MOLECULAR REPRODUCTION AND DEVELOPMENT 74:851–859 (2007)
Equine Zona Protein Synthesis and ZP Structure
During Folliculogenesis, Oocyte Maturation,
and Embryogenesis
SABINE KÖLLE,1* CLOTILDE S. DUBOIS,2 MAUD CAILLAUD,2 CÉCILE LAHUEC,2 FRED SINOWATZ,3
2
AND GHYLÈNE GOUDET
1
Institute of Veterinary Anatomy, University of Giessen, Giessen, Germany
2
INRA-CNRS-University of Tours-Haras Nationaux, Nouzilly, France
3
Institute of Veterinary Anatomy II, University of Munich, Munich, Germany
ABSTRACT In the equine, the zona pellucida
(ZP) is the major barrier to successful in vitro fertiliza-
tion. Therefore the aim of our studies was to analyze
species-specific features of the equine ZP in regard to
structure and glycoprotein ZPB and ZPC expression
sites during oocyte development and embryogenesis.
The equine ZP revealed high immunological cross-
reactivity to porcine ZPB and ZPC. In the ovary, the
distribution of ZPB and ZPC was co-localized and
correlated with the developmental stage of the follicle.
ZPB and ZPC expression started in the oocyte of the
late primordial and primary follicle. In the secondary
follicle, both the oocyte and the cumulus cells con-
tributed to ZPB and ZPC synthesis. After in vivo
maturation the oocyte stopped ZPB and ZPC produc-
tion whereas the cumulus cells continued synthesis.
Contrary, in vitro matured (IVM) cumulus-oocyte-
complexes (COCs) revealed a reverse expression
pattern. This was correlated to alterations in the
distribution, number, and size of pores in the ZP. In
the zona, N-acetylglucosamine residues were co-
localized with ZPC. The acellular glycoprotein capsule
surrounding early equine embryos was negative for
ZPB and ZPC. Our results imply that in the horse ZPB
and ZPC glycoprotein expression is differentially reg-
ulated during folliculogenesis, oocyte maturation, and
embryogenesis. Contrary to the bovine and porcine,
zona protein synthesis during in vivo maturation is
completely overtaken by the cumulus cells implying
that in the horse these cells are crucial for zona
integrity. During IVM, the cumulus cells lose their
ability to synthesize glycoproteins leading to alterations
in the zona structure. Mol. Reprod. Dev. 74: 851–
859, 2007. ß 2007 Wiley-Liss, Inc.
Key Words: equine zona pellucida; ZPB; ZPC; N-
acetylglucosamine; scanning electron microscopy;
confocal laser scanning microscopy
INTRODUCTION
Very scarce studies have been performed on the
composition and structure of the equine ZP. Up to now,
ß 2007 WILEY-LISS, INC.
three zona glycoproteins with apparent molecular
masses of 60, 75, and 90 kDa (Miller et al., 1992)
have been identified. It has been demonstrated that
equine ZP glycoproteins share common antigens with
bovine and porcine ZP glycoproteins (Liu and Shivers,
1982; Miller et al., 1992), which are named ZPA, ZPB,
and ZPC (Harris et al., 1994). Especially cloning and
characterization of the ZPC gene and cDNA from a
variety of mammalian species such as dog, cat, pig, and
cattle have shown a high homology of this gene (Harris
et al., 1994; McLeskey et al., 1998).
During equine in vitro fertilization the major barrier
to successful fertilization is the zona pellucida (ZP),
since the sperms bind to the ZP but are not able to
penetrate. As a consequence, in the horse, in vitro
fertilization (IVF) rates are lower than 30% (Palmer
et al., 1991; Dell’Aquila et al., 1996; Alm et al., 2001).
These low fertilization rates are distinctly improved by
zona drilling or partial zona dissection (Choi et al., 1994;
Azuma et al., 1995). Intracytoplasmic sperm injection
(ICSI) is the most successful technique of equine-
assisted reproduction because it bypasses the critical
fertilization events of ZP binding and penetration
(Squires et al., 1996; Dell’Aquila et al., 1997; Li et al.,
2000).
In most mammals, the glycoprotein ZPC plays a
central role in the fertilization process. It provides
receptors for the species-specific binding of spermatozoa
and is involved in the induction of the sperm acrosome
reaction (reviewed by Prasad et al., 2000; Wassarman
et al., 2004; Wassarman, 2005). Because of common
antigen determinants in different species, antibodies
directed against ZPC can prevent sperm binding to the
zona and induce infertility without species-specificity
Grant sponsor: Haras Nationaux, France; Grant sponsor: Region
Centre, France.
*Correspondence to: Dr. Sabine Kölle, Institute of Veterinary
Anatomy II, University of Giessen, Frankfurter Street 98, 35392
Giessen, Germany. E-mail: sabine.koelle@vetmed.uni-giessen.de
Received 15 December 2005; Accepted 11 February 2006
Published online 24 January 2007 in Wiley InterScience
(www.interscience.wiley.com).
DOI 10.1002/mrd.20501
Molecular Reproduction and Development. DOI 10.1002/mrd
852 S. KÖLLE ET AL.
(Dunbar, 1990; Dunbar, 1995). In the horse, the
mechanisms of sperm binding, sperm penetration, and
fertilization of the oocyte have not been clarified. In the
mouse, b1,4-galactosyltransferase (GalT) located in
the sperm plasma membrane binds to N-terminal N-
acetylglucosamine residues on ZPC oligosaccharides
(reviewed by Nixon et al., 2001; Shi et al., 2001) and is
thought to induce the acrosome reaction by GalT
clustering (Villalobo and Gabius, 1998). After the pene-
tration of the first spermatozoon, both the ZPA and the
ZPC proteins are modified so that the ZP serves as a site
of secondary block to polyspermy (Dean, 1992; Wassar-
man et al., 2005). Regarding the carbohydrate residues,
this phenomenon can be explained by the global release
of hexosaminidase from the cortical granules, which
destroys the GalT recognition site on ZPC (Nixon et al.,
2001) and accounts for the loss of ZPC’s sperm-binding
activity following fertilization (Miller et al., 1993).
However, recent genetic data in mice indicate that not
a single protein or carbohydrate, but the supramolecu-
lar structure of the ZP determines the loss of sperm-
binding activity. Thus, the cleavage of ZPA seems to
modulate the three-dimensional structure of the ZP,
thus inhibiting sperm binding after fertilization (Dean,
2003; Hoodbhoy and Dean, 2004).
The zona glycoprotein ZPC is not only involved in the
fertilization process but also affects growth and differ-
entiation of the oocyte. Mice, which are homozygous
nulls for ZPC not only lack the ZP, but also show
misshapen and developmentally retarded oocytes (Was-
sarman et al., 2004). There is increasing evidence that
ZP proteins also play a role in granulosa cell differentia-
tion (Prasad et al., 2000). Consequently the female mice
of ZPC knockout mice are completely infertile (Liu et al.,
1996; Rankin et al., 1996).
The origin of mammalian ZP glycoproteins has been
controversial for a long time. There has been recent
evidence that glycoproteins are synthesized by:
(a) either the oocyte alone; or (b) by the follicle cells; or
(c) by both the oocyte and the follicle cells (reviewed by
Sinowatz et al., 2001a). Thus, in mice possessing a thin
ZP, glycoproteins are exclusively produced by the oocyte
(Bleil and Wassarman, 1980a,b; Flechon et al., 1984),
whereas in the cat the ZP is exclusively synthesized by
granulosa cells (Jewgenow and Rudolph, 2001). In other
species such as pig, cow, dog, rabbit, and monkey reveal-
ing a well-developed zona both the oocyte and the follicle
cells contribute to ZP glycoprotein synthesis (Dunbar
et al., 1994; Sinowatz et al., 1995; Grootenhuis et al.,
1996; Kölle et al., 1996; Martinez et al., 1996; Kölle et al.,
1998; Blackmore et al., 2004). Generally the ability of
synthesizing ZP proteins is strongly correlated to the
developmental capacity of the cumulus-oocyte-complex
(Kölle et al., 1998). This is corroborated by findings that
the mean thickness of the ZP is greater in ovulated
oocytes than in oocytes matured in vitro (Funahashi
et al., 2000). Besides the ability of zona glycoprotein
synthesis the three-dimensional structure of the ZP
is thought to influence successful fertilization of the
oocyte.
In the horse, however, information on species-specific
features of the ZP especially in regard to structure and
topography of equine glycoprotein expression sites is
lacking. Therefore the aim of our studies was to analyze
the developmental expression of the zona glycoproteins
ZPB and ZPC in close correlation to the zona structure
during folliculogenesis, oocyte maturation, and embry-
ogenesis by confocal laser scanning microscopy, light
microscopy, and scanning electron microscopy.
MATERIALS AND METHODS
Preparation of Ovaries
Ovaries of 43 horses (breeds: Bavarian Horse, Han-
noverian Horse, Holstein Horse) aged from 2 to 17 years
were obtained from the local slaughterhouse. Immedi-
ately after slaughtery pieces of ovarian medulla as
well as the ovulation fossa were fixed in Bouin’s fluid
overnight, dehydrated in graded series of ethanols and
embedded in paraffin. Sections (5 mm) were cut on a
Leica microtome (Leica, Bensheim, Germany).
Collection of Cumulus-Oocyte-Complexes (COCs)
Ovaries of horses collected from a local slaughter-
house were transported to the laboratory in 0.9% NaCl
at 378C. Cumulus-oocyte-complexes (COCs) were aspi-
rated from follicles using a 18.5 gauge needle at 50 mm
Hg vacuum pressure before and after ovarian slicing.
In Vitro Maturation of COCs
In vitro maturation (IVM) was performed in tissue
culture medium 199 (TCM 199, Sigma, Saint Quentin
Fallavier, France) supplemented with Earle’s salts and
20% fetal calf serum (Sigma) and 50 ng/ml Epidermal
Growth Factor (Sigma). Maturation took place in a
humidified atmosphere of 5% CO2 in air at 38.58C for
30 hr.
Collection of Preovulatory
COCs Matured In Vivo
COCs matured in vivo were obtained from adult
cycling pony mares (aged 2–10 years) by transvaginal
ultrasound-guided aspiration. Ovarian activity was
regularly assessed by rectal ultrasonography. When
the largest follicle reached 33 mm in diameter, ovulation
was induced by intravenous injection of 25 mg of crude
equine gonadotropin (CEG) (Duchamp et al., 1987). The
largest follicle was punctured 34 hr after induction of
ovulation as previously described (Goudet et al., 1997).
During follicle puncture, mares were sedated with deto-
midine (Domosedan, 0.3 ml/mare, i.v., Pfizer, Pocé sur
Cisse, France) and the rectum was relaxed with
propantheline bromide (60 mg/mare, i.v., Sigma). After
follicular fluid aspiration, follicles were flushed with
PBS containing heparin (5 IU/ml, Sanofi, Paris, France)
at 378C. Aspirated fluids were examined with a stereo-
microscope and COCs were collected. After the puncture
sessions, the mares received an antibiotic injection
(Intramicine, 4 g benzylpenicillin procaı̈ne/mare, and
 Molecular Reproduction and Development. DOI 10.1002/mrd
ZPB AND ZPC EXPRESSION AND ZP STRUCTURE IN THE HORSE
4
106 IU dihydrostreptomycin/mare, i.m., Ceva,
Libourne, France).
Recovery of Embryos
Collection of embryos was performed from two mares
in three different experiments. As soon as clinical signs
of estrus occurred, the mares were inseminated two to
three times with fresh semen of stallions of proven
fertility. On days 8, 9, and 10 after insemination, blasto-
cysts were obtained by uterine flushing under epidural
anesthesia. In total, one 8-day-old embryo, two 9-day-old
embryos, and one 10-day-old blastocyst were obtained.
Production of Polyclonal Antibodies
Polyclonal antibodies to HPLC-purified pZPB and
pZPC were produced according to Sinowatz et al. (1995).
These antibodies were validated by Topper et al. (1997)
by two-dimensional polyacrylamide gel electrophoresis
and have been widely used to localize ZPB and ZPC in
the porcine (Sinowatz et al., 1995) and bovine (Kölle
et al., 1998). As demonstrated by Bousquet et al. (1987)
common antigens are shared by equine, porcine, and
bovine ZP. Using polyacrylamide gel electrophoresis
Miller et al. (1992) were able to demonstrate that
anti-porcine ZP antibodies recognize the equine ZP
glycoproteins.
Confocal Laser Scanning
Microscopy of Denuded Oocytes
Localization of ZPB, ZPC, and N-acetylglucosamine
residues was compared in oocytes before maturation as
well as after maturation in vivo and in vitro by using
confocal laser scanning microscopy. COCs were rinsed
in PBS and mechanically stripped by using a small glass
pipette. To determine the nuclear stage, denuded
oocytes were stained with 1 mg/ml bisbenzimide fluor-
escent dye (Hoechst 33342, Sigma) in PBS. Merely
immature oocytes revealing a germinal vesicle and
mature oocytes having reached the metaphase II stage
were used for further experiments. Each immunohis-
tochemistry was performed twice. Localization of N-
acetyglucosamine residues was assessed by double
staining. For the localization of the ZPB protein, 10
immature oocytes, 7 oocytes after in vivo maturation,
and 10 oocytes after in vitro maturation were examined.
For the localization of the ZPC protein, 10 immature
oocytes, 6 oocytes after in vivo maturation, and 12
oocytes after in vitro maturation were analyzed.
The oocytes were fixed in 2.5% paraformaldehyde in
PBS for 20 min at 378C, rinsed in PBS containing
0.05% NaN3 (sodium azide, Prolabo, Fontnay sous Bois,
France) and 1 mM phenylmethanesulfonyl fluoride
(PMSF, Serva, Amstetten, Germany), and kept at 48C
in PBS containing 0.05% NaN3 and 1 mM PMSF till
further preparation. Nonspecific binding was blocked by
treatment with PBS/2% bovine serum albumin (BSA,
Sigma). In the following the oocytes were incubated with
the chicken polyclonal anti-ZPB and ZPC overnight in
dilutions of 1:200 and 1:500, respectively. After washing
853
in PBS/2% BSA, specimens were treated with a tetra-
methylrhodamine isothiocyanate (TRITC)-conjugated
rabbit anti-chicken IgG (Sigma) in a dilution of 1:200 in
PBS/2% BSA. In the following 10 ng/ml of FITC-
conjugated wheat germ agglutinin (WGA, Sigma) was
applied to detect N-acetylglucosamine residues. After
washing steps oocytes were mounted in a mixture of
Moviol V4-88 (133 mg/ml, Hoechst, Frankfurt, Ger-
many) and n-propyl gallate (5 mg/ml, Sigma). Oocytes
were kept in darkness at 48C till examination.
Controls were performed by (a) omission of the ZPB
and ZPC antibody, respectively, (b) omission of the
secondary antibody. Regarding lectin histochemistry an
additional control was performed by preincubation of
the lectin with 1 M or 0.5 M N-acetylglucosamine in
PBS. The oocytes were analyzed using the confocal
laser-scanning microscope Olympus IX 81 (Olympus,
Germany). For detection of FITC an argon ion laser
adjusted at 488 nm wavelength was used. For detection
of TRITC, a helium–neon ion laser adjusted at 543 nm
was applied.
Immunohistochemistry and
Light Microscopy of COCs
Forty-three equine ovaries, 28 immature COCs as
well as 12 COCs matured in vivo and 12 COCs matured
in vitro, and 4 8–10 days old embryos were subjected to
immunohistochemistry using the olyclonal anti-ZPB
and anti-ZPC antibodies. Immature COCs as well as
COCs matured in vitro were removed from the medium,
washed in PBS twice and fixed in Bouin’s fluid for 8 min.
COCs obtained ex vivo were rinsed in PBS for three
times and fixed in Bouin’s fluid for 8 min. Eight to
10 days old embryos were washed in PBS twice and
fixed in Bouin’s fluid for 20 min. After fixation all
specimens were dehydrated in graded series of pure
ethanols (30%–100%) and embedded in paraffin. Sec-
tions (5 mm) were mounted on 3-aminopropyl-ethoxysi-
lane-coated slides. Sections were deparaffinized in
xylene and rehydrated in graded series of ethanol.
Endogenous peroxidase was eliminated by incubation
in 7.5% H2O2 in distilled water for 10 min. Nonspecific
binding was blocked with Dako Protein Block Reagent
(Dako, Hamburg, Germany). The ZPB and ZPC anti-
bodies were used in dilutions of 1:1,000 at 48C overnight.
The secondary antibody was biotinylated rabbit anti-
chicken IgG (Biotrend, Köln, Germany, dilution 1:300).
After incubation of the slides with avidin-streptavidin-
biotin horse radish peroxidase complex (Dako) for 1 hr at
208C (dilution 1:150 in PBS-1% BSA), proteins were
detected by 0.05% diaminobenzidine in PBS containing
1% H2O2.
To optimize morphology, two embryos as well as eight
immature COCs, six COCs after in vitro maturation,
and six COCs after in vivo maturation were washed in
cacodylate buffer (0.2 M sodium cacodylate, pH 7.2).
After fixation in Karnovsky’s fluid (2.5% glutaralde-
hyde, 2% formaldehyde in 0.1 M cacodylate buffer),
embryos and COCs were postfixed in 1% OsO4 and 1.5%
KFe(CN)6. After dehydration in a graded series of
Molecular Reproduction and Development. DOI 10.1002/mrd
854 S. KÖLLE ET AL.
ethanols, the embryos and COCs were embedded in
Epon (Polysciences, Eppelheim, Germany). Semithin
sections (1 mm) were mounted on polylysine-coated
slides. The Epon was removed from the sections by
treatment with methanol/benzol for 3 min, with metha-
nol for 2 min, and with acetone for 2 min. After a washing
step in distilled water, the immunohistochemical pro-
cedure was started by incubation in 10% H2O2 for
10 min. Incubations with the Protein Block Reagent,
first and secondary antibody, avidin-streptavidin-biotin
horseradish peroxidase complex and diaminobenzidine/
H2O2 were performed as described above.
Controls were performed by (a) omission of the ZPB
and ZPC antibody, respectively, (b) omission of the
secondary antibody, (c) replacement of the ZPB/ZPC
antibody by preimmune chicken serum. Porcine ovaries
known to be rich in ZPB and ZPC were used as positive
controls.
Scanning Electron Microscopy
of COCs and Denuded Oocytes
Immature COCs, IVM COCs, and COCs matured
in vivo (preovulatory COCs) were removed from the
flushing fluid and medium, respectively, and were
washed twice in Soerensen buffer at pH 7.4 (1:5 solution
of 0.07 M KH2PO4 and 0.07 M Na2HPO42H2O). The
COCs were then fixed in 1% glutaraldehyde in Soer-
ensen buffer at 48C for 24 hr. After further washes in
Soerensen buffer, the COCs were dehydrated in an
ascending series of acetone (10%, 20%, 30%, 40%, 50%,
and 60%: twice, 5 min each; 70%, 80%, and 90%: 1 hr
each; 100%: 12 hr). In the following, COCs were dried in
a Union Point Dryer CPD 030 (Bal-Tec, Walluf,
Fig. 1. Topography of ZPC expression sites in the course of
folliculogenesis. Bar ¼ 100 mm. a: ZPC synthesis starts in the oocyte
and the follicular cells of the late primary follicle. Inlay: No signal is
visible in the corresponding negative control. b: In the secondary
Germany) by using liquid CO2 as transitional fluid.
After being dried, specimens were coated with 12 nm
gold–palladium by a Union SCD 040 sputtering device
(Bal-Tec). Scanning-electron microscopic analyses was
performed with the Zeiss scanning electron microscope
DSM 950 at magnifications from 50
to 10,000
. After
analysis, COCs were mechanically denuded and coated
with gold–palladium for a second time thus enabling us
to examine the structure of the ZP.
RESULTS
Localization of ZPC
The topography of ZPC expression revealed charac-
teristic changes in the course of folliculogenesis. ZPC
synthesis started in the oocytes and follicular cells of the
late primary follicle (Fig. 1a). In the secondary follicle,
the ZP revealed strong staining for ZPC which was
organized in two thin layers at the most inner und most
outer surface of the ZP (Fig. 1b). In this follicular stage
both the oocyte and scattered follicular cells synthesized
the ZPC protein (Fig. 1b). In the tertiary follicle, how-
ever, the oocyte stopped production of ZPC (Fig. 1c).
Thus, the cumulus cells still synthesized ZPC, whereas
the mature oocyte was negative for ZPC (Fig. 1c).
Synthesis of ZPC in the cumulus cells was generally
highest in the cumulus cells of the corona radiata and
the adjacent 3–4 cumulus cell layers (Fig. 1c). Controls
were regularly negative for ZPC (Fig. 1c: Inlay, Fig. 1d).
In isolated COCs, the expression of ZPC was co-
localized with that of ZPB (see Fig. 2). Immature COCs
obtained by ovarian slicing showed the same ZPC
expression pattern as in secondary follicles. Thus,
follicle the ZP is labeled in the inner and outer layer. Both the oocyte
and scattered follicular cells express ZPC. c: In the tertiary follicle the
oocyte stops ZPC synthesis whereas the follicular cells continue ZPC
protein production. d: No staining is visible in the negative control.
 Molecular Reproduction and Development. DOI 10.1002/mrd
ZPB AND ZPC EXPRESSION AND ZP STRUCTURE IN THE HORSE
Fig. 2. Localization of ZPB in COCs before and after in vivo and
in vitro maturation. a: In the isolated immature COC both the oocyte
and the cumulus cells express ZPB. The cytoplasmic projections of the
cumulus cells reaching the zona pellucida (ZP) show distinct labeling
for ZPB. b: No signal is visible in the negative control. c: In the in vivo
the ZP, the oocyte and the follicle cells were labeled. The
COCs obtained after in vivo maturation did not express
ZPC in the oocyte any more, but continued ZPC syn-
thesis in the cumulus cells. After in vitro maturation the
COCs revealed a reverse ZPC expression pattern as
compared to those matured in vivo. Thus, the IVM
oocyte continued ZPC synthesis, whereas the majority of
the cumulus cells did not express ZPC any more. In two
single individuals, the in vivo matured oocyte continued
to synthesize both ZPC and ZPB.
Early embryonic development in the horse was
characterized by the formation of an acellular glycopro-
tein capsule between the trophectoderm and the under-
lying ZP between the days 6 and 7 (Fig. 3a). On day 7–8,
the embryo hatched from the ZP. In all 8–10 days old
Fig. 3. Expression of ZPB in a 9-day-old equine ex vivo embryo.
Bar ¼ 540 mm. a: View on a 9-day-old equine embryo flushed from the
uterus. b: The capsule of the blastocyst is negative for ZPB.
855
matured, preovulatory COC the cumulus cells still synthesize ZPB,
whereas the oocyte is negative for ZPB. d: After in vitro maturation, the
oocyte shows a low amount of ZPB synthesis whereas the majority of the
cumulus cells do not produce ZPB any more. The ZP is irregular in
shape and thickness.
hatched blastocysts neither the glycoprotein capsule nor
the trophectoderm or ICM cells revealed any signals for
ZPC (Fig. 3b). Controls were regularly negative for ZPC.
Localization of ZPB
In the ovary, topography of ZPB synthesis during
folliculogenesis resembled that of ZPC (see Fig. 1). Thus,
ZPB synthesis started in the oocytes of single late
primordial follicles and continued in the oocytes and
follicular cells of the primary follicle. In the secondary
follicle, the ZP, the cytoplasm of the oocyte, and the
follicular cells were distinctly labeled for ZPB. A very
strong signal was seen in the outer and the inner layer of
the zona. In tertiary and preovulatory follicles, merely
scattered follicular cells showed ZPB synthesis, whereas
the oocyte stopped ZPB protein production.
The isolated COCs underwent distinct changes after
in vitro and in vivo maturation. Isolated immature
COCs surrounded by 12–25 layers of densely packed
cumulus cells revealed a clear signal for ZPB in the
oocyte, in the ZP and in the follicular cells (Fig. 2a). The
cytoplasmic processes of the follicular cells reaching into
the ZP regularly revealed a strong staining for ZPB
(Fig. 2a). After in vivo maturation, the oocyte stopped
ZPB synthesis, whereas the follicular cells adjacent to
the oocyte showed continuous high ZPB expression
(Fig. 2c). After in vitro maturation the COCs revealed a
completely different pattern of ZPB synthesis. There
was still a low amount of ZPB synthesis in the oocyte,
Molecular Reproduction and Development. DOI 10.1002/mrd

856 S. KÖLLE ET AL.

whereas the cumulus cell did not express ZPB any more
(Fig. 2d) The ZP was irregular in shape and thickness
(Fig. 2d). The controls did regularly not show any signal
(Fig. 2b).
8 to 10 days old blastocysts were negative for ZPC
(Fig. 3) and ZPB.

Topography of N-Acetylglucosamine Residues

As shown by confocal laser scanning microscopy, both
immature and mature oocytes displayed a strong signal
for N-acetylglucosamine residues which was uniformly
dispersed throughout the ZP (Fig. 4a: surface section,
Fig. 4b: equatorial section). No difference in the
topography of the N-acetylglucosamine residues was
found between in vivo and in vitro matured (IVM)
oocytes. All over the ZP, the N-acetylglucosamine
residues were co-localized with ZPC (Fig. 4c: surface
section, Fig. 4d: equatorial section). The controls did not
display any signal.

Morphology of the COC

and Structure of the ZP
The scanning-electron microscopic studies demon-
strated that the zona surface of immature oocytes
clearly differed from that of matured oocytes. Further-
more there were distinct differences in the zona archi-
tecture of oocytes matured in vitro and in vivo. The ZP of
immature oocytes displayed a spongy appearance with
numerous, regularly arranged circular or elliptical

Fig. 5. Scanning electron microscopy of immature COCs as well as of

COCs after in vitro and in vivo maturation. a: The ZP of the immature
oocyte displays a spongy appearance with numerous, regularly
arranged small pores. Bar ¼ 20 mm. b: Processes of the corona radiata
cells reach into the circular or elliptical pores (arrows). Bar ¼ 2 mm.
c: After in vitro maturation, the cumulus cells appear disorganized
and the zona surface appears smooth with only few pores. Bar ¼ 20 mm.
d: The pores of the ZP appear irregular in form and size. Bar ¼ 5 mm.
e: After in vivo maturation the ZP is covered by a huge mass of
intercellular matrix. Bar ¼ 50 mm. f: The intercellular matrix covers
both the cumulus cells and the zona surface. Bar ¼ 20 mm.

small pores (Fig. 5a,b). Processes of the corona radiata

Fig. 4. Co-localization of N-acetylglucosamine residues and ZPC in
the zona of an immature equine oocyte by confocal laser scanning
microscopy. Bar ¼ 30 mm. a, b: N-acetylglucosamine residues are
dispersed throughout the ZP. Section in the surface plane (a) and in
the equatorial plane (b). c, d: ZPC is synthesized in the ZP and in
the cytoplasm of the oocyte. In the ZP, ZPC and N-acetylglucosamine
residues are co-localized. Section in the surface plane (c) and in the
equatorial plane (d).
cells reaching into the pores were regularly seen
(Fig. 5b, arrows). The pores generally showed a
centripetal narrowing architecture, as the diameter of
the pores decreased toward the inner layer of the ZP
(Fig. 5b). In oocytes matured in vitro, however, the zona
surface appeared smooth with only few pores of
irregular size (Fig. 5c,d). The cumulus cells appeared
disorganized and the cells of the corona radiata merely
sent few processes through the ZP (Fig. 5c,d). Contrary
during in vivo maturation the cumulus cells sent
numerous processes through the ZP. They were reg-
ularly distributed, connected between each other by
long, filiform processes and embedded in a huge mass of
extracellular matrix (Fig. 5e,f). This huge extracellular
matrix was so tightly attached to the oocyte (Fig. 5e) that
it could not mechanically be removed without destroying
 Molecular Reproduction and Development. DOI 10.1002/mrd
ZPB AND ZPC EXPRESSION AND ZP STRUCTURE IN THE HORSE
the oocyte after drying—therefore scanning electron
microscopic analysis of the ZP of the preovulatory oocyte
was not possible. As shown by confocal laser scanning
electron microscopy of natively denuded oocytes, how-
ever, the ZP of preovulatory equine oocytes was
characterized by regularly distributed, wide and deep
circular and elliptical pores.
DISCUSSION
Our study was designed to investigate the topography
of zona glycoprotein ZPB and ZPC expression sites in the
equine ovary, in the oocyte before and after in vivo and
in vitro maturation and in the embryo in correlation to
equine zona structure. As equine oocytes and embryos
are very scarce, difficult to obtain and fragile, new
techniques such as immunohistochemistry on epon-
embedded single oocytes and embryos had to be
established. The technique of scanning electron micro-
scopy had to be refined to maintain proper morphology.
Using optimized techniques we were able to demon-
strate that porcine ZPB and ZPC antibodies reveal
distinct cross-reaction with the equine ZP during
folliculogenesis and oocyte maturation. Although the
sequence data of equine zona glycoproteins are still
unknown, these results confirm that equine ZPB and
ZPC show a high homology to porcine and bovine ZPB
and ZPC. In mice, ZP proteins have been reported to be
coordinately expressed only in the oocytes (Flechon
et al., 1984; Sinowatz et al., 1995; Jewgenow and
Rudolph, 2001). Contrary in the horse, both the oocyte
and the follicular cells contribute to ZPB and ZPC
protein synthesis in a time-specific pattern. The neces-
sity of follicle cells being involved in the synthesis of zona
glycoproteins is substantiated by the fact that the rate of
protein synthesis by the oocyte is too low to account for
the oocyte’s weight plus that of its zona (Schultz et al.,
1979). The equine ZP is distinctly thicker as compared to
the mouse zona so that the contribution of the follicular
cells—especially of the corona radiata cells—seems to be
essential for the development of a functional ZP. This is
corroborated by the fact that strong expression of ZPB
and ZPC was seen in the cytoplasmic projections of the
follicle cells reaching the ZP. Expression of ZP mRNA
and/or protein in granulosa cells has already been
demonstrated in several species such as the pig
(Sinowatz et al., 1995; Kölle et al., 1996), cow (Kölle
et al., 1998), dog (Blackmore et al., 2004), rabbit (Dunbar
et al., 1994; Grootenhuis et al., 1996); marmoset
(Grootenhuis et al., 1996), rhesus monkey (Grootenhuis
et al., 1996; Martinez et al., 1996) as well as in the
human (Grootenhuis et al., 1996). Contrary to the
porcine and bovine, the equine preovulatory, in vivo
matured oocyte stops ZPB as well as ZPC synthesis so
that their production is completely overtaken by the
granulosa cells. This is corroborated by the findings that
in the secondary follicle distinct staining for ZPB and
ZPC is seen both in the inner and outer layer of the ZP,
whereas in the tertiary and preovulatory follicle, the
signal for ZPB and ZPC is mainly located in the outer
layer of the ZP. Thus, in the horse, the oocyte seems to
857
undergo species-specific metabolic changes during
maturation which may—in part—explain why in vitro
maturation techniques established in the bovine and
porcine merely insufficiently work in the equine. In two
individuals examined, however, the preovulatory,
matured oocyte did not stop ZPB and ZPC glycoprotein
synthesis. Further studies are necessary to elucidate
whether the ongoing production of ZPB and ZPC in the
oocyte is correlated with retarded maturation, pro-
grammed cell death, or even altered fertility.
Distinct differences in the topography of ZPB and ZPC
expression sites were found between oocytes matured
in vitro as compared to in vivo. As mentioned above, in
vivo maturation was characterized by the oocyte stop-
ping ZPB and ZPC synthesis and the cumulus cells
overtaking the glycoprotein production. Contrary COCs
matured in vitro revealed a reverse expression pattern.
Thus, the oocyte continued ZPB and ZPC synthesis,
whereas the cumulus cells stopped the production of
these glycoproteins. This was correlated to conspicuous
morphological findings. The ZP did not show any more
regularly distributed, deep, and centripetal narrowing
pores, but revealed a reduced number of irregularly
distributed and flat pores. Similarly the cumulus cells
appeared disorganized and sent merely few processes
through the ZP. This points to the strong interaction
between cumulus cells and ZP and implies that the
viability of an equine COC is strongly correlated with
the ability of the cumulus cells to synthesize ZP
glycoproteins. This is corroborated by the findings that
in the bovine COCs cultured with media and supple-
ments, which enhance fertilization rates and blastocyst
development reveal increased ability of synthesizing
ZPC (Kölle et al., 1998). Contrary to the bovine and
porcine, the cumulus cells overtake total glycoprotein
production during oocyte maturation. Thus, in the
horse, the role of the cumulus cells seems to be more
privotal for zona integrity as compared to other domestic
species. This is substantiated by the fact that the
cumulus cells of equine COCs matured in vivo are
covered by excessive amounts of extracellular matrix
which might play a role in early signaling pathways
between the oocyte and the oviductal cells. As a con-
sequence, molecules supporting equine cumulus cell
function will distinctly improve success rates of in vitro
maturation and fertilization.
The ZP does not only play an essential role for the
growth and differentiation of the oocyte, and for
fertilization, but is also involved in early embryonic
development. In the horse, an additional acellular
glycoprotein capsule between the trophectoderm and
the ZP is synthesized between days 6 and 7 after
ovulation. As shown by Stout et al. (2005) and Tremo-
leda et al. (2003) the formation of this capsule is
instrumental to hatching and embryonic survival in
vivo. Our studies showed that ZPB and ZPC glycopro-
teins are not involved in the formation of this embryonic
capsule pointing to completely different structure and
function of the ZP and the embryonic capsule during
early development.
Molecular Reproduction and Development. DOI 10.1002/mrd
858 S. KÖLLE ET AL.
Our studies showed that in the horse—similarly to the
mouse—the ZPC protein possesses N-acetylglucosa-
mine residues, which are uniformly distributed
throughout the ZP. This points to the possibility that
also in the equine species the N-acetylglucosamine
residues might play a role in sperm binding. This is
corroborated by the finding that sperm from a variety of
mammalian species express b1,4-galactosyltransferase
on their surface (Larson and Miller, 1997; Tengowski
et al., 2001) and that equine sperm are able to bind to
bovine oocytes (Sinowatz et al., 2003). The finding that
the localization of N-acetylglucosamine residues in
oocytes matured in vivo and in vitro do not differ implies
that the low success rates of equine in vitro fertilization
are basically not due to alterations of N-acetylglucosa-
mine residues but to structural features of the ZP.
Further additional investigations will specifically
focus on zona constituents derived from the oviduct,
which may specifically support equine sperm-oocyte
interaction.
In summary, our studies have shown that in the horse
ZPB and ZPC glycoprotein expression are differentially
regulated during folliculogenesis, oocyte maturation,
and embryogenesis in a time-specific and species-
specific manner. Whereas in the porcine and bovine
both the oocyte and the cumulus cells contribute to zona
synthesis during ooccyte maturation, the cumulus cells
in the equine overtake total glycoprotein synthesis in
the tertiary follicle. In the course of in vitro maturation,
the cumulus cells loose the ability to synthesize ZP
proteins. This results in alterations of the structure of
the ZP implying that in the horse the cumulus cells play
a crucial role for zona integrity.
ACKNOWLEDGMENTS
We thank Prof. Dr. Braun, Institute for Gynecology
and Obstetrics of the University of Munich, for kindly
providing equine embryos. The excellent technical
assistance of Ms. Gudrun Boie and Ms. Christine
Neumueller is gratefully acknowledged. We thank Guy
Duchamp and the staff of the experimental study of
INRA for technical assistance during the follicular
punctures and Pierre-Yves Sizaret for the technical
help in confocal laser scanning microscopy.
REFERENCES
Alm H, Torner H, Blottner S, Nürnberg G, Kanitz W. 2001. Effect of
sperm cryopreservation and treatment with calcium ionophore or
heparin on in vitro fertilization of horse oocytes. Theriogenology
56:817–829.
Azuma T, Choi YH, Hochi S, Oguri N. 1995. Effect of glucose in the
culture medium on development of horse oocytes matured and
microfertilized in vitro. Reprod Fertil Dev 106:1067–1071.
Blackmore DG, Baillie LR, Holt JE, Dierkx L, Aitken RJ, McLaughlin
EA. 2004. Biosynthesis of the canine zona pellucida requires the
integrated participation of both oocytes and granulosa cells. Biol
Reprod 71:661–668.
Bleil JD, Wassarman PM. 1980a. Structure and function of the zona
pellucida: Identification and characterization of the proteins of the
mouse oocyte’s zona pellucida. Dev Biol 76:185–202.
Bleil JD, Wassarman PM. 1980b. Synthesis of zona pellucida proteins
by denuded and follicle-enclosed mouse oocytes during culture
in vitro. Proc Natl Acad Sci USA 77:1029–1033.
Bousquet D, Guillomot M, Betteridge KJ. 1987. Equine zona pellucida
and capsule: Some physicochemical and antigenic properties.
Gamete Res 16:121–132.
Choi YH, Okada Y, Hochi S, Braun J, Sato K, Oguri N. 1994. In vitro
fertilization rate of horse oocytes with partially removed zonae.
Theriogenology 42:795–802.
Dean J. 1992. Biology of mammalian fertilization: Role of the zona
pellucida. J Clin Invest 89:1055–1059.
Dean J. 2003. Reassessing the molecular biology of sperm-egg
recognition with mouse genetics. BioEssays 26:29–38.
Dell’Aquila ME, Fusco S, Lacalandra GM, Maritato F. 1996. In vitro
maturation and fertilization of equine oocytes recovered durino the
breeding season. Theriogenology 45:547–560.
Dell’Aquila ME, Cho YS, Minoia P, Traina V, Lacalandra GM, Maritato
F. 1997. Effects of follicular fluid supplementation of in vitro matura-
tion medium on the fertilization and development of equine oocytes
after in vitro fertilization or intracytoplasmic sperm injection. Hum
Reprod 12:2766–2772.
Duchamp G, Bour B, Combarnous Y, Palmer E. 1987. Alternative
solutions to hCG induction of ovulation in the mare. J Reprod Fertil
Suppl 35:221–228.
Dunbar BS. 1990. Ovarian antigens and infertility. Am J Reprod
Immunol 21:28–31.
Dunbar BS. 1995. Role of ovarian antigens in fertility and infertility. In:
Kurpisz M, Fernandez N, editors. Immunology of human reproduc-
tion. Oxford: Bios Scientific. pp 115–132.
Dunbar BS, Avery S, Lee V, Prasad S, Schwahn D, Schwoebel E,
Skinner S, Wilkins B. 1994. The mammalian zona pellucida: Its
biochemistry, immunochemistry, molecular biology and develop-
ment expression. The mammalian zona pellucida: Its biochemistry,
immunochemistry, molecular biology and development expression
Reprod Fertil Dev 6:331–347.
Flechon JE, Avlok A, Kopecny V. 1984. Dynamics of the zona pellucida
formation by mouse oocyte: An autoradiographic study. Biol Cell
51:403–406.
Funahashi H, Ekwall H, Rodriguez-Martinez H. 2000. Zona reaction in
porcine oocytes fertilized in vivo and in vitro as seen with scanning
electron microscopy. Biol Reprod 63:147–1442.
Goudet G, Bezard J, Duchamp G, Gerard N, Palmer E. 1997. Equine
oocyte competence for nuclear and cytoplasmic in vitro maturation:
Effect of follicle size and hormonal environment. Biol Reprod 57:232–
245.
Grootenhuis AJ, Philipsen HL, de Breet-Grijsbach JT, van Duin M.
1996. Immunocytochemical localization of ZP3 in primordial follicles
of rabbit, marmoset, rhesus monkey and human ovaries using
antibodies against human ZP3. J Reprod Fertil Suppl 50:43–54.
Harris JD, Hibler DW, Fonteno GK, Hsu KT, Yurewicz EC, Sacco AG.
1994. Cloning and characterization of zona pellucida genes and
cDNAs from a variety of mammalian species: The ZPA, ZPB and ZPC
gene families. DNA Seq 4:361–393.
Hoodbhoy T, Dean J. 2004. Insights into the molecular basis of sperm-
egg recognition in mammals. Reproduction 127:417–422.
Jewgenow K, Rudolph M. 2001. Timing and location of zona pellucida
synthesis during oogenesis in domestic cats—An ultrastructural
immunohistochemical investigation. J Reprod Fertil Suppl 57:
23–29.
Kölle S, Sinowatz F, Boie G, Totzauer I, Amselgruber W, Plendl J. 1996.
Localization of the mRNA encoding the zona protein ZP3a in the
porcine ovary, oocyte and embryo by non-radioactive in situ
hybridization. Histochem J 28:441–447.
Kölle S, Sinowatz F, Boie G, Palma G. 1998. Differential expression of
ZPC in the bovine ovary, oocyte and embryo. Mol Reprod Dev 49:435–
443.
Larson JL, Miller DJ. 1997. Sperm from a variety of mammalian
species express b1,4-galactosyltransferase on their surface. Biol
Reprod 57:442–453.
Li X, Morris LHA, Allen WR. 2000. Effects of different activation treat-
ments on fertilization of horse oocytes by intracytoplasmic sperm
injection. J Reprod Fertil 119:253–260.
Liu IK, Shivers CA. 1982. Antibodies to the zona pellucida in mares.
J Reprod Fertil Suppl 32:309–313.
Liu C, Litscher ES, Mortillo S, Sakai Y, Kinloch RA, Stewart CL,
Wassarman PM. 1996. Targeted disruption of the mZP3 gene results
 Molecular Reproduction and Development. DOI 10.1002/mrd
ZPB AND ZPC EXPRESSION AND ZP STRUCTURE IN THE HORSE
in production of eggs lacking a zona pellucida and infertility in female
mice. Proc Natl Acad Sci USA 93:5431–5436.
Martinez ML, Fontenot GK, Harris JD. 1996. The expression and
localization of zona pellucida glycoproteins and mRNA in cynomol-
gus monkeys (Macaca fascicularis). J Reprod Fertil Suppl 50:
35–41.
McLeskey SB, Dowds C, Carballada R, White RR, Saling PM. 1998.
Molecules involved in mammalian sperm-egg interaction. Intl Rev
Cytol 177:57–113.
Miller CC, Fayrer-Hosken RA, Timmons TM, Lee VH, Caudle AB,
Dunbar BS. 1992. Characterization of equine zona pellucida
glycoproteins by polyacrylamide gel electrophoresis and immunolo-
gical techniques. J Reprod Fertil 96:815–825.
Miller DJ, Gong X, Decker G, Shur BD. 1993. Egg cortical granule N-
acetylglucosaminidase is required for the mouse zona block to
polyspermy. J Cell Biol 123:1431–1440.
Nixon B, Quingxian L, Wassler MJ, Foote CI, Ensslin MA, Shur BD.
2001. Galactosyltransferase function during mammalian fertiliza-
tion. Cells Tissues Organs 168:46–57.
Palmer E, Bezard J, Magistrini M, Duchamp G. 1991. In vitro
fertilization in the horse. A retrospective study. J Reprod Fertil
Suppl 44:375–384.
Prasad SV, Skinner SM, Carino C, Wang N, Cartwright J, Dunbar BS.
2000. Structure and function of the proteins of the mammalian zona
pellucida. Cells Tissues Organs 166:148–164.
Rankin T, Familiari M, Lee E, Ginsburg A, Dwyer N, Blanchette-
Mackie J, Drago J, Westphal H, Dean J. 1996. Mice homozygous for
an insertial mutation in the ZP3 gene lack a zona pellucida and are
infertile. Development 122:2903–2910.
Schultz RM, Letourneau GE, Wassarman PM. 1979. Program of early
development in the mammal: Changes in patterns and absolute rates
of tubulin and total protein synthesis during oogenesis and early
oogenesis in the mouse. Dev Biol 68:341–359.
Shi S, Amindari S, Paruchuru K, Skalla D, Burkin H, Shur BD, Miller
DJ. 2001. Cell surface b1,4-galactosyltransferase activates G
protein-dependent exocytotic signalling. Development 128:645–
654.
Sinowatz F, Amselgruber W, Töpfer-Petersen E, Totzauer I, Calvete JJ,
Plendl J. 1995. Immunocytochemical characterization of porcine
859
zona pellucida during follicular development. Anat Embryol 161:
196–205.
Sinowatz F, Kölle S, Töpfer-Petersen E. 2001a. Biosynthesis and
expression of zona pellucida glycoproteins in mammals. Cells Tissues
Organs 168:24–35.
Sinowatz F, Töpfer-Petersen E, Kölle S, Palma G. 2001b. Functional
morphology of the zona pellucida. AHE 30:257–263.
Sinowatz F, Wessa E, Neumüller C, Palma G. 2003. On the species
specificity of sperm binding and sperm penetration of the zona
pellucida. Reprod Dom Anim 38:141–146.
Squires EL, Wilson JM, Kato H, Blaszczyk A. 1996. A pregnancy after
intracytoplasmic sperm injection into equine oocytes matured
in vitro. Theriogenology 45:306.
Stout TA, Meadows S, Allen WR. 2005. Stage-specific formation of the
equine blastocyst capsule is instrumental to hatching and to
embryonic survival in vivo. Anim Reprod Sci 87:269–281.
Tengowski MW, Wassler MJ, Shur BD, Schatten G. 2001. Subcellular
localization of beta 1,4-galactosyltransferase on bull sperm and its
function during sperm-egg interactions. Mol Reprod Dev 58:236–
244.
Topper EK, Kruijt L, Calvete J, Mann K, Töpfer-Petersen E, Woelders
H. 1997. Identification of bovine zona pellucida glycoproteins. Mol
Reprod Dev 46:344–350.
Tremoleda JL, Stout TAE, Lagutina I, Lazzari G, Bevers MM,
Colenbrander B, Galli C. 2003. Effects of in vitro production on
horse embryo morphology, cytosceletal characteristics and blasto-
cyst capsule formation. Biol Reprod 69:1895–1906.
Villalobo A, Gabius HJ. 1998. Signaling pathways for transduction of
initial message of the glycocode into cellular response. Acta Anat
161:110–120.
Wassarman PM. 2005. Contribution of mouse egg zona pellucida
glycoproteins to gamete recognition during fertilization. J Cell Phys
204:388–391.
Wassarman PM, Jovine L, Litscher ES, Qi H, Williams Z. 2004. Egg-
sperm interactions at fertilization in mammals. Eur J Obstet Gynecol
Reprod Biol 1158:57–60.
Wassarman PM, Jovine L, Qi H, Williams Z, Darie C, Litscher ES. 2005.
Recent aspects of mammalian fertilization research. Mol Cell
Endocrinol 234:95–103.