green J, Bixby JB, Szafranska B, Demartini JC, Hecht S,et al. The diversity and evolutionary relationships of thepregnancy-associated glycoproteins, an aspartic proteinase sub-

family consisting of many trophoblast-expressed genes. ProcNatl Acad Sci USA 1997b;94:12809–16.

Zoli AP, Beckers JF, Woutters-Ballman P, Falmagne P, Ectors F. Purifi-cation and characterization of a bovine pregnancy-associatedglycoprotein. Biol Reprod 1991;45:1–10.