Cellular localisation of the pregnancy-associated glycoprotein family (PAGs) in the...

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General and Comparative Endocrinology 155 (2008) 422–431

Cellular localisation of the pregnancy-associated glycoproteinfamily (PAGs) in the synepitheliochorial placenta of the European bison

Marta Majewska a, Grzegorz Panasiewicz a, Bozena Szafranska a,*, Zygmunt Gizejewski b,Mariusz Majewski c, Krzysztof Borkowski d

a Department of Animal Physiology, University of Warmia and Mazury in Olsztyn, Oczapowskiego Str 1A/222, 10-719 Olsztyn-Kortowo, Polandb Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, 10-747 Olsztyn, Poland

c Division of Clinical Physiology, Department of Functional Morphology, Faculty of Veterinary Medicine,

University of Warmia and Mazury in Olsztyn, Polandd Olympus Poland Ltd., Suwak Str 3, 02-676 Warsaw, Poland

Received 17 May 2007; revised 19 July 2007; accepted 20 July 2007Available online 8 August 2007

Abstract

This paper describes the cellular immuno-localisation of the PAG family in synepitheliochorial (cotyledonary) placenta of the Euro-pean bison (Eb). Uteri were harvested from pregnant wild Eb (n = 4; 45–150 days post coitum – dpc); and additionally from cattle (30,45 dpc) and pigs (42 dpc) – both domestic species were used as positive controls for cellular PAG immunodetection. Placentas were sec-tioned, fixed, dehydrated and subjected to double fluorescent immunohistochemistry (dF-IHC) with the use of Alexa 488 fluorochrom(A488) and propidium iodide (PI). Native positive EbPAG signals were detected by heterologous (ht; cross-species) dF-IHC with pri-mary rabbit anti-PAG polyclonals against native or recombinant porcine PAG antigens (anti-pPAG); then visualised with secondaryanti-rabbit goat immunoglobulins – conjugated to A488. Our htdF-IHC indicated an unequivocal localisation to the mono- and bi-nuclear trophectoderm (chorionic epithelium) cells expressing the PAGs (A488 – green) among all placental cells, in which PI (red)stained nuclei. This is the first paper reporting the EbPAG family expression examined by htdF-IHC at the feto-maternal interface inwild Pecoran species. The cross-reactivity of anti-pPAG polyclonals with the EbPAGs suggests that shared epitopes are present in thesemolecules. It seems that the EbPAG family, which is robustly expressed in mono- and bi-nucleated trophectoderm cells, is associatedwith events taking place during placenta development. Our study also provided a proficient ht-system to identify various PAGs thatcould be useful as prenatal protein markers for pregnancy diagnoses, which is essential for effective reproductive management of endan-gered mammals.� 2007 Elsevier Inc. All rights reserved.

Keywords: Chorion; European bison; PAG; Pecoran; Pregnancy; Trophectoderm; Wisent

1. Introduction

The European bison (Eb; Bison bonasus L.), alsoknown as a Wisent, has been classified on the Red Listof Threatened Species (RLTS) in the endangered cate-gory (EN A2ce, C2a). In contrast, the American bison(Amb) are related taxa that include the Plains (Bisonbison bison) and Wood bison (Bison bison athabascae).

0016-6480/$ - see front matter � 2007 Elsevier Inc. All rights reserved.

doi:10.1016/j.ygcen.2007.07.008

* Corresponding author. Fax: +48 89 5233937.E-mail address: [email protected] (B. Szafranska).

These subspecies have been generally considered to beat a lower risk of extinction and are classified in the con-servation-dependent category (RLTS: LR/cd). Bothextant bison species belong to the Bovidae family (Artio-

dactyla order; Ruminantia/Pecoran suborder) along with16 other species representing nine related mammal tribes(Gatesy et al., 1992; Atanasov, 2005). Both the Eb andthe Amb species develop synepiteliochorial placentaltypes during nine-month gestation periods (Krasinskiand Raczynski, 1967; Vervaecke and Schwarzenberger,2006).

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M. Majewska et al. / General and Comparative Endocrinology 155 (2008) 422–431 423

Successful implantation requires coordinated develop-ment of a conceptus (embryo with its placental membranes)that is competent to attach to the maternal endometrial(END) epithelium. A distinguishing feature of most syne-piteliochorial placentas of domestic and wild ruminants(Pecoran) is the presence of placentomes (PL) that areformed by the development of embryo-originated chorioniccotyledons (CT) that are intimately attached to maternal-originating endometrial caruncles (CAR). A variety ofembryonic molecules are temporally produced by pre-pla-cental trophoblastic (TRF) cells as well as by the maternaltissues to which they associate. The result is a pivotal anddiscerning ‘cross-talk’ between the attaching conceptusand the maternal uterine END epithelium (Wooding,1992; Wooding and Flint, 1994; Wooding et al., 2005;Hashizume et al., 2007). Several distinct proteins originatefrom the developing placental trophectoderm (TRD – cho-rionic epithelium). These are generally known as major‘‘embryonic signals’’ that are involved in conceptus-mater-nal interactions (Ealy et al., 2004; Green et al., 2005b; Cha-krabarty et al., 2006; Chen et al., 2006; Szafranska et al.,2006b; Panasiewicz et al., 2007; Szafranska et al., 2007).

Pregnancy-associated glycoprotein (PAG) gene familymembers (Szafranska et al., 1995; Garbayo et al., 2000;Xie et al., 1997; Green et al., 1998, 1999, 2000; Panasiewiczet al., 2004a; Serrano et al., 2006; see Szafranska et al.,2006a) have been identified by complementary DNA(cDNA) cloning from conceptuses and placentas of severaleutherians, including cattle, sheep, goats and pigs. ThePAGs belong to the aspartic proteinase superfamily (EC3.4.23), which also includes many catalytically activeenzymes such as the pepsins (A, C and F). More recently,distinct PAG cDNAs have also been cloned from the pla-centa of the white-tail deer (Brandt et al., 2007). Thisreport is the first one describing molecular cloning of PAGsbelonging to the Cervidae family of the Ruminantia subor-der. Currently, there are at least 66 known PAG cDNAsthat have been identified and deposited in public databases.Not surprisingly, 26 native PAGs have been identified indomestic species as well (see Szafranska et al., 2006a),and three novel AmbPAG proteins (UniProtKB ofEMBL-EBI database; Acc. Nos. P84916, P84917 andP84918) that were purified from bison placental extracts(Kiewisz et al., 2007).

Our previous studies on the European bison (Eb)described the localisation of EbPAG mRNA (identifiedby ISH with porcine PAG cDNA probes) and the N-gly-codiversity of secreted EbPAGs released by cultured CTexplants (Szafranska et al., 2005a). Various secretory mul-tiple protein forms of placental EbPAGs and bovine PAGs(glycosylated and deglycosylated) were identified by heter-ologous and homologous Western analyses with the use ofanti-bovine and anti-porcine PAG sera. However, a cellu-lar localisation of EbPAGs has yet to be performed. Theobjective of this study was to identify the cellular localisa-tion of EbPAGs by double fluorescent detection in theplacenta of the European bison during pregnancy.

2. Materials and methods

2.1. Animals and placental tissue harvesting

Uteri of adult European bison (n = 4) were harvested post mortem

from pregnant females (370–450 kg), seasonally eliminated in the forestof the Bialowieza National Park (BNP) in Poland, with the agreementsof the governmental and local ethical authorities. The uteri harvestingwas delayed approximately 3 h (after hunting; due to a forest searchingand animal transport to special BNP facilities) until it was stored at�20 �C and transported to the UWM laboratory.

The stage of pregnancy of the Eb females (45, 65, 85 and 150 days post

coitum; dpc) was estimated by the use of a standardised crown-rumplength (crl) measurement of domestic cattle embryos (Noden and DeLahunta, 1985). The Eb uteri were separated by mechanical dissectionof placentomes (PL: cotyledon/caruncle) from the inter-PL regions, andstored at �70 �C.

In addition, placental tissues were also harvested from pregnantdomestic cows (Bt – Bos taurus; 30 and 45 dpc) and pigs (Ss – Sus scrofa;42 dpc) that were commercially sacrificed at a local slaughterhouse. TheseBt and Ss placental samples, immediately frozen after harvesting, wereused as species-specific positive controls for bovine or porcine PAG immu-nodetection (Szafranska et al., 1995; Majewska et al., 2005, 2006; Sza-franska et al., 2005a).

2.2. Placenta sectioning and morphological staining

Frozen placental Eb, Bt and Ss tissues were cut at �25 �C (Leica CM3050, Germany) and the sections (5–7 lm) were prepared as previouslydescribed for different Artiodactyla species (Szafranska and Panasiewicz,2002; Szafranska et al., 2005a). Briefly, sections were mounted on Super-frost�/Plus slides (Fisher Scientific, USA), fixed with 4% paraformalde-hyde (w/v) in PBS for 5 min, rinsed in PBS (3 · 3 min) and stained withhematoxylin and eosin (H/E) or subjected to fluorescent immunohisto-chemistry (F-IHC).

2.3. Cross-species specificity of the anti-pPAG polyclonal antibodies

Rabbit anti-porcine PAG polyclonals, that were raised against severalnative antigens secreted in vitro (anti-pPAG-P) or against a recombinantpPAG2 antigen (anti-pPAG2L-REC), were selected for this study. Thesewere chosen among 10 that were produced and characterised previouslyto be reactive toward different pPAGs in a pregnancy-stage-dependentmanner (Szafranska et al., 2002, 2003, 2004; Panasiewicz et al., 2004a,b). Generally, the polyvalent anti-pPAG-P polyclonals were able to iden-tify various epitopes of multiple dominant native PAG forms that are gly-cosylated post-translationally. In contrast, the anti-pPAG2L-RECpolyclonals were able to identify only some PAG forms, probably becausethe antibody was raised against a non-glycosylated recombinant pPAG2antigen. The specificity of these two anti-pPAG polyclonals (anti-pPAG-P and anti-pPAG2L-REC) was previously defined by multiple homolo-gous Western blotting of secretory native PAGs in pigs (Szafranskaet al., 2003, 2004), or by heterologous Western blotting of secretory PAGsof domestic cattle and wild European bison (Majewska et al., 2005; Sza-franska et al., 2005a). During our present study, both anti-pPAG polyclo-nals were used for heterologous cellular immunodetection of the PAGfamily in placental sections of the European bison.

2.4. Double fluorescent staining

Cellular PAG detection in the Eb placenta was performed by doublefluorescent immunohistochemistry (dF-IHC) as was previously described(Majewska et al., 2006). Briefly, placental sections were blocked with2.5% ovalbumin solution (4–5 h/37 �C), washed in PBS (3 · 10 min/RT)and incubated (18–24 h/4 �C) with selected heterologous primary anti-pPAG-P (1:300) or anti-pPAG2L-REC (1:100) polyclonal antibodies.The placental sections of domestic cattle and pigs – both used as positive

424 M. Majewska et al. / General and Comparative Endocrinology 155 (2008) 422–431

controls for PAG immunodetection – were similarly treated. Negativecontrols consisted of diluted normal rabbit serum (NRS; 1:100–300)and/or the complete omission of the primary anti-pPAG polyclonals.The placental sections were washed in PBS (3 · 10 min) and incubated(1.5 h/37 �C) with secondary anti-rabbit goat immunoglobulins (1:1000),conjugated with Alexa 488 dye (A488; 495ext/519em nm), as fluorochrom(Molecular Probe, USA). After washing in PBS (3 · 10 min), specimenswere counterstained (1 min/RT) with propidium iodine (PI; 0.6 lg/ml)for nuclei staining. Sections were washed again and coverslipped withantifade solution (20 mM Tris–HCl pH 8.0, 90% glycerol, 2.3% Dabco)for fluorescence protection. Microscopic dF-IHC examination (BX51,Olympus, Japan) verified the localisation of chorionic cells containingthe immunoreactive PAG signals. Morphological and fluorescent imageswere archived (AnalySIS Software ver. 3.2; Olympus, Japan) and analysed(ACDSee 7.0; USA) before initial selection and final figure preparations.

3. Results

Morphological staining (H/E) demonstrated develop-mental changes of synepitheliochorial placenta in wildEuropean bison (Fig. 1). The heterologous dF-IHC (Figs.2 and 3) with the use of anti-pPAG-P (identifying the entirePAG family) and anti-pPAG2L-REC polyclonals (identi-

Fig. 1. Histo-morphological staining of bovine (A–C) and European bisomagnifications: 40· (D, G, J), 125· (A), 250· (B), 400· (E, F, H, I, K, L)trophectodermal cotyledons, in which arrowheads indicate binucleated trophec

fying PAG2-like epitopes) indicated placental localisationof the EbPAG proteins within chorionic cells in the Euro-pean bison placenta (45–150 dpc). In addition, the PAGproteins were also detected in placental sections of domes-tic cattle (bPAG) and pigs (pPAG), both species used aspositive controls for the PAG family immunodetection.Double fluorescent staining (A488/PI) identified thechorionic localisation of the examined EbPAGs as well asthe control bPAGs and pPAGs (A488; green) among allplacental cells with nuclei stained by PI (red).

3.1. Placenta of positive control species

Standard morphological staining (H/E) of synepithelio-chorial placenta sections of domestic cow (45 dpc) demon-strated structural development of the placentome, i.e.embryonic unit of the TRD layer folds forming the CT,as well as endometrial epithelium grouped in the CAR ofthe maternal uterus (Fig. 1A–C). A very characteristic net-work of embryonic chorionic folds perfectly matches the

n (D–L) placental sections (45–150 days of pregnancy; dpc). Opticaland 500· (C). CAR – uterine endometrial caruncles; CT – embryonic

todermal cells digitally magnified (4· in B insert; and 2· in other inserts).

Fig. 2. Placental localisation of the bPAG family (positive control) within trophectoderm cells of domestic cattle (A–F; 30–45 dpc) and the EbPAG familyof the European bison (G–O; 45–150 dpc) by double fluorescent IHC with the use of polyvalent polyclonals against various porcine secretory nativeantigens (anti-pPAG-P) visualized by Alexa 488 (green), among all placental cells, in which fluorescent propidium iodine (red) stained nuclei.Magnifications: 40· (A, J, M), 50· (G), 100· (H, K, N), 200· (B) and 400· (C–F, I, L, O). CT – embryonic cotyledons; CAR – uterine caruncles; END –maternal endometrial tissue; Neg – negative controls.


uterine CAR structure. Within the CT region, someenlarged and binucleated TRD cells (BNC; indicated byarrowheads and demonstrated in the inserts) were identi-fied (Fig. 1B and C).

The htF-IHC detection with polyvalent anti-pPAG-Ppolyclonals revealed the presence of the bPAG proteinswithin chorionic cells forming the placentomal CT (30and 45 dpc) in domestic cows (Fig. 2A–F). The bPAG sig-nals were not detected in END tissues (Fig. 2A). The stron-gest bPAG signals were localised within the CT regions(Fig. 2A and B), especially in the enlarged or multinucle-

ated TRD cells (Fig. 2C–F). Similarly, the bPAG proteinswere also effectively localised with the use of anti-pPAG2L-REC polyclonals, in the placental sections of domestic cat-tle (data not shown) and pigs (Fig. 3M–O).

3.2. Placenta of the European bison

The use of H/E staining of synepitheliochorial placentalEb sections revealed many developmental changes in theplacentome structures (Fig. 1D–L). The CT and CARstructures were morphologically transformed as the

Fig. 3. Placental localisation of the EbPAG family within trophectoderm cells of the European bison during selected pregnancy stages (A–F, 45 dpc; G–L,

85 dpc) and control porcine PAG family (M–O, 42 dpc) visualized by double fluorescence with the use of potentially monovalent polyclonals againstrecombinant porcine antigen (anti-pPAG2L-REC) visualized by Alexa 488 (green), among all placental cells, in which fluorescent propidium iodine (red)stained nuclei. Magnifications: 40· (A, D, J), 100· (B, G, H), 200· (C, E, K, M–O) and 400· (F, I, L). CT – embryonic cotyledons; CAR – uterinecaruncles; int-CT – intercotyledonary regions; END – maternal endometrial tissue; Neg – negative controls.


pregnancy advanced (45–150 dpc). Intensive developmentof the CT and the CAR tissues during pregnancy progres-sion caused morphological changes of these structures thatseemed to be more condensed. Proliferating cells created amore compact layer of underlying CAR cells. During rapiddevelopment of the chorionic villi (grouped within CT), thelength and branching of the TRD layer folds intensivelyincreased. In the CT regions, there was slight interdigita-tion of the TRD villi within CAR stroma during early preg-nancy (45 dpc) and then markedly increased duringintensive placenta (85 dpc) development (Fig. 1D–I). In a

fully formed CT structure during mid-pregnancy(150 dpc), violent proliferation of the TRD cells, increasingvillus folding, resulted in the forming of a dense network ofTRD structures within the placentome (Fig. 1J–L). Two-CT cell types comprise the TRD structure: cuboidalmono-nucleated cells (MNC), and locally dispersed andenlarged BNC cells. In the placentome, the BNC (indicatedby arrowheads and inserts) dominated mainly within inten-sively developed chorionic villi of the CT folds, and werelocated near the dense CAR structure of the uterine endo-metrium (Fig. 1F, H, I, K and L).


The use of anti-pPAG-P polyclonals revealed thelocalisation of the EbPAG protein family (Fig. 2G–O)and the bPAGs (Fig. 2A–F) as a control for antibody spec-ificity in ruminants. The dominant signals of the EbPAGprotein family were clearly identified in the TRD cell layer,especially located in the folds of developed CT that, byimmunofluorescence, were easy distinguishable from thelayer of uterine CAR (Fig. 2G–O). The EbPAG signalswere not detected either in the maternal CAR tissues orthe endometrial stroma (Fig. 2J–N).

The use of anti-pPAG2L-REC polyclonals unquestion-ably (due to recombinant pPAG2 antigen) confirmed thecellular localisation of the EbPAG protein family expressedwithin the CT and inter-CT/paraplacental regions(Fig. 3A–L). The EbPAG immunodetection during earlierpregnancy stages (45–85 dpc) resembles fluorescent signalsof the pPAG family detection within porcine placental sec-tions (Fig. 3M–O) that were used as an alternative positivecontrol of the PAG expression. However, the EbPAG sig-nals (45 dpc; Fig. 3A) seemed to be slightly stronger thanthe pPAG signals (42 dpc; Fig. 3M). During mid-preg-nancy of the Eb (150 dpc) decreased expression wasobserved and the fluorescent EbPAG signals (representingnon-glycosylated PAG epitopes) were relatively weaker(data not show).

Generally, the strongest fluorescent signals were identi-fied in the developed placentomal regions cumulating secre-tory forms of the EbPAG proteins that were localised inthe TRD cells within apical (uterine-directed) regions ofthe CT villi folds (Fig. 3J). Relatively higher fluorescencewas also detected in the cytoplasm of several enlargedTRD cells (Fig. 3F). The anti-pPAG2L-REC polyclonalsalso revealed the cellular localisation of the EbPAG pro-teins in the inter-CT regions of the TRD layer (Fig. 3J–L).

4. Discussion

This is the first study reporting the immuno-localisationof the PAG family within chorionic cells in the Europeanbison placenta. Our morphological examination of the Ebplacentas, as well as the control Bt sections, revealed anumber of developmental changes of the placentomes,including the CT and the CAR structures in both thesePecoran species (the Ruminantia suborder of the Artiodac-

tyla order). The cellular localisation of the EbPAG familyexpression was presently identified by htF-IHC with twotypes of anti-PAG polyclonal sera that had been raisedagainst various glycosylated and non-glycosylated porcineantigens. The use of both anti-pPAG polyclonals, polyva-lent (anti-pPAG-P) and potentially monovalent (anti-pPAG2L-REC) during htF-IHC/dF-IHC permitted theidentification of cellular PAG family expression specificitywithin chorionic cells.

The ht-immunodetection of the EbPAG family waspossible due to the high resemblance of the amino acidsequences determining important evolutionary similaritiesof the PAG epitopes identified previously in several

domestic (Xie et al., 1997; Green et al., 1998; Garbayoet al., 1998, 2000; Panasiewicz et al., 2004a, b; Szafranskaet al., 2006a) and wild eutherian species (Brandt et al.,2007). The high epitope homology of the PAG familyhas previously been indicated by heterologous and/orhomologous Western/PAGE analyses of pregnancy-stagedependent specific secretions of porcine and bovinePAG forms with the use of various selected anti-PAGpolyclonals (Szafranska et al., 1995, 2003, 2004, 2005a;Majewska et al., 2006). Lately, numerous EbPAG andbPAG protein forms produced in vitro by various TRDexplants harvested from the CT and the inter-CT regions(paraplacental) of the Eb and the Bt placentas were alsoidentified by our ht-detection system with the use ofselected anti-pPAG polyclonals (Majewska et al., 2005).Moreover, specific N-glycodiversity of the EbPAG family,secreted by the CT explants, has also been identified byprevious Western/PAGE comparison of molecular massesof native glycosylated and deglycosylated secretoryEbPAG forms – examined by the use of N-glycanase Ftreatment (Szafranska et al., 2005a). This N-glycodiversityof the EbPAG family was recognised by various glycosyl-ation/deglycosylation patterns of secretory forms pro-duced in vitro during 18–72 h culture of CT explantsharvested from mid-pregnant Eb females (150 dpc). Simi-lar data concerning the highest N-glycodiversity were alsoobtained in another study of domestic late-pregnant rumi-nants (Klisch et al., 2006). Increased oligosaccharide con-tent of the secretory pPAG forms was also indicatedduring early, middle and late pregnancy, but in the samestudy, the highest N-glycodiversity occurred during earlyimplantation period (Szafranska et al., 2004). Thus, thepresent data concerning cellular localisation of theEbPAG family expression resemble our previous studiesrevealing N-glycodiversity of multiple EbPAG formsand chorionic mRNA expression coding the EbPAGfamily in European bison placenta development.

In this examination of Eb placental sections, we identi-fied the morphology changes during placentomal develop-ment as the pregnancy advanced. The embryonic TRDvilli folds strongly grouped within the CT that were firmlycompacted to the maternal uterine END tissue in the CAR,both forming the synepitheliochorial placentome structuresof the Eb taxon. We have observed intensive developmentof the TRD layer during fast placenta formation in the firsthalf of Eb pregnancy (45–150 dpc). In the placentomalregions, embryonic chorionic villi expressing the fluores-cent EbPAGs (A488; green) perfectly matched to thematernal CAR structure (PI; red). Such development ofthe Eb placentomes, being also fundamental units of vari-ous domestic ruminant placentas (Wooding, 1992; Woo-ding and Flint, 1994; Hashizume et al., 2007), may alsoplay a major role in properly nourishing the Eb embryo/foetus. Thus, this study increased general knowledge con-cerning various expressions of the embryonic TRF/TRDmolecules in the synepiteliochorial placenta of the Ebtaxon.


Our examination of the placental Eb proteomes with theuse of polyvalent anti-pPAG-P polyclonals revealed thepresence of the EbPAG family within the TRD cells, espe-cially located in developed CT folds. Similarly, in the con-trol bovine placenta, htF-IHC performed with the sameanti-pPAG-P polyclonals revealed the presence of severalbPAG forms within the TRD cells, located in the CT andin the inter-CT/paraplacental regions. Presently usedanti-pPAG2L-REC polyclonals entirely confirmed the spe-cific cellular identification of the EbPAG family withincytoplasm of enlarged TRD cells, located mainly in theCT regions. The presently identified cellular expression ofthe EbPAG family (Figs. 2 and 3) also corresponds withthe EbPAG mRNA localisation in placental sections, iden-tified previously in this taxon by in situ hybridisation withthe use of porcine PAG probes (Szafranska et al., 2005a).However, the slightly stronger EbPAG signals (45 dpc;Fig. 3A) than the porcine PAG signals (42 dpc; Fig. 3M)suggests a higher multiplicity of the EbPAG family in thesynepitheliochorial Eb placenta. Our present data of cellu-lar EbPAG expression correspond also with bPAG familyexpression examined previously by various molecularmethods in domestic cattle (Zoli et al., 1992a; Xie et al.,1997; Wooding et al., 2005; Klisch et al., 2006) and wilddeer species (Brandt et al., 2007). Moreover, our presenthtF-IHC, used for EbPAG family expression, performedon the placental sections (delayed recovering after hunting)seemed to be comparable to bPAG family expression(recovered immediately after slaughter). Thus, it may beexpected that a number of transcribed EbPAG genes canbe relatively similar or higher to at least 100 of the PAGgenes estimated in the bovine and ovine genomes (Xieet al., 1997). However, our further study of the Eb genomefor EbPAG gene number estimation will require specificpre-selection of females and males for better conservationof endangered Eb taxon. So far, it is known only thatSwedish bulls represent distinct EbPAG genotypes that dif-fer from genotypes of most Polish males possessing vesti-gial/regressive uteri (80%), probably due to limitedreproduction of very related Polish individuals (Szafranskaet al., 2005b).

The existence of the BNCs is very characteristic for thesynepitheliochorial type of the Ruminantia/Pecoran pla-centa (Wooding, 1992; Hoffman and Wooding, 1993; Kli-sch et al., 2006; Hashizume et al., 2007). Fromapproximately 19 dpc, the BNCs are able to migrate andfuse with uterine END epithelium cells, allowing temporalformation of syncytial foetal-maternal trinucleated cellsthat play many important endocrine functions. Presentlyit is known that cytoplasmic secretory granules of BNCcontain several glycoproteins, including multiple PAGfamily members (Zoli et al., 1992a; Xie et al., 1997; Woo-ding et al., 2005; Hashizume et al., 2007). These granules,released after TRD fusion with uterine epithelial END cellsare able to release their content (including the PAGs)directly into maternal tissues and peripheral blood circula-tion (Zoli et al., 1992a, b). Moreover, the BNC are created

by uni-nucleated TRD cell polyploidisation, during two-acytokinetic mitosis (Klisch et al., 1999a, b). During thefirst cell mitosis, the MNC became BNC with two diploidalnuclei, but during the second acytokinetic mitosis, thechromosomes of both nuclei create a large metaphaseplate, which develop BNC with two tetraploidal nuclei.Presumably, the polyploidisation of embryonic-originatedBNC cells permitted an increased number of many genecopies that will be transcribed. Such a polyploidisation ofgenetic materials has been observed in the placentomal cellsof cattle (Klisch et al., 1999a, 2004), water buffaloes (Carv-alho et al., 2006) and alpaca (Klisch et al., 2005a). How-ever, in pigs, multinucleated TRD cells (up to ninenuclei) have been identified during in vitro cultures only(Szafranska and Panasiewicz, 2002). The ploidality estima-tion revealed that some of the ruminant TRD cells are tet-raploidal, but other cells are with flat nuclei revealing aoctoploidal nuclei number, however, with a huge diversityof total nuclear volume (Klisch et al., 1999a, b). It seemsthat each genome multiplication presumably determinatesintensive expression of the PAG family members in theseTRD cells. Such a hypothesis, so far, may be confirmedby genetic multiplication (pro-gene duplication and posi-tive selection) during the evolution of the PAG gene family(Hughes et al., 2000, 2003), and various library-screeningsand Southern analyses that permitted us to expect at least ahundred of PAG genes in ruminant species (Xie et al.,1997; Szafranska et al., 2006a).

Our results directly indicated a high resemblance of thePAG epitopes (entire EbPAG family members) revealed byhtF-IHC/dF-IHC (with the use of anti-pPAG-P andanti-pPAG2L-REC polyclonals). Such a high epitoperesemblance caused many difficulties during the previouspurification and identification of miscellaneous highly-gly-cosylated native PAG forms in many examined eutherianspecies, in which only a few N-terminal PAG proteinsequences were recognised (Szafranska et al., 2006a), espe-cially due to unique placental oligosaccharide side chainstructures of the PAG molecules (Atkinson et al., 1993;Klisch et al., 2006). Other studies concerning the histo-chemical examination of various lectins (Lehmann et al.,1992; Jones et al., 1994) revealed that some of them mightspecifically label cytoplasmic granules of the BNC. Thus,such lectin-bounding has been utilised for beneficial proteinisolation and sequencing of different bovine PAG forms(Klisch et al., 2005b), which were previously identified asdistinct polypeptide PAG precursors (as AP zymogens)by bovine placental library screening with the use of differ-ent PAG cDNA probes (Xie et al., 1997; Green et al.,2000). During our study, presently used polyvalent anti-pPAG-P (against many native glycosylated antigens – thePAG family) and anti-pPAG2L-REC polyclonals (againstantigens without unique oligosaccharide side chain epi-topes) permitted us to detect various forms of EbPAGsduring cotyledonary placenta development. However,relatively similar expression of the EbPAGs (with anti-pPAG-P) was observed during all examined pregnancy


stages, and slightly decreased EbPAG signals (with anti-pPAG2L-REC), especially during advanced bison gesta-tion (150 dpc). These data can only be partially explainedby great N-glycodiversity of bPAGs (by lectin histochemis-try) that was identified in domestic cattle before parturition(Klisch et al., 2006).

The plasmatic PAG forms are detectable in maternalblood from about the third week of pregnancy until thepostpartum period, according to usable anti-PAG poly-clonal specificity (Butler et al., 1982; Sasser et al., 1986;Zoli et al., 1992b; Kiracofe et al., 1993; Green et al.,2005a). The plasmatic postpartum PAG protein half-lifeis slightly diversified; 4.5 days in the ewes, 9 days in cowsand 3.6–7.5 days in goats (Haugejorden et al., 2006). Besidesuch a short half-life of the plasmatic PAGs circulatingafter previous gestation, all the above-mentioned nativePAG forms purified from placental tissues are required asRIA/ELISA protein standards for various pregnancy diag-noses. The pregnancy diagnostic PAG tests are very usefulfor effective reproduction management of many domestic(Green et al., 2005a; see Sousa et al., 2006) and wild endan-gered eutherian ungulate species (see Szafranska et al.,2006a). Presumably, our studies describing various PAGforms purified from the placenta of American bison (Kie-wisz et al., 2007) and additionally from European bison(Kiewisz et al., unpublished data) will allow the improve-ment of diagnostic testing for both bison species. Previ-ously, such pregnancy testing was performed only withthe use of the heterologous standard (R-37) that was iso-lated from domestic cattle placenta (Haigh et al., 1991).

In conclusion, this is the first study reporting the cellularlocalisation of EbPAG family expression within variouschorionic cells (MNC and BNC) during the developmentof placentomal proteomes as pregnancy advanced. Ourdata increased general knowledge on very dynamic struc-tural changes of the synepitheliochorial placenta type,throughout intensive cotyledonary development in theendangered European bison. These results indicate thatthe EbPAG family is robustly expressed in various chori-onic cells and can be pivotally involved in the regulationof intensive placenta development of this endangered wildtaxon. This paper also demonstrates useful tools for effi-cient cellular PAG family identification in various wildand endangered mammals. Further experiments on euthe-rian species are required to understand the nature of vari-ous possible interactions between embryo-originatedPAG proteins and many uterine molecules.

Acknowledgments

This study is a part of PhD thesis (M. Majewska) sup-ported by the State Committee for Scientific Research inPoland (UWM528-0206.0206 and UWM528-0206.805 pro-jects) granted to corresponding author. The authors thankDr. Jonathan A. Green (University of Missouri-Columbia,USA) for his helpful suggestions and comments regardingthe manuscript.

References

Atanasov, A.T., 2005. Possible metabolism-body weight effect on prolon-gation and reduction of the pregnancy duration. Med. Hypotheses. 64,1247–1248.

Atkinson, Y.H., Gogolin-Ewens, K.J., Hounsell, E.F., Davies, M.J.,Brandon, M.R., Seamark, R.F., 1993. Characterization of placenta-tion-specific binucleate cell glycoproteins possessing a novel carbohy-drate. J. Biol. Chem. 268, 26679–26685.

Brandt, G.A., Parks, T.E., Killian, G., Ealy, A.D., Green, J.A., 2007. Acloning and expression analysis of pregnancy-associated glycoproteinsexpressed in trophoblasts of the white-tail deer placenta. Mol. Reprod.Dev.. doi:10.1002/mrd.20669.

Butler, J.E., Hamilton, W.C., Sasser, R.G., Ruder, C.A., Hass,G.M., Wiliams, R.J., 1982. Detection and partial characterizationof two bovine pregnancy-associated proteins. Biol. Reprod. 26,925–933.

Carvalho, A.F., Klisch, K., Miglino, M.A., Pereira, F.T.V., Bevilacqua,E., 2006. Binucleate trophoblast giant cells in the water buffalo(Bubalus bubalis) placenta. J. Morphol. 267, 50–56.

Chakrabarty, A., MacLean II, J.A., Hughes, A.L., Roberts, R.M., Green,J.A., 2006. Rapid evolution of the Trophoblast Kunitz domainproteins (TKDPs) – a multigene family in ruminant ungulates. J.Mol. Evol. 63, 274–282.

Chen, Y., Green, J.A., Antoniou, E., Ealy, A.D., Mathialagan, N.,Walker, A.M., Avalle, M.P., Rosenfeld, C.S., Hearne, L.B., Roberts,R.M., 2006. Effect of interferon-tau administration on endometrium ofnonpregnant ewes: a comparison with pregnant ewes. Endocrinology147, 2127–2137.

Ealy, A.D., Wagner, S.K., Sheils, A.E., Whitley, N.C., Kiesling, D.O.,Johnson, S.E., Barbato, G.F., 2004. Identification of interferon-tauisoforms expressed by the peri-implantation goat (Capra hircus)conceptus. Domest. Anim. Endocrinol. 27, 39–49.

Garbayo, J.M., Remy, B., Alabart, J.L., Folch, J., Wattiez, R., Falmagne,P., Beckers, J-F., 1998. Isolation and partial characterization of apregnancy-associated glycoprotein family from the goat placenta. Biol.Reprod. 58, 109–115.

Garbayo, J.M., Greek, J., Manikkam, M., Beckers, J.-F., Kiesling, D.O.,Ealy, A.D., Roberts, R.M., 2000. Caprine pregnancy-associatedglycoproteins (PAGs): their cloning, expression and evolutionaryrelationship to other PAG. Mol. Reprod. Dev. 57, 311–322.

Gatesy, J., Yelon, D., DeSalle, R., Vrba, E.S., 1992. Phylogeny of theBovidae (Artiodactyla, Mammalia), based on mitochondrial ribosomalDNA sequences. Mol. Biol. Evol. 9, 433–446.

Green, J.A., Xie, S., Roberts, R.M., 1998. Pepsin-related moleculessecreted by trophoblast. Rev. Reprod. 3, 62–69.

Green, J., Xie, S., Szafranska, B., Newman, A., Gan, X., McDowell, K.,Roberts, R.M., 1999. Identification of a new aspartic proteinaseexpressed by the outer chorionic cell layer of the equine placenta. Biol.Reprod. 60, 1069–1077.

Green, J.A., Xie, S., Quan, X., Bao, B., Gan, X., Mathialagan, N.,Beckers, J.F., Roberts, R.M., 2000. Pregnancy-associated bovine andovine glycoproteins exhibit spatially and temporally distinct expressionpatterns during pregnancy. Biol. Reprod. 62, 1624–1631.

Green, J.A., Parks, T., Avalle, M.P., Telugu, B.P., McLain, A.L.,Peterson, A.J., McMillan, W., Mathialagan, N., Hook, R.R., Xie, S.,Robert, R.M., 2005a. The establishment of an ELISA for thedetection of pregnancy-associated glycoproteins (PAGs) in the serumof pregnant cows and heifers. Theriogenology 63, 1481–1503.

Green, M.P., Spate, L.D., Bixby, J.A., Ealy, A.D., Roberts, R.M., 2005b.A comparison of the anti-luteolytic activities of recombinant ovineinterferon-alpha and -tau in sheep. Biol. Reprod. 73, 1087–1093.

Haigh, J.C., Gates, C., Ruder, A., Sasser, R., 1991. Diagnosis ofpregnancy in wood bison using a bovine assay for pregnancy-specificprotein B. Theriogenology 36, 749–754.

Hashizume, K., Ushizawa, K., Patel, O.V., Kizaki, K., Imai, K., Yamada,O., Nakano, H., Takahashi, T., 2007. Gene expression and mainte-

http://dx.doi.org/10.1002/mrd.20669


nance of pregnancy in bovine: roles of trophoblastic binucleate cell-specific molecules. Reprod. Fert. Dev. 19, 79–90.

Haugejorden, G., Waage, S., Dahl, E., Karlberg, K., Beckers, J.F.,Ropstad, E., 2006. Pregnancy-associated glycoproteins (PAG) inpostpartum cows, ewes, goats and their offspring. Theriogenology66, 1976–1984.

Hoffman, L.H., Wooding, F.B.P., 1993. Giant and binucleate trophoblastcells of mammals. J. Exp. Zool. 266, 559–577.

Hughes, A.L., Green, J.A., Garbayo, J.M., Roberts, R.M., 2000. Adaptivediversification within a large family of recently duplicated, placentallyexpressed genes. Proc. Natl. Acad. Sci. USA 97, 3319–3323.

Hughes, A.L., Green, J.A., Piontkivska, H., Roberts, RM., 2003. Asparticproteinase phylogeny and the origin of pregnancy-associated glyco-proteins. Mol. Biol. Evol. 20, 1940–1945.

Jones, C.J.P., Koob, B., Stoddart, R.W., Hoffmann, B., Leiser, R., 1994.Lectin-histochemical analysis of glycans in ovine and bovine near-termplacental binucleate cells. Cell Tissue Res. 278, 601–610.

Kiewisz, J., Melo de Sousa, N., Beckers, J.-F., Vervaecke, H.,Panasiewicz, G., Szafranska, B., 2007. Isolation of pregnancy-associated glycoproteins from placenta of the American bison(Bison bison) at first half of pregnancy. Gen. Comp. Endocr. 155,164–175.

Kiracofe, G.H., Wright, J.M., Schalles, R.R., Ruder, C.A., Parish, S.,Sasser, R.G., 1993. Pregnancy-specific protein B in serum ofpostpartum beef cows. J. Anim. Sci. 71, 2199–2205.

Klisch, K., Hecht, W., Pfarrer, C., Schuler, G., Hoffmann, B., Leiser, R.,1999a. DNA content and ploidy level of bovine placentomal tropho-blast giant cells. Placenta 20, 451–458.

Klisch, K., Pfarrer, C., Schuler, G., Hoffmann, B., Leiser, R., 1999b.Tripolar acytokinetic mitosis and formation of feto-maternal syncytiain the bovine placentome: different modes of the generation ofmultinuclear cells. Anat. Embryol. 200, 229–237.

Klisch, K., Thomsen, P.D., Dantzer, V., Leiser, R., 2004. Genomemultiplication is a generalised phenomenon in placentomal andinterplacentomal trophoblast giant cells in cattle. Reprod. Fertil.Dev. 16, 301–306.

Klisch, K., Bevilacqua, E., Olivera, L.V., 2005a. Mitotic polyploidizationin trophoblast giant cells of the alpaca. Cells Tissues Organs 181, 103–108.

Klisch, K., Sousa, N., Beckers, J.F., Leiser, R., Pich, A., 2005b. Pregnancyassociated glycoprotein-1, -6, -7, and -17 are major products of bovinebinucleate trophoblast giant cells at midpregnancy. Mol. Reprod. Dev.71, 453–460.

Klisch, K., Boos, A., Friedrich, M., Herzog, K., Feldmann, M., Sousa, N.,Beckers, J., Leiser, R., Schuler, G., 2006. The glycosylation ofpregnancy-associated glycoproteins and prolactin-related protein-I inbovine binucleate trophoblast giant cells changes before parturition.Reproduction 132, 791–798.

Krasinski, Z., Raczynski, J., 1967. The reproduction biology of Europeanbison living in reserves and in freedom. Acta Theriologica 12, 407–444.

Lehmann, M., Russe, I., Sinowatz, F., 1992. Detection of lectin bindingsites in the trophoblast of cattle during early pregnancy. Anat. Histol.Embryol. 21, 263–270.

Majewska, M., Panasiewicz, G., Dabrowski, M., Gizejewski, Z., Beckers,J.F., Szafranska, B., 2005. Multiple forms of pregnancy-associatedglycoproteins released in vitro by porcine chorion or placentomal andinterplacentomal explants of wild and domestic ruminants. Reprod.Biol. 5, 185–203.

Majewska, M., Panasiewicz, G., Majewski, M., Szafranska, B., 2006.Localization of chorionic pregnancy-associated glycoprotein family inthe pig. Reprod. Biol. 6, 205–230.

Noden, D.W., De Lahunta, A., 1985. Embryology of Domestic Animals:Normal Development and Congenital Malformations. LippincottWilliams & Wilkins, 384 pp., ISBN: 0683065459.

Panasiewicz, G., Majewska, M., Szafranska, B., 2004a. TrophoblasticcDNA cloning of porcine Pregnancy-Associated Glycoprotein genes(pPAG) and in silico analysis of coded polypeptide precursors. Reprod.Biol. 4, 131–141.

Panasiewicz, G., Majewska, M., Szafranska, B., 2004b. The involvementof luteinizing hormone (LH) and pregnancy-associated glycoproteinfamily (PAG) in pregnancy maintenance in the pig. Reprod. Biol. 4,143–163.

Panasiewicz, G., Majewska, M., Romanowska, A., Dajnowiec, J.,Szafranska, B., 2007. Radiocompetition of secretory pregnancy-associated glycoproteins as chorionic ligands with luteal and uterinegonadotrophin receptors of pregnant pigs. Anim. Reprod. Sci. 99,285–298.

Sasser, R.G., Ruder, C.A., Ivani, K.A., Butler, J.E., Hamilton, W.C.,1986. Detection of pregnancy by radioimmunoassay of a novelpregnancy-specific protein in serum of cows and a profile of serumconcentrations during gestation. Biol. Reprod. 35, 936–942.

Serrano, B., Yaniz, J., Lopez-Gatius, F., Garbayo, J.M., 2006.Pregnancy-associated glycoprotein (PAG) cloning and expression inpre-implantational ovine conceptuses. Reprod. Domest. Anim. 41(Suppl. 2), 104.

Sousa, N.M., Ayad, A., Beckers, J.F., Gajewski, Z., 2006. Pregnancy-associated glycoproteins (PAG) as pregnancy markers in the rumi-nants. J. Physiol. Pharmacol. 57 (Suppl. 8), 153–171.

Szafranska, B., Panasiewicz, G., 2002. The placental expression of theporcine pregnancy-associated glycoprotein (pPAG) gene family exam-ined in situ and in vitro. Anim. Reprod. Sci. 72, 95–113.

Szafranska, B., Xie, S., Green, J., Roberts, R.M., 1995. Porcinepregnancy-associated glycoproteins: new members of the asparticproteinase gene family expressed in the trophectoderm. Biol. Reprod.53, 21–28.

Szafranska, B., Ziecik, A., Okrasa, S., 2002. Primary antisera againstselected steroids (E17b, E1, A4, T, B, F) or proteins (pLH, pPAG) andsecondary antisera against c-globulins – an available tool for studies ofreproductive processes. Reprod. Biol. 2, 187–204.

Szafranska, B., Panasiewicz, G., Majewska, M., Beckers, J.F., 2003.Chorionic expression of heterogeneous products of the PAG (preg-nancy-associated glycoprotein) gene family secreted in vitro through-out embryonic and foetal development in the pig. Reprod. Nutr. Dev.43, 497–516.

Szafranska, B., Majewska, M., Panasiewicz, G., 2004. N-glycodiversity ofthe pregnancy-associated glycoprotein family (PAG) produced in vitro

by trophoblast and trophectoderm explants during implantation,placentation and advanced pregnancy in the pig. Reprod. Biol. 4, 67–89.

Szafranska, B., Panasiewicz, G., Dabrowski, M., Majewska, M.,Gizejewski, Z., Beckers, J.F., 2005a. Chorionic mRNA expressionand N-glycodiversity of pregnancy-associated glycoprotein family(PAG) of the European bison (Bison bonasus). Anim. Reprod.Sci. 88, 225–243.

Szafranska, B., Panasiewicz, G., Majewska, M., Dajnowiec, J.,Kuber, K., Bociek, K., Gizejewski, Z., Goczkowski, M., 2005b.Amplicon-polymorphism of pregnancy-associated glycoproteingene (PAG) comparison of Swedish population of Europeanbison and Polish males with regressive uterus. In: XXVIIth

Congress of the International Union of Game Biologists, Han-nover, Germany, Book of Extended Abstracts, pp. 246–248,ISBN: 3-88412-431-5.

Szafranska, B., Panasiewicz, G., Majewska, M., 2006a. Biodiversity ofmultiple pregnancy-associated glycoprotein (PAG) family: gene clon-ing and chorionic protein purification in domestic and wild eutherians(Placentalia) – a review. Reprod. Nutr. Dev. 5, 481–502.

Szafranska, B., Panasiewicz, G., Majewska, M., 2006b. Porcine preg-nancy-associated glycoprotein family (pPAGs) – as in vitro-producedchorionic ligands for luteal and uterine gonadotropin receptors.Reprod. Biol. 6 (Suppl. 1), 105–111.

Szafranska, B., Panasiewicz, G., Majewska, M., Romanowska, A.,Dajnowiec, J., 2007. Pregnancy-associated glycoprotein family(PAG) – as chorionic signaling ligands for gonadotropin receptors ofcyclic animals. Anim. Reprod. Sci. 99, 269–284.

Vervaecke, H., Schwarzenberger, F., 2006. Endocrine and behavioralobservations during transition of non-breeding into breeding


season in female American bison (Bison bison). Theriogenology66, 1107–1114.

Wooding, F.B.P., 1992. Current topic: the synepitheliochorial placenta ofruminants: binucleate cell fusions and hormone production. Placenta13, 101–113.

Wooding, F.B.P., Flint, A.P.F., 1994. Placentation. In: Lamminig, G.E.(Ed.), Marshall’s Physiology of Reproduction. Chapman Hall, Lon-don, pp. 233–429.

Wooding, F.B., Roberts, R.M., Green, J.A., 2005. Light andelectron microscope immunocytochemical studies of the distri-bution of pregnancy-associated glycoproteins (PAGs) throughoutpregnancy in the cow: possible functional implications. Placenta26, 807–827.

Xie, S., Green, J., Bixby, J.B., Szafranska, B., Demartini, J.C., Hecht, S.,Roberts, R.M., 1997. The diversity and evolutionary relationships ofthe pregnancy-associated glycoproteins, an aspartic proteinase sub-family consisting of many trophoblast-expressed genes. Proc. Natl.Acad. Sci. USA 94, 12809–12816.

Zoli, A.P., Demez, P., Beckers, J.F., Reznik, M., Beckers, A., 1992a. Lightand electron microscopic immunolocalization of bovine pregnancy-associated glycoprotein in the bovine placentome. Biol. Reprod. 46,623–629.

Zoli, A.P., Guilbault, L.A., Delabaut, P., Orates, W.E.B., Beckers, J.F.,1992b. Radioimmunoassay of a bovine pregnancy-associated glyco-protein in serum: its application for pregnancy diagnosis. Biol.Reprod. 46, 83–92.

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