Differential expression and localization of glycosidic residues in in vitro- and in vivo-matured cumulus-oocyte complexes in equine and porcine species

RESEARCH ARTICLE Molecular Reproduction & Development 81:1115–1135 (2014)

Differential Expression and Localization of Glycosidic Residues in In Vitro- and In Vivo-Matured Cumulus-Oocyte Complexes in Equine and Porcine Species
GIANLUCA ACCOGLI,1 CECILE DOUET,2,3,4,5 BARBARA AMBRUOSI,2,3,4,5 NICOLA ANTONIO MARTINO,1 1 MANUEL FILIOLI URANIO, STEFAN DELEUZE,6 MARIA ELENA DELL’AQUILA,1 SALVATORE DESANTIS1*,  GHYLENE GOUDET2,3,4,5*

AND

1

Section of Veterinary Clinics and Animal Productions, Department Emergency and Organ Transplantation (DETO), University of Bari Aldo Moro, Bari, Italy 2 INRA, UMR 85, Physiologie de la Reproduction et des Comportements, Nouzilly, France 3 CNRS, UMR 7247, Nouzilly, France 4
Franois Rabelais, Tours, France Universite 5 IFCE, Nouzilly, France 6
de Me
decine ve
te
rinaire, De
partement des Sciences Cliniques-Clinique Equine, Universite
de Lie ge, Lie ge, Belgium Faculte

SUMMARY Glycoprotein oligosaccharides play major roles during reproduction, yet their function in gamete interactions is not fully elucidated. Identification and comparison of the glycan pattern in cumulus-oocyte complexes (COCs) from species with different efficiencies of in vitro spermatozoa penetration through the zona pellucida (ZP) could help clarify how oligosaccharides affect gamete interactions. We compared the expression and localization of 12 glycosidic residues in equine and porcine in vitro-matured (IVM) and preovulatory COCs by means of lectin histochemistry. The COCs glycan pattern differed between animals and COC source (IVM versus preovulatory). Among the 12 carbohydrate residues investigated, the IVM COCs from these two species shared: (a) sialo- and bN-acetylgalactosamine (GalNAc)-terminating glycans in the ZP; (b) sialylated and fucosylated glycans in cumulus cells; and (c) GalNAc and N-acetylglucosamine (GlcNAc) glycans in the ooplasm. Differences in the preovulatory COCs of the two species included: (a) sialoglycans and GlcNAc terminating glycans in the equine ZP versus terminal GalNAc and internal GlcNAc in the porcine ZP; (b) terminal galactosides in equine cumulus cells versus terminal GlcNAc and fucose in porcine cohorts; and (c) fucose in the mare ooplasm versus lactosamine and internal GlcNAc in porcine oocyte cytoplasm. Furthermore, equine and porcine cumulus cells and oocytes contributed differently to the synthesis of ZP glycoproteins. These results could be attributed to the different in vitro fertilization efficiencies between these two divergent, large-animal models.

Mol. Reprod. Dev. 81: 1115
1135, 2014. ß 2014 Wiley Periodicals, Inc. Received 22 July 2014; Accepted 9 October 2014



Corresponding author: INRA, UMR de Physiologie de la Reproduction et des Comportements 37380 Nouzilly, France. E-mail : [email protected]

ne Goudet Salvatore Desantis and Ghyle authors have the same importance in this paper. Grant sponsor: ONEV project MIUR; Grant number: PONa3_00134
n.254/R&C 18/05/ 2011; Grant sponsor: Institut Franais du Cheval et de l’Equitation’ (IFCE);
Franois Grant sponsor: ‘‘Universite Rabelais de Tours’’, France Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/mrd.22432

Abbreviations: COCs, cumulus-oocyte complexes; Cp, compact; Exp, expanded; Fuc, fucose; GalNAc, bN-acetylgalactosamine; Glc, glucose; GlcNAc, Nacetylglucosamine; IVM, in vitro maturation; KOH, potassium hydroxide; Man, mannose; ZP, zona pellucida

ß 2014 WILEY PERIODICALS, INC.

Molecular Reproduction & Development INTRODUCTION Fertilization involves a highly coordinated sequence of cellular interactions between the sperm and the egg, resulting in the formation of a diploid zygote and, ultimately, a new individual. Early work on lower marine animals suggested that sperm-egg binding relied primarily on carbohydrate recognition (Monroy, 1965; Woodward et al., 1985). Similar observations were later reported in mice, where research on sperm-zona pellucida (ZP) binding identified carbohydrate recognition in the ZP via lectin-like proteins in the sperm plasma membrane (reviewed in Clark and Dell, 2006; Clark, 2011). Yet, the mouse may not be the best model to study fertilization in mammals. Murine ZP is comprised of three glycoproteins: ZPA/ZP2, ZPC/ZP3, and ZP1. This combination of glycoproteins, however, is very specific and has not been found in any other mammals: dog, cat, cattle, and pig ZPs contain ZPA/ZP2, ZPB/ZP4, and ZPC/ZP3 while human, primate, horse, rat, rabbit, and hamster ZPs contain ZPA/ZP2, ZPB/ZP4, ZPC/ZP3, and ZP1 (Goudet et al., 2008; Izquierdo-Rico et al., 2009; Stetson et al., 2012). These differences in ZP composition may account for differences in the glycoprotein matrix and carbohydrate pattern, which can modify the ZP structure and may consequently impact how sperm attach to and penetrate the ZP (Mugnier et al., 2009; Wassarman and Litscher, 2012). Thus, mechanistic insight to how murine sperm-ZP interactions occur may not be directly transferred to other mammals; rather, studies in large-animal models could provide new insights that are directly applicable to large domestic mammals and human. Which oligosaccharide side chains are associated with glycoproteins can fundamentally regulate critical steps of mammalian reproduction, such as oocyte maturation (El€pfer-Petersen Mestrah and Kan, 2002; Rath et al., 2005; To et al., 2008), sperm-egg binding, and fertilization (Tulsiani €pfer-Petersen, 1999; Clark et al., 1997; Dell et al., 1999; To and Dell, 2006). The glycan composition of glycoproteins depends on physiological conditions of organs and their contents (Varki et al., 2009), including ovarian follicles. Cortical granules of hamster preovulatory and ovulated oocytes, for example, exhibit differential expression of some glycoconjugates (El-Mestrah and Kan, 2001, 2002). The oligosaccharide pattern of the ZP also changes between the germinal-vesicle and metaphase-II stages of €pfer-Petersen et al., porcine oocytes (Rath et al., 2005; To 2008). In addition, immature-viable (compact [Cp]) and atretic (expanded [Exp]) equine cumulus-oocyte complexes (COCs) display distinct oligosaccharide patterns that may account for their different in vitro maturation and developmental competence (reviewed in Desantis et al., 2009). Lectins have a specific binding affinity for the sugar residues of glycoconjugates, making them useful tools to characterize the glycan pattern as well as to investigate the cell differentiation and functional maturation (Spicer and Schulte, 1992; Sharon and Lis, 2003). Oligosaccharide chains of glycoproteins are classified into two families: N- and O-linked oligosaccharides. The N-linked group

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carries a reducing, terminal N-acetylglucosamine (GlcNAc) conjugated to the nitrogen residue of asparagine. The Olinked (mucin-type) oligosaccharides, on the other hand, contain a reducing, terminal N-acetylgalactosamine (GalNAc) linked to the hydroxyl group of serine or threonine. These amino-acidic residues can also link fucose (Fuc), glucose (Glc), mannose (Man), and N-acetylglucosamine (GlcNAc) to form other classes of O-linked glycans (reviewed in Lowe and Marth, 2003). Histochemical techniques using lectins have been widely employed to characterize the oligosaccharide pattern of the glycoproteins present in the ovarian follicles of lower vertebrates (Fang and Welsch, 1995; Prisco et al., 2003; Farias et al., 2006; Rodler, 2011; Scillitani et al., 2011; Accogli et al., 2012) as well as of several mammalian species (see Desantis et al., 2009). In mammals, lectin histochemistry has been also applied to investigate the glycan pattern in isolated antral oocytes of humans (Talevi et al., 1997;
nez-Movilla et al., 2004), cow (Verini-Supplizi et al., Jime 1996; de Paz et al., 2001; Velsquez et al., 2007), pig (Parillo et al., 2003; Pastor et al., 2008), and equine (Desantis et al., 2009), as well as in the postovulatory oocytes from mouse
s et al., 1999, 2000), rat (Avile
s et al., 2000) and (Avile
s et al., 2000; El-Mestra and Kan, 2001). hamster (Avile In vitro- and in vivo-matured (preovulatory) COCs are currently used in reproductive technologies. Due to differences between in vivo and in vitro maturation (IVM) conditions, however, some variations in their glycoprofiles are anticipated. Comparative studies on the glycan pattern of in vitro- versus in vivo-matured COCs could help identify key mechanistic elements at play during fertilization, and would provide new insight to the shared versus species-specific involvement of oligosaccharides. In this study, we compared equine and porcine COCs because of their contrasting efficiencies of in vitro sperm penetration of the ZP. In pigs, in vitro fertilization rates are higher than 80%, and the reported levels of polyspermy in these situations often exceed 50% (Abeydeera and Day, 1997; Funahashi and Day, 1997; Day, 2000; Nagai et al., 2006). In the equine, in vitro fertilization rates are lower than 60%, and polyspermy is very rare (Palmer et al., 1991; Dell’Aquila et al., 1996; Alm et al., 2001; Hinrichs et al., 2002; McPartlin et al., 2009; Ambruosi et al., 2013). This difference in efficiency could be attributed to the specific components required for ZP penetration, so comparison of these two models could help identify elements that distinguish their mechanisms of fertilization. We, thus characterized and localized the oligosaccharide sequences of glycoproteins in in vitro- and in vivo-matured equine and porcine COCs using a panel of lectins frequently used in glycohistochemistry.

RESULTS The glycan patterns of equine and porcine COCs were evaluated for in vitro- and in vivo-matured samples, including: equine in vitro-matured COCs (IVM-metaphase-II stage, n ¼ 57 Cp COCs and 43 Exp COCs) and in vivomatured metaphase-II stage COCs (n ¼ 18), and porcine

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COCs (IVM-metaphase-II stage, n ¼ 80) and in vivo-matured metaphase-II stage COCs (n ¼ 70). The lectin histochemistry was considered reliable based on the absence of staining produced in the negative-control procedures (Fig. 1). In addition, potassium hydroxide (KOH)-sialidase treatment abolished the reactivity for MAL II and SNA, but did not significantly modify WGA staining. The lectins used and their sugar specificities can be found in Table 1. Summaries of results can be found for equine COCs in Table 2 and Figure 2, and for porcine COCs in Table 3 and Figure 9. The subsections that follow cover the histochemistry associated with the identification of specific sugar residues on COCs from each animal.

Equine Sialic acid residues (MAL II and SNA). Glycans terminating in NeuNAca2,3Galb1,3( NeuNAca2
6) GalNAc were revealed using MAL II (Fig. 3A
C). This terminal residue was detected in cumulus cells of both IVM and preovulatory COCs, specifically in the outer region of the ZP from IVM COCs and in the whole ZP of preovulatory oocytes, although the intensity of staining was stronger in the inner region. MAL II binding was also observed in cortical granules of IVM Exp oocytes (Fig. 3B). Pre-treatment with potassium hydroxide (KOH)-sialidase to remove neuraminic acid abolished MAL II binding. The area occupied by MAL II binders was significantly lower in cumulus cells and significantly higher in the ZP of preovulatory COCs compared to IVM COCs (Fig. 2A
B). SNA, which is specific for NeuNAca2,6Gal/GalNAc terminal residues, produced the same staining pattern as MAL II (Fig. 3D
F), although the ooplasm showed higher stain-

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ing intensity in IVM Exp oocytes than in preovulatory ones (Figs. 3E
F). SNA binding sites disappeared after KOHsialidase treatment. The density of SNA binding sites was significantly higher in cumulus cells and the ooplasm, and lower in the ZP of IVM Exp COCs (Fig. 2).

Terminal galactose residues (PNA and RCA120). Terminal Galb1,3GalNAc residues were revealed by PNA staining in the cumulus cells of IVM Cp COCs and, to a lesser extent, in preovulatory COCs (Figs. 2A and 4 A,C) as well as in the outer region of the ZP from IVM Cp COCs (Fig. 4A) and in cortical granules of preovulatory oocytes (Fig. 4C). No binding sites were observed in IVM Exp COCs, however (Fig. 4B). Terminal Gab1,4GlcNAc bound RCA120 in cumulus cells from IVM Exp COCs and preovulatory COCs (Fig. 4D
F), the ZP of in vivo-matured oocytes (Fig. 4F) and the ooplasm of IVM Exp COCs, which also possessed binding sites in their cortical granules (Fig. 4E). Quantification revealed a significantly higher percentage of RCA120 binders in cumulus cells of IVM Exp COCs than in in vivo-matured ones (Fig. 2A).

Terminal N-acetylgalactosamine residues (HPA, SBA, DBA). HPA revealed terminal aGalNAc in the cumulus cells of all analysed COCs and in the cytoplasm of IVM oocytes (Fig. 5A
C). The area occupied by positive cumulus cells was significantly higher in IVM Cp COCs than IVM Exp and preovulatory COCs (Fig. 2A). SBA, which is specific for terminal a/bGalNAc, showed different staining intensity among the cumulus cells of the investigated COCs (Figs. 5D
F). This lectin bound the

Figure 1. Negative staining of equine and porcine COCs. (A) Equine preovulatory COC incubated with lectin-free substrate medium (Tris-buffered saline followed by incubation in a solution of 0.05% diaminobenzidine and 0.003% H2O2). (B) Porcine preovulatory COC incubated with a 0.5 M galactoseRCA120 medium. Scale bars, 75 mm.

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TABLE 1. Lectin Used, Their Sugar Specificities and the Inhibitory Sugar Used in Control Experiments Lectin Abbreviation

Source of lectin

Conc (mg/ml)

Sugar specificity NeuNAca2
3Galb1
3 ( NeuNAca2
6)GalNAc NeuNAca2,6Gal/GalNAc terminal Galb1,3GalNAc terminal Gab1,4GlcNAc terminal aGalNAc terminal a/bGalNAc terminal GalNAca1,3(LFuca1,2) Galb1,3/4GlcNAcb1 terminal/internal aMan>aGlc terminal/internal bGlcNAc >>NeuNAc terminal D-GlcNAc terminal L-Fuca1,2Galb1, 4GlcNAcb terminal a L-Fuc

MAL II

Maackia amurensis

15

SNA PNA* RCA120 HPA SBA* DBA

Sambucus nigra Arachis hypogaea Ricinus communis Helix pomatia Glycine max Dolichos biflorus

15 25 25 25 25 25

Con A WGA

Canavalia ensiformis Triticum vulgaris

15 25

GSA II UEAI*

Griffonia simplicifolia Ulex europaeus

20 25

LTA

Lotus tetragonolobus

25

Inhibitory sugar

References

NeuNAc

Geisler and Jarvis, 2011

NeuNAc Galactose Galactose GalNAc GalNAc GalNAc

Shibuya et al., 1987 Lotan et al., 1975 Baenziger and Fiete, 1979 Roth, 1984 €m et al., 1977 Hammarstro Sharon and Lis, 2003

Mannose GlcNAc

Goldstein and Hayes, 1978 Debray et al., 1981

GlcNAc Fucose

Shanker Iyer et al., 1976 Sugii and Kabat, 1982

Fucose

Pereira and Kabat, 1974

Fuc, fucose; Gal, galactose; GalNAc, N-acetylgalactosamine; Glc, glucose; GlcNAc, N-acetylglucosamine; Man, mannose; NeuNAc, N-acetyl neuraminic (sialic) acid. *HRP-conjugated lectins. All other lectins were biotinylated.

entire ZP of IVM Cp (Fig. 5d) and the outer region of IVM Exp COCs (Fig. 5E
F). The ooplasm of IVM Exp was stained more intensely than IVM Cp COCs (Figs. 5D
E), and did not react with preovulatory oocytes (Fig. 5D). The density of SBA binders was significantly higher in the cumulus cells of IVM COCs compared to preovulatory COCs (Fig. 2A), and in the ooplasm of IVM Exp compared to IVM Cp COCs (Fig. 2C). DBA revealed the presence of terminal GalNAca1,3 (LFuca1,2) Galb1,3/4GlcNAcb1 only in cumulus cells of IVM Exp COCs (data not shown).

Terminal/Internal Mannose residues (Con A). Con A (Fig. 6A
B) revealed high-mannose glycans in the cumulus cells from all types of COCs and in the cytoplasm of IVM Cp COCs and preovulatory oocytes. The area

occupied by positive cumulus cells was significantly higher in the in vivo-matured COCs than in IVM complexes (Fig. 2A).

N-acetylglucosamine residues (KOH-sialidaseWGA, GSA II). KOH-sialidase-WGA, which is specific

for terminal/internal bGlcNAc, reacted only with the IVM Exp COCs. Staining was observed in the cumulus cells, ZP, and the ooplasm (Fig. 7A). GSA II, which identifies terminal D-GlcNAc glycans, stained the cumulus cells of preovulatory COCs more intensely than IVM COCs, and reacted with the ZP of preovulatory oocytes and the ooplasm of all investigated COCs (Fig. 7B
C). The area occupied by GSA II binding sites was significantly lower in the cumulus cells of preovulatory compared with IVM COCs (Fig. 2A).

TABLE 2. Lectin-Binding Pattern of Equine In Vitro- and In Vivo-Matured COCs Cumulus cells Lectin MAL II KOH-s-MAL II SNA KOH-s-SNA PNA RCA120 HPA SBA DBA Con A KOH-s-WGA GSA II UEA I LTA

Zona pellucida

Ooplasm

IVM Cp

IVM Exp

PREOV

IVM Cp

IVM Exp

PREOV

IVM Cp

IVM Exp

PREOV

þ
þ
þ
þ þ
þ
þ þ þ

þ
þ

þ þ þ þ þ þ þ þ þ

þ
þ
þ þ þ þ
þ
þ



þo
þo
þo

þ



þo


þo
þo



þo

þ




þ/þi
þ/þi

þ




þ









þ þ
þ
þ þ þ

þcg
þ

þ/þcg þ þ

þ þ þ þ



þsc
þcg



þ
þ þ þ

cg, cortical granules; co, cortical ooplasm; Cp, COCs retrieved with compact cumulus investment; Exp, COCs recovered with expanded cumulus; KOH-s, potassium hydroxide-sialidase pretreatment; i, inner region of the ZP; IVM, in vitro-matured COCs; o, outer region of the ZP; PREOV, preovulatory COCs; sc, scattered granularpositive staining pattern;
, negative reaction; þ, positive reaction.

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Figure 2. Lectin-positive area in (A) cumulus cells, (B) the zona pellucida, and (C) the ooplasm of equine IVM compact (Cp) and expanded (Exp) and in vivo-matured, preovulatory COCs. The values are expressed as means  standard error. Identical lowercase letters among bars represent statistical insignificance (P > 0.05). RCA, RCA120; s-WGA, KOH-sialidase-WGA.

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TABLE 3. Lectin Binding Pattern of Porcine In Vitro- and In Vivo-Matured COCs Cumulus cells Lectin MAL II KOH-s-MAL II SNA KOH-s-SNA PNA RCA120 HPA SBA DBA Con A KOH-s-WGA GSA II UEA I LTA

Zona pellucida

Ooplasm

IVM

PREOV

IVM

PREOV

IVM

PREOV

þ
þ

þ



þ

þ

þ
þ

þ


þ þ þ
þ

þ
þi,o

þ
þ
þ þ




þ



þ
þ

þ




þcg
þ

þ
þsc
þ þ þ
þ/þcg



þ

þ


þ þ þ
þ/þcg

cg, cortical granules; i, inner region of the ZP; IVM, in vitro matured COCs; KOH-s, potassium hydroxide-sialidase pretreatment; o, outer region of the ZP; PREOV, preovulatory COCs; sc, scattered granular-positive staining pattern;
, negative reaction; þ, positive reaction.

Terminal Fucose residues (UEA I, LTA). UEA I revealed terminal L-Fuca1,2Galb1,4GlcNAcb in the cumulus cells of IVM COCs, in the outer region of ZP of IVM Cp COCs (Figs. 8A
B), and in the oocytes of three types of COCs with a minor amount in IVM Exp COCs (Fig. 8A
C). The stained area was significantly higher in the cumulus

cells of IVM Cp compared to IVM Exp COCs, and in the ooplasm of IVM Cp and preovulatory COCs compared to IVM Exp complexes (Figs. 2A
B). Other aL-Fuc-terminating glycans were detected using LTA. LTA binders were observed in the cumulus cells of IVM COCs and in the oocyte of all investigated COCs

Figure 3. Localization of sialic acid-terminating glycans using (A
C) MAL II and (D
F) SNA in equine IVM and preovulatory COCs. cc, cumulus cells; Cp, compact COC; Exp, expanded COC; o, oocyte; *, ZP; arrow, cortical granules; arrowhead, inner region of the ZP; double arrowheads, outer region of the ZP. Scale bars, 75 mm. The inset (B) shows the magnified zone of the boxed region. Inset scale bar, 20 mm.

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Figure 4. Localization of Galb1,3GalNAc- and Galb1,4GlcNAc-terminating glycans in equine IVM and preovulatory COCs using (A
C) PNA and (D
F) RCA120, respectively. cc, cumulus cells; Cp, compact COC; Exp, expanded COC; o, oocyte; *, ZP; arrow, cortical granules. Scale bars, 75 mm. Insets (C and E) show the magnified zone of the boxed regions. Inset scale bar, 15 mm.

(Fig. 8D
F). The staining intensity of the in vitro-matured oocytes was higher than in vivo-matured ones. The area occupied by LTA binders was significantly higher in cumulus cells of IVM Exp compared to IVM Cp COCs, whereas it was significantly lower in the ooplasm of IVM Exp and preovulatory COCs (Fig. 2).

Porcine Sialic acid residues (MAL II and SNA). MAL II reacted with cumulus cells and the ZP of both COCs types, as well as with cortical granules of IVM COCs (Fig. 10A
B). After KOH-sialidase treatment, however, no positive reaction was observed. The area of stained cumulus cells was significantly higher in IVM COCs compared with the in vivomatured COCs (Fig. 9A). SNA showed affinity for cumulus cells and the ooplasm (Fig. 10C
D), and with the outer and inner regions of the ZP from IVM COCs (Fig. 10C). KOH-sialidase treatment removed SNA binding sites. As with MAL II, cumulus cells were significantly more reactive for SNA in IVM COCs (Fig. 9A).

Terminal Galactose residues (PNA and RCA120). PNA did not react with the investigated COCs, whereas RCA120 stained the cumulus cells, the ZP, and oocytes of both IVM and preovulatory COCs (Fig. 11A
B). The

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staining density of cumulus cells was significantly higher from IVM than preovulatory COCs (Fig. 9A).

Terminal N-acetylgalactosamine residues (DBA, HPA, SBA). HPA and DBA were unreactive with the investigated COCs. SBA, however, did stain the cytoplasm of IVM oocytes and the ZP of both the IVM and preovulatory COCs (Fig. 11C
D). Quantification of the stained ZP area did not show significant differences between these two types of COCs.

Terminal/Internal Mannose residues (Con A). Con A bound the cumulus cells of preovulatory COCs, the ZP from IVM COCs, and the oocyte cytoplasm from both COC types (Fig. 11E
F). The area occupied by Con A binders was significantly higher in the ooplasm of preovulatory compared with IVM COCs (Fig. 9C). N-acetylglucosamine residues (KOH-sialidaseWGA, GSA II). KOH-sialidase-WGA reacted with cumulus cells, the ZP, and the ooplasm of all COCs, showing stronger staining in the cumulus cells and ooplasm of IVM compared to preovulatory COCs (Fig. 12A
B). The area positive for KOH-sialidase-WGA binders was

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Figure 5. Localization of GalNAc-terminating glycans using (A
C) HPA and (D
F) SBA in equine IVM and preovulatory COCs. Cp, compact COC; Exp, expanded COC; *, ZP. Scale bars, 75 mm.

significantly higher in the cumulus cells of IVM compared to preovulatory COCs (Fig. 9A). GSA II bound the cumulus cells of preovulatory COCs and stained more strongly in the oocytes of IVM than in vivomatured COCs (Fig. 12C
D). The area positive for GSA II staining was significantly higher in the ooplasm of IVM compared to preovulatory COCs (Fig. 9C).

Terminal Fucose residues (UEA I, LTA). No binding was observed for UEA I, whereas LTA reacted with cumulus cells, cortical granules, and the cytoplasm of the investigated oocytes (Fig. 12E). No significant difference was observed in the positive-stained area for either cumulus cells or ooplasm from IVM compared to preovulatory COCs (Fig. 9A
C).

Figure 6. Localization of mannosylated glycans in equine IVM COCs using Con A. cc, cumulus cells; Cp, compact COC; Exp, expanded COC; o, oocyte. Scale bars, 75 mm.

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Figure 7. Localization of GlcNAc-containing glycans with (A) KOH-sialidase-WGA (s-WGA) and (B
C) GSAII in equine IVM and preovulatory COCs. cc, cumulus cells; Cp, compact COC; Exp, expanded COC; o, oocyte; *, ZP. Scale bars, 75 mm.

DISCUSSION In the present study, we analyzed the in situ localization of oligosaccharide chains in equine and porcine COCs after IVM or in vivo maturation. Figure 13 summarizes the differences in the distribution and staining intensity. The comparison between in vitro- and in vivo-matured COCs pointed out differences in the presence or distribution of some carbohydrates that could be connected with the lower

competence of IVM COCs to undergo fertilization and development, as well as interspecies differences that might affect the efficiency of the in vitro penetration of sperm through the ZP.

Cumulus Cells The complexity of glycan profiles differed dramatically between the two species studied. Equine cumulus cells

Figure 8. Localization of aL-Fuc-terminating glycans with (A
C) UEAI and (D
F) LTA in equine IVM and preovulatory COCs. cc, cumulus cells; Cp, compact COC; Exp, expanded COC; o, oocyte; double arrowheads, outer region of the ZP. Scale bars, 75 mm.

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Figure 9. Lectin-positive area in (A) cumulus cells, (B) the zona pellucida, and (C) the ooplasm of porcine

IVM and preovulatory COCs. The values are expressed as means  standard deviation. Asterisk indicates statistical significance (P < 0.05). RCA, RCA120; s-WGA, KOH-sialidase-WGA.

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Figure 10. Localization of sialic acids revealed using (A
B) MAL II and (C
D) SNA in porcine IVM and preovulatory COCs. cc, cumulus cells; o, oocyte; *, ZP; arrow, cortical granules; arrowhead, inner region of the ZP; double arrowheads, outer region of the ZP. Scale bars, 75 mm.

consistently possessed O-linked glycans terminating with NeuNAca2,3Galb1,3GalNAc and/or GalNAc (MAL II, HPA, SBA) as well as N-linked glycans terminating with NeuNAca2,6Gal/GalNAc (Con A and SNA) (Spicer and Schulte, 1992; Geisler and Jarvis, 2011). GlcNAc generally terminated both O- and N-linked oligosaccharides (GSA II) (Varki et al., 2009). Notably, cumulus cells from equine IVM COCs were characterized by fucosylated oligosaccharides (UEA I and LTA); further maturation to Exp IVM COCs additionally enriched for O-linked glycans with terminal GalNAca1,3(LFuca1,2) Galb1,3/4GlcNAcb1 (DBA) and/or internal bGlcNAc residues (KOH-sialidase-WGA). In contrast, porcine cumulus cells possessed fewer overall residue types. A notable distinction between in vitro- and in vivo-matured COCs was the presence of high mannose Nlinked glycans and terminal D-GlcNAc residues (Con A, GSA II) in only preovulatory populations. These results suggest that the glycan profile of cumulus cells is (1) subjected to species-specific modification during late oocyte maturation; (2) commonly represented by O- and N-linked sialoderivates; and (3) more complex in equine than in porcine COCs. The quantitative expression of glycans is overall lower in preovulatory COCs of both species, except for

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Con A binders, whose abundance increased in equine preovulatory cumulus cells. The demonstrated intra- and interspecific differences likely contribute to the differences in sperm behavior since cumulus cells functionally support oocyte growth and maturation as well as active synthesize and secretion specific ZP glycoproteins within porcine and equine antral follicles (Parillo et al., 2003; Desantis et al., 2009). Furthermore, Lay et al. (2013) recently demonstrated that cumulus cells contribute to ZP glycosylation during maturation of porcine oocytes, and that this process is necessary for sperm penetration. Synthesis of ZP glycoproteins
s in the follicular cells has also been reported in rats (Avile €lle et al. 1998), dogs (Blackmore et al., et al., 1994), cows (Ko 2004; Parillo et al., 2005), and mice (Xie et al., 2010).

Zona Pellucida The ZP is a relatively thick and viscous glycoprotein coat that surrounds all mammalian oocytes. Ovulated with the oocyte, this extracellular structure is responsible for binding spermatozoa to the oocyte and inducing the acrosome reaction. This study revealed a different glycan pattern in the ZP of the equine and porcine IVM and preovulatory COCs.

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ACCOGLI

ET AL.

Figure 11. Localization of glycans terminating with Gab1,4GlcNAc or GalNAc, and terminal/internal mannose in porcine IVM and preovulatory COCs using (A
B) RCA120, (C
D) SBA, and (E
F) Con A, respectively. cc, cumulus cells; o, oocyte; *, ZP. Scale bars, 75 mm.

The ZP of all investigated equine COCs expressed a2,3and 2,6-linked NeuNAc in O- and N-glycans, respectively (Geisler and Jarvis, 2011). This suggests that sialoglycoproteins are a common constituent of the equine ZP. Interestingly, the topography of these sialoglycoproteins differed in the ZP of IVM versus preovulatory COCs, occupying the outer

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region of the ZP in IVM COCs versus the entire ZP

mostly in the inner region

of in vivo-matured COCs. This suggests that negatively charged glycans contribute structurally to distinct ZP region, which can thereby affect sperm penetration. Additionally, the ZP of Cp and Exp IVM COCs differed from the ZP of preovulatory COCs by the presence of terminal

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GLYCOSIDIC RESIDUES

IN

EQUINE

AND

PORCINE COCs

Figure 12. Localization of GlcNAc (a,b,c,d) and aL-Fuc (e) in porcine IVM and preovulatory COCs using (A
B) KOH-sialidase-WGA (s-WGA) or (B
D) GSA II and (E) LTA, respectively. cc, cumulus cells; o, oocyte; arrow, cortical granules; *, ZP. Scale bars, 75 mm.

bGalNAc (SBA binders) (enriched in IVM COCs) and terminal Gab1,4GlcNAc and/or GlcNAc (RCA120 and GSA II) (enriched in in vivo-matured COCs). We even observed differences within the IVM group, which showed the ZP glycan composition of Galb1,3GalNAc and fucosylated residues in the outer region of Cp COCs whereas Exp ones showed uniform distribution of internal GlcNAc residues (KOH-sialidase-WGA). This differential expression of glycoproteins in the ZP of IVM and preovulatory COCs clearly indicate that

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IVM conditions do not appropriately mimic the in vivo maturation process. Indeed, the significant increase in MAL II and SNA binders that we observed on the ZP of in vivo-matured equine oocytes suggests that sialoglycoconjugates play a crucial role at the end of oocyte maturation, and thus may participate in sperm-ZP interactions. A previous analysis of the ZP in immature COCs (Desantis et al., 2009) and our study both report the disappearance of Con A binding sites in the ZP of all matured COCs,

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equine IVM compact (Cp) COCs compared with in vivo-matured COCs, and (D) equine IVM expanded (Exp) COCs compared with in vivo matured-COCs. R, amino acid sequence to which the sugar residue is linked; R’, unknown carbohydrate sequence to which the sugar residue is linked; >, increased expression of the sugar residue;