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Annual Review of Biochemistry

Zona Pellucida Proteins,
Fibrils, and Matrix
Eveline S. Litscher and Paul M. Wassarman
Department of Cell, Developmental, and Regenerative Biology, Icahn School of Medicine at
Mount Sinai, New York, NY 10029, USA; email: [Link]@[Link],
[Link]@[Link]

Annu. Rev. Biochem. 2020. 89:695–715 Keywords

The Annual Review of Biochemistry is online at
mammalian eggs, zona pellucida, ZPD proteins, Ig-like fold, fibrils, matrix,
[Link]
amyloids
[Link]
105310 Abstract
Copyright © 2020 by Annual Reviews.
The zona pellucida (ZP) is an extracellular matrix that surrounds all mam-
All rights reserved
malian oocytes, eggs, and early embryos and plays vital roles during oogene-
sis, fertilization, and preimplantation development. The ZP is composed of
three or four glycosylated proteins, ZP1–4, that are synthesized, processed,
secreted, and assembled into long, cross-linked fibrils by growing oocytes.
ZP proteins have an immunoglobulin-like three-dimensional structure and
a ZP domain that consists of two subdomains, ZP-N and ZP-C, with ZP-N
of ZP2 and ZP3 required for fibril assembly. A ZP2–ZP3 dimer is located
periodically along ZP fibrils that are cross-linked by ZP1, a protein with a
proline-rich N terminus. Fibrils in the inner and outer regions of the ZP
are oriented perpendicular and parallel to the oolemma, respectively, giving
the ZP a multilayered appearance. Upon fertilization of eggs, modification
of ZP2 and ZP3 results in changes in the ZP’s physical and biological prop-
erties that have important consequences. Certain structural features of ZP
proteins suggest that they may be amyloid-like proteins.

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Contents
1. THE ZONA PELLUCIDA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 696
1.1. Nature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 696
1.2. Oocyte Growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 697
1.3. General Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 698
2. ZONA PELLUCIDA PROTEINS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 698
2.1. Nature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 698
2.2. Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 699
2.3. Conservation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 700
2.4. Gene Targeting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 700
3. ZONA PELLUCIDA DOMAIN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 701
3.1. Nature of Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 701
3.2. Subdomain Organization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 701
3.3. Polymerization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 702
4. ORGANIZATION OF ZP1/4 DOMAINS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 703
4.1. ZP1 Proline-Rich Regions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 703
4.2. ZP-N1 Subdomain and Dimer Formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 704
5. STRUCTURE OF ZONA PELLUCIDA PROTEINS . . . . . . . . . . . . . . . . . . . . . . . . . . 705
5.1. X-Ray Diffraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 705
5.2. Structural Features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 705
6. STRUCTURE OF ZONA PELLUCIDA FIBRILS AND MATRIX . . . . . . . . . . . . . 706
6.1. Structural Features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 706
6.2. Changes Postfertilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 707
7. ZONA PELLUCIDA PROTEINS AS AMYLOIDS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 708
7.1. Nature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 708
7.2. Amyloids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 709

1. THE ZONA PELLUCIDA

1.1. Nature
The term zona pellucida (ZP) first gained widespread use in 1841, when it was used to describe
the relatively thick transparent zone surrounding mammalian eggs. However, 14 years earlier, Karl
Ernst von Baer, the first person to observe human eggs, referred to the transparent zone as “zona
pellucida” in his 1827 pamphlet titled De ovi mammalium et hominis genesi (1).
The ZP performs various functions during mammalian oogenesis, fertilization, and preim-
plantation development (Figure 1). First, it supports the health, nutrition, and growth of
mammalian oocytes during the final stages of oogenesis as they prepare to mature and become
unfertilized eggs. Second, it regulates species-restricted fertilization of eggs by sperm and helps
prevent polyspermy. Third, it protects preimplantation-stage embryos as they traverse the female
reproductive tract, the fallopian tube, on their way to the uterus. A ZP remains around an embryo
until the blastocyst stage, when the embryo hatches from the ZP and implants in the uterus,
which in humans occurs on day 6–10 postfertilization. Shortly after the eggs are fertilized, the ZP
undergoes changes that affect its physical and biological properties; these changes are particularly
important in preventing polyspermy and protecting preimplantation embryos in the fallopian
tube.

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a Fully grown mouse oocyte b Unfertilized mouse egg

ZP
Sperm

ZP

N
GV

20 µm 20 µm
Figure 1
Light micrographs of the mouse zona pellucida (ZP). (a) A light micrograph (Nomarski differential interference contrast) of a fully
grown mouse oocyte showing the ZP, germinal vesicle (GV; nucleus), and nucleolus (N). (b) A light micrograph (Nomarski differential
interference contrast) of an unfertilized mouse egg incubated in the presence of free-swimming sperm. Sperm are shown bound to the
ZP.

The ZP of mammalian oocytes, eggs, and embryos is a viscoelastic, fibrillar extracellular ma-
trix (ECM) whose surface is covered with pores and is quite permeable to macromolecules. The
width of the ZP varies relatively little, from approximately 2 μm for chicken eggs to 22 μm for
human eggs (an ∼10-fold difference), whereas the diameter of eggs varies enormously, from ap-
proximately 85 μm for mouse eggs to 30 mm for chicken eggs (an ∼350-fold difference). Today
we know that the ZP of all mammalian eggs consists of either three or four glycosylated proteins,
called ZP1–4, each with a unique polypeptide chain that includes a ZP domain (ZPD) consisting
of two subdomains, ZP-N and ZP-C, and a linker region (2). Eggs from fish, amphibia, reptiles,
and birds are also surrounded by an ECM, called the vitelline envelope (VE) or ZP, that consists
of several ZPD proteins that are closely related to ZP1–4 (2).

1.2. Oocyte Growth

A ZP first appears around mouse oocytes during their 2–3-week growth phase while arrested
in the first meiotic prophase in the ovary (Figure 2). The pool of nongrowing oocytes (∼7,500
oocytes per mouse ovary) is the sole source of unfertilized eggs in sexually mature adults (3).
By day 5 postpartum, nearly all oocytes are arrested in the late diplotene or dictyate stage of
meiosis, with chromosomes present as bivalents consisting of four chromatids. Oocyte growth is
regulated within the ovary; the number of oocytes entering their growth phase is a function of the
size of the pool of nongrowing oocytes. Fully grown oocytes resume meiosis, complete the first
meiotic reductive division (meiotic maturation) with separation of homologous chromosomes and
emission of the first polar body, and become unfertilized eggs just prior to ovulation. Fertilized
eggs complete meiosis with separation of chromatids and emission of a second polar body.
Small nongrowing mouse oocytes ∼12 μm in diameter (0.9 pL in volume) do not have a ZP,
but as soon as they begin to grow, a very thin ZP appears and continues to thicken throughout
oocyte growth. Initially, the ZP appears as diffuse fibrils in localized pockets around the surface of

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Fully grown Unfertilized egg Fertilized egg

oocyte
Nongrowing Growing oocyte
oocyte
GV

No ZP Patches ZP
ZP First Second
polar body polar body
1 2 3 4 5 6 7 8
Meiotic Fertilization
maturation

Figure 2
Schematic representation of zona pellucida (ZP) production during oocyte growth in mice (2–3 weeks). (● 1 ) Nongrowing oocytes lack a

ZP. (●2 ) As soon as they begin to grow, they lay down patches of ZP fibrils (blue). (●3 ) The fibrils coalesce into a ZP matrix (blue)

relatively early in oocyte growth. (● 4 –●6 ) The ZP matrix continues to thicken throughout oocyte growth (blue). The ZP remains around

(●
6 ) fully grown oocytes [germinal vesicle (GV) present; orange], (● 7 ) unfertilized eggs (dissolution of the GV and emission of first polar

body; yellow), and (●8 ) fertilized eggs (emission of second polar body; yellow).

growing oocytes. Shortly thereafter, the pockets of fibrils coalesce to form a uniform ECM around
oocytes (4). For example, as mouse oocytes grow from approximately 40 to 80 μm in diameter (33
to 270 pL in volume), their ZP increases on average from approximately 1.6 to 6.2 μm in width
(3, 4). The ZPs of eggs from different mammals vary in width, ranging from less than 2 μm for
platypus eggs to 13 μm for baboon eggs. The width of the ZP is directly correlated with protein
content, which varies from ∼1 to 30 ng per ZP, depending on the mammalian species. The mouse
egg ZP contains ∼3.5 ng protein or ∼54 pg/pL volume.

1.3. General Properties

The ZP is quite porous and permeable to small viruses 20–30 nm in diameter as well as to proteins
with a molecular weight (MW) of up to 500 kDa, including antibodies and enzymes (5). The ZP
can be dissolved under a variety of conditions, including at low pH or at elevated temperatures, and
in the presence of reducing agents such as mercaptoethanol or dithiothreitol; detergents such as
lithium diiodosalicylate; or proteases such as pronase, chymotrypsin, or elastase. For example, the
mouse oocyte’s ZP is completely dissolved at 50°C and neutral pH, and the rate of dissolution is
fastest at low (∼1 h in 10 mM ammonium acetate) and slowest at high (∼7 h in 100 mM ammonium
acetate) ionic strength (4). Notably, the ZP can be dissolved under conditions that do not result in
breaking of covalent bonds, which suggests that the structural integrity of the ECM is maintained
by noncovalent interactions.

2. ZONA PELLUCIDA PROTEINS

2.1. Nature
The mammalian egg ZP consists of either three (e.g., mouse) or four (e.g., human) proteins, called
ZP1–4, that migrate as very broad bands on SDS-PAGE (sodium dodecyl sulfate–polyacrylamide
gel electrophoresis) (2, 6, 7). The breadth of the bands reflects heterogeneous glycosylation and
other modifications of unique ZP1–4 polypeptides. Mouse ZP2 (120 kDa MW; 77-kDa polypep-
tide) and ZP3 (83 kDa MW; 44-kDa polypeptide) are present at a ratio of approximately 1:1 in
the ZP and represent 80% or more of ZP protein (∼2.8 ng). ZP1 (200 kDa MW; two 75-kDa

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ZPD CFCS

mZP1 N N1 N C C 623 aa

mZP2 N N1 N2 N3 N C C 713 aa

mZP3 N N C C 424 aa

hZP4 N N1 N C C 540 aa

Signal sequence N ZP-N subdomain Linker CTP

Trefoil domain C ZP-C subdomain TMD

Figure 3
Schematic representation of the organization of mouse zona pellucida (ZP) proteins mZP1–3 and human ZP
protein hZP4. In each case, the polypeptide has a signal sequence (magenta) at the N terminus; a ZP domain
(ZPD) consisting of ZP-N (green) and ZP-C (cyan) subdomains and a linker region (blue); a consensus furin
cleavage site (CFCS) (arrow); transmembrane domain (TMD) (yellow); and cytoplasmic tail (black) in the
C-terminal propeptide (CTP). mZP1 and hZP4 also have a trefoil domain (brown) adjacent to the ZPD.
mZP2 [713 amino acids (aa)] has three additional ZP-N subdomains, N1–N3 (green), between the N
terminus of the polypeptide and the ZPD. mZP1 (623 aa) and hZP4 (540 aa) have one additional ZP-N
subdomain, N1 (green), between the N terminus of the polypeptide and the trefoil domain. mZP3 is the
smallest of the three mouse ZP proteins (424 aa) and consists primarily of a ZPD. Notably, mZP1 and mZP2
have only three or four aa between the ZPD and the CFCS (red), whereas mZP3 has 47 aa, which include the
sperm-combining site and represent a region of positive Darwinian selection during evolution (red) (7).

polypeptides) accounts for less than 20% of ZP protein (∼0.7 ng). ZP2 and ZP3 are monomers,
whereas ZP1 is a dimer composed of identical subunits held together by a disulfide bond. ZP1 and
ZP4 are homologous proteins, but ZP1 has a single intermolecular disulfide, whereas ZP4 does
not. In some mammals (e.g., rat and human) both ZP1 and ZP4 are present, in others (e.g., cow)
only ZP4 is present (ZP1 is present as a pseudogene), and in still others (e.g., mouse) only ZP1 is
present (ZP4 is present as a pseudogene).
Mouse ZP1–3 are synthesized as 623-, 713-, and 424-amino-acid (aa) polypeptides, respec-
tively, and are heterogeneously glycosylated with asparagine (N )-linked and/or serine/threonine
(O)-linked oligosaccharides that are often sialylated and sulfated (Figure 3) (7). Nascent mam-
malian ZP proteins have an N-terminal signal sequence (∼20–30 aa) that targets them to the se-
cretory pathway, a ZPD (∼270 aa) and a short internal hydrophobic patch (IHP), and a C-terminal
propeptide (CTP) required for secretion of ZP proteins. The CTP has a consensus furin cleavage
site (CFCS; Arg–X–X–Arg or Arg–X–Arg/Lys–Arg), a short external hydrophobic patch (EHP), a
hydrophobic transmembrane domain (TMD; ∼20 aa), and a short cytoplasmic tail. ZP1 and ZP4
also have a cloverleaf or trefoil domain (∼45 aa), a three-loop compact structure with six cysteine
(Cys) residues present as three intramolecular disulfides linked 1→5, 2→4, and 3→6 (8).

2.2. Synthesis
Among mammals and amphibians, ZP proteins are synthesized solely by growing oocytes in the
ovary (2, 6, 7). Messenger RNA encoding ZP proteins is undetectable in nongrowing oocytes

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but increases to hundreds of thousands of copies in midstage and fully grown oocytes. The abso-
lute rate of protein synthesis increases approximately 40-fold during mouse oocyte growth, from
∼1 pg/h per nongrowing oocyte to ∼40 pg/h per fully grown oocyte. Synthesis of ZP proteins
represents ∼2–7% of total protein synthesis by growing mouse oocytes. In contrast, among fish
and birds, ZP proteins are synthesized by both oocytes and the liver, and nascent ZP proteins are
transported in circulating blood to the oocyte for uptake and assembly into a ZP. Since many fish
release several tens of thousands of small eggs at a time and birds have extremely large, yolk-laden
eggs, perhaps having two sites for ZP protein synthesis in fish and birds reflects the necessity of
synthesizing large amounts of ZP proteins in a relatively short time.
In growing mouse oocytes, nascent ZP proteins are localized to unusually large secretory
vesicles (SVs) that are ∼2.3 μm in diameter, in comparison to SVs of somatic cells that are
∼0.1–0.2 μm in diameter (9). SVs that contain nascent ZP1–4 originate from the oocyte’s Golgi
apparatus and are doughnut shaped with an empty cavity or lumen in the middle, and nascent ZP
proteins associate with the SV membrane by insertion of their TMD. Loaded SVs fuse with the
oocyte’s oolemma, and nascent ZP proteins are processed and deposited solely into the innermost
layer of the ZP such that the matrix thickens from the inside to the outside (9).

2.3. Conservation
ZP1–3 from different mammals are well conserved, exhibiting ∼60–98% sequence identity (2, 6).
The amphibian and bird ZP consists of four to six proteins, called ZP1–4, ZPd, and ZPax, and
in many fish the ZP consists of two to four proteins, called ZP1, ZP3, ZPax, and variants of ZP1
and ZP3. ZP3 is the smallest ZP protein, and it is likely that all ZP proteins are derived from an
ancestral ZP3 gene, possibly by an initial duplication event hundreds of millions of years ago.
Sequences of vertebrate ZP1–4 have been compared with orthologs from humans, and al-
though aa sequences between species vary in length, there is a high degree of sequence identity
in overlapping sequences (2, 6). The average percent identity with human ZP1–4 suggests that
organisms ranging from trout to chimpanzee can be divided into four groups. Group I contains
trout, with 33% average identity; group II contains frog, chicken, and possum, with 43–51% aver-
age identity; group III contains mouse, rat, cow, pig, and dog, with 64–69% average identity; and
group IV contains macaque and chimpanzee, with 93–99% average identity. Investigators believe
that proteins with sequence identities of 40% or more perform the same function (e.g., ZP pro-
teins in groups II, III, and IV) and that those with identities of 25–40% perform similar functions
(e.g., ZP proteins in group I) (10).

2.4. Gene Targeting

In mice and humans, ZP1–4 are encoded by single-copy genes located on different chromo-
somes (mouse ZP1-9, ZP2-7, ZP3-5, and pseudogene ZP4-13; human ZP1-11, ZP2-16, ZP3-7, and
ZP4-1) (11, 12). Fewer than 200 nucleotides of the ZP3 5 -flanking sequence are sufficient to tar-
get expression of ZP3 or foreign proteins such as firefly luciferase exclusively to growing mouse
oocytes (7).
Gene targeting has been used to establish mouse lines in which ZP genes have been inacti-
vated by either homologous recombination or insertional mutagenesis. First, female mice that are
homozygous null for either ZP2 (ZP2−/− ) or ZP3 (ZP3−/− ) produce eggs that lack a ZP and are
infertile due to a scarcity of growing oocytes in their ovaries and ovulated eggs in their oviducts
(7, 13–15). The presence of both ZP2 and ZP3 is absolutely required for assembly of a ZP around
growing oocytes. Notably, purified ZP2 and ZP3 polymerize into homomeric fibrils on their own

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in vitro (16). Second, female mice that are heterozygous null for ZP3 (ZP3+/− ) are as fertile as
wild-type mice, but their eggs have a thin ZP (width, ∼2.7 μm; wild-type width, ∼6.2 μm) that
contains approximately one-half the amount of ZP2 and ZP3 found in the ZP of wild-type eggs
(17). Apparently the width of the ZP is not a critical parameter for binding of free-swimming
sperm and fertilization of eggs. Third, female mice that are homozygous null for ZP1 (ZP1−/− )
exhibit reduced fertility due to early embryonic loss of preimplantation embryos (18) as a result
of having a ZP that is insufficiently cross-linked and, consequently, extremely porous and fragile.

3. ZONA PELLUCIDA DOMAIN

3.1. Nature of Proteins
A structural element present in all ZP proteins and in a wide variety of other proteins was initially
defined by pattern-based sequence analysis and named the ZPD (Figure 3) (19). It was proposed
that the ZPD has a common tertiary structure and plays a common biological role (2, 7). Many
ZPD proteins are present in ECMs, and mutations in the ZPD can lead to severe human patholo-
gies such as deafness, vascular and renal disease, cancer, and infertility. [It has been suggested that
the two ZPD subdomains, ZP-N and ZP-C, should be treated as independent domains and that
the term ZPD should be changed to ZP module (20).]
The ZPD arose more than 600 million years ago, consists of ∼270 aa, and has eight conserved
Cys residues present as four intramolecular disulfides (2, 21). ZP1–4 are prototypical ZPD pro-
teins; however, a ZPD is present in hundreds of other proteins (e.g., cuticlin, endoglin, hensin,
muclin, nompA, oikosin, tectorin, uromodulin) with diverse functions (e.g., receptors, mechanical
transducers, matrix components, cell shape control) in a wide variety of organisms (from jellyfish
to humans). Proteins that possess the same domains are thought to share an ancestor and have
functional features in common (10).

3.2. Subdomain Organization

A comparison of ZPD sequences for ZP1–4 from platypus to human reveals an average percent
identity of 73% and percent similarity of 90%. Nascent ZPD proteins almost invariably con-
tain a single TMD composed of hydrophobic aa that anchors the proteins to a membrane (e.g.,
SV membrane and oolemma) and is missing from secreted ZPD proteins. The ZPD is a bipar-
tite structure consisting of an N-terminal subdomain (ZP-N; ∼100 aa) and a C-terminal sub-
domain (ZP-C; ∼145 aa) that are separated by a short (∼25-aa) protease-sensitive linker region
(Figure 3) (Table 1) (2, 21). Each ZPD subdomain has four conserved Cys residues present as
two intramolecular disulfides, with ZP-N disulfides linked 1→4 and 2→3 and ZP-C disulfides
linked 5→7 and 6→8. ZP1 and ZP2 have two additional Cys residues, called a and b, in their
ZP-C domain that form an a→b linked disulfide.
ZP1 and ZP4 contain an additional ZP-N subdomain, called N1. In most vertebrates, ZP2
contains three ZP-N subdomains, called N1, N2, and N3, as extensions at its N terminus (2, 21,
22). According to structure-based sequence alignments, ZP-N1 has four conserved Cys residues
linked 1→4 and 2→3, whereas N2 and N3 have two conserved Cys residues linked 1→4; ZP-N3
has four Cys residues, but only two are conserved. Perhaps a single 1→4 linkage is sufficient for
structural stability of the ZP-N subdomain. ZP3 has only the ZP-N of the ZPD and does not have
any additional copies as extensions at its N terminus.
For different ZPD proteins, the linker between the ZP-N and ZP-C subdomains can be ei-
ther flexible, as for ZP1–4, or rigid (short), as for the ZPD protein uromodulin. Considering

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Table 1 The ZPD of ZP1–4a

Polypeptide (number
Protein of amino acids) ZPDb ZP-Nc ZP-Cd Linkere
mZP1 623 272 (271–542) 99 149 24
hZP1 638 271 (279–549) 99 148 24
cZP1 934 273 (624–896) 98 151 24
mZP2 713 267 (364–630) 96 148 23
hZP2 745 267 (371–637) 96 148 23
cZP2 695 267 (348–614) 96 148 23
mZP3 424 260 (45–304) 96 135 29
hZP3 424 259 (45–303) 97 133 29
cZP3 437 260 (57–316) 97 134 29
hZP4 540 275 (188–462) 99 151 25
cZP4 543 274 (197–470) 99 150 25

a
Domain comparisons of ZP1–4 orthologs using sequence alignments generated by Clustal Omega.
b
Number of amino acids in the ZPD (amino acid positions in the ZPD).
c
Number of amino acids in the ZP-N subdomain.
d
Number of amino acids in the ZP-C subdomain, including the internal hydrophobic patch.
e
Number of amino acids in the linker region between the ZP-N and ZP-C subdomains.
Abbreviations: c, chicken; h, human; m, mouse; ZPD, zona pellucida domain.

the two ZPD subdomains as separate units, the sequential order is always ZP-N→ZP-C, never
ZP-C→ZP-N; however, a ZP-N can be present in the absence of a ZP-C. Several proteins have
been identified that have a ZP-N subdomain present in the absence of ZP-C (e.g., Plac1, Oosp1,
and papillote), but no proteins have been found that have a ZP-C subdomain in the absence of a
ZP-N (2, 21). This observation suggests that the role of ZP-C is dependent on its partner ZP-N,
which is a conserved domain necessary for polymerization of extracellular proteins into fibrils (23,
24).

3.3. Polymerization
Nascent ZP proteins have two hydrophobic patches, the EHP in the CTP and the IHP in the
ZPD (Figure 4). These patches interact with each other in nascent ZP proteins and lock the
proteins in a conformation that is incompatible with polymerization (21, 25, 26). The EHP–IHP
interaction prevents formation of ZP fibrils within growing oocytes prior to excision of the CTP
at its CFCS and unlocking of the ZPD. The CFCS must be cleaved for normal levels of secretion
of ZP proteins by growing oocytes, and when the CFCS is not cleaved, nascent ZP proteins ac-
cumulate in the endoplasmic reticulum. The EHP and IHP are functionally related to each other
and, together with the CFCS and TMD, control incorporation of nascent ZP proteins into the ZP
by coupling of proteolysis and polymerization (9, 21, 25). Cleavage of inhibitory sequences from
protein precursors with concomitant exposure of polymerization elements is thought to regulate
polymerization of several other kinds of proteins, including fibrinogen, fibrillin, and tau protein.
Notably, the ZP of fully grown mouse oocytes varies considerably in width, from approximately
4.3 to 8.1 μm, which suggests that ZP assembly may be a stochastic process during oocyte growth.
Not all nascent ZP protein is incorporated into the ZP during oocyte growth; as much as half
of nascent ZP protein passes through the ZP and leaves the oocyte unassembled under certain
conditions (27).

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3 4
1 2 Activation of ZPD Polymerization
Precursor Cleavage at CFCS
ZP-C
IHP
N
ZP-N ZP-C
ZP-C ZP-C IHP ZP-N
IHP
IHP
ZP-N EHP CFCS ZP-N
EHP × CFCS
N
EHP
ZP-N
ZP-C ZP-C
N
N ZP-N
N D N D D ZP-N
TM TM TM
Plasma membrane ZP-C IHP
Extracellular N
C C C space

N ZP-N subdomain
TMD IHP

C ZP-C subdomain CFCS EHP

Figure 4
Schematic representation of a general mechanism for assembly of nascent zona pellucida (ZP) proteins. In all ZP domain (ZPD)
precursor proteins, the ZPD, consisting of two subdomains, ZP-N (green) and ZP-C (cyan), is followed by a C-terminal propeptide
(CTP) that contains a basic cleavage site, such as a consensus furin cleavage site (CFCS, magenta), as well as an external hydrophobic
patch (EHP, purple) and a transmembrane domain (TMD, yellow) anchor site. (● 1 ) Precursors do not polymerize within the cell, either as

a result of direct interaction between the EHP and internal hydrophobic patch (IHP, gray) or because they adopt a conformation
dependent on the presence of both hydrophobic patches. (● 2 ) C-terminal processing at the CFCS by a proprotein convertase (cleaved at

CFCS; marked by a cross) leads to dissociation of mature proteins from the EHP and (● 3 ) activation of the ZPD for assembly

[(●4 ) polymerization] into fibrils and matrix.

4. ORGANIZATION OF ZP1/4 DOMAINS

4.1. ZP1 Proline-Rich Regions
Proline (Pro) residues account for 6.3% of aa in proteins, of which 4.4% are generally present in
trimers or longer repeats (28). Vertebrate ZP1 contains an N-terminal region that is rich in Pro
residues (i.e., contains long spans of multiple but not necessarily consecutive Pro residues). Pro-
rich regions (PRRs) are referred to as low-complexity domains and often form left-handed helical
structures called polyproline type II (PPII) helices (29–31). For ZP1, these PRRs stretch over
100 aa residues or more, and Pro can account for 17% (human), 21% (mouse), or 37% (trout) of
the residues (Table 2) (32–34). The PRRs are located between the N-terminal ZP-N1 subdomain
and the adjacent trefoil domain, except in fish that lack an N-terminal ZP-N1 subdomain and have
a PRR at the N terminus (35). ZP4 proteins are shorter versions of ZP1 that lack the long PRR but
otherwise have a comparable domain organization. PPII is not a standard secondary structure, and
predictions of the structure of PRR-containing proteins without high-resolution structural data
are only suggestive (36). A PPII helix is a short structure consisting of three to six aa residues,
and consecutive helices can form a relatively flexible rod in an extended helical structure or at
terminal regions of a protein. This flexibility may be important for creating elasticity, supporting
self-assembly, and promoting intermolecular interactions (31, 37, 38). In this context, note that
squamate reptiles, such as geckos, lizards, and snakes, produce eggs with either flexible or hard
eggshells. The two groups of eggshells can be distinguished from one another by a significant
difference in the number of Pro residues present; flexible eggshells have much higher levels of
Pro than hard eggshells (39).

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Table 2 PRRs of ZP1 and ZP4

PRR length (number of Number of proline
Species amino acids)a residues (%)
ZP1
Mouse 100 21 (21)
Human 100 17 (17)
Chicken 456 61 (13)
Cobra 544 107 (20)
Trout 145 54 (37)
Sharkb 208 68 (33)
ZP4
Human 14 2
Cow 12 3
Chicken 14 3
Sharkb 5 1

a
For mouse, human, chicken, cow, and cobra, the sequence between ZP-N1 and the trefoil domain; for trout and shark, the
sequence between the signal sequence and the trefoil domain.
b
There are two predicted ZP1/ZP4 sequences for shark (Callorhincus milii). We refer to the longer sequence, 616 amino
acids, as ZP1 and to the shorter sequence, 442 amino acids, as ZP4.
Abbreviations: PRR, proline-rich region; ZP, zona pellucida.

4.2. ZP-N1 Subdomain and Dimer Formation

ZP1 in tetrapods, such as humans, chickens, and snakes, has a single N-terminal ZP-N1 subdomain
(34, 40, 41), whereas in fish, such as trout and sharks, it does not have an N-terminal ZP-N1
subdomain but does have a PRR (see the preceding section) (33, 42, 43). ZP-N1 of ZP1 is unusual
in that it has five Cys residues, as opposed to the ZP-N of the ZPD, which has four Cys residues;
however, it adopts the same immunoglobulin (Ig)-like fold as the ZP-N subdomain, with two
disulfides linked 1→4 and 2→3 (Figure 5). The fifth Cys residue (Cys69 in mouse and Cys77
in human) of ZP-N1 is likely used to covalently link the two subunits of the ZP1 homodimer.
If the ZP1 homodimer cross-links ZP fibrils, the two flexible PRR rods of ZP1 could serve as
elastic regions between ZP fibrils and give the ECM its stretchability. ZP-N1 of ZP4 does not

SH
ZP-N1
Human a
S–S
Mouse
Chicken Cys Cys Cys Cys Cys
Snake S–S
ZP1

ZP-N1 b
Human S–S
Cow Cys Cys Cys Cys
Chicken
ZP4 S–S

Figure 5
Schematic representation of the positions of the Cys residues and disulfide linkages of ZP-N1 of (a) ZP1 and
(b) ZP4. (a) Relative positions of the five Cys residues (blue), the two disulfide (S–S) linkages (light green), and
the free sulfhydryl (SH) (red, arrow). (b) Relative positions of the four Cys residues (blue) and the two S–S
linkages (light green). Each dot represents an amino acid.

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have a fifth Cys residue; in chicken the two subunits of ZP4 may be held together by noncovalent
interactions between their ZP-N1 subdomains. Furthermore, ZP4 may not have the elasticity
presumably present in ZP1 since it is missing an N-terminal PRR.

5. STRUCTURE OF ZONA PELLUCIDA PROTEINS

5.1. X-Ray Diffraction
The three-dimensional structures of several ZPD proteins have recently been determined by X-
ray diffraction. These include the following: the ZP-N subdomain of mouse ZP3 at 2.3-Å resolu-
tion (44), full-length chicken ZP3 at 2.0-Å resolution (45), the ZP-C subdomain of rat betaglycan
at 2.0-Å resolution and mouse TGFR-3 at 2.7-Å resolution (46, 47), the protease-resistant core
of human uromodulin at 3.2-Å resolution (48), the ZP-C subdomain of mouse ZP2 at 2.25-Å
resolution (48, 49), mollusk VE receptor for lysin at ∼2-Å resolution (49), the ZP-N and ZP-C
subdomains of human endoglin at 2.7-Å resolution (50), and homodimers of chicken ZP1 at 2.7-Å
resolution (51).

5.2. Structural Features

X-ray diffraction studies of ZPD proteins have revealed that both the ZP-N and ZP-C subdomains
adopt an Ig-like fold despite the complete absence of sequence identity (Figure 6). The structures
of ZP-N and ZP-C resemble C-type and V-type Ig-like domains, respectively (52). However,

a ZP3 ZP-N C-Type Ig

N N
D C N E
A B E C2 F G C4 A
A B E D C F G
B C D
C1 C1 C2
G F
C3 E'
C
C E'
C

b ZP2 ZP-C V-Type Ig

N C8 N
Cb N
C6
C'' C C''
C5 C7a E
A B E D C' C F G A
A B E D C' C F G A'
B
G
A' F D C1 C2
A' C
C''
C C'
C
C

Figure 6
Topology of ZP-N and ZP-C and their relationship with immunoglobulin (Ig)-like domains. (a) Comparison
of ZP3 ZP-N with C-type Ig-like domains. The β-strands are labeled according to standard Ig terminology;
helices are indicated by rectangles. Opposing β-sheets 1 and 2 are colored blue and green, respectively, with
termini circled. The additional E strand is in orange, and disulfides are in magenta. (b) Comparison of ZP2
ZP-C with V-type Ig-like domains. Features are indicated as in panel a, except for the additional A and
C /C strands, which are in yellow and red, respectively. Figure adapted with permission from L. Jovine
(52, figure 4).

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certain structural features make the ZPDs distinct from other known Ig-like domains, such that
they represent new Ig superfamily subtype structures.
The ZP-N subdomain consists of an antiparallel sandwich of two β-sheets made up of eight
strands of polypeptide, designated the A, B, D, and E and C, E , F, and G strands, that enclose
a hydrophobic core of buried residue side chains, with two disulfides that have 1→4 and 2→3
connectivity and clamp both sides of the sandwich (Figure 6) (52). Similar to the structure of
the ZP-N subdomain, the ZP-C subdomain consists of a β-sandwich comprising two stacked
β-sheets, one with four strands and the other with six strands, that approximate a Greek key–like
motif, which is characteristic of Ig-like domains (53). In this context, the ancestral gene for the Ig
superfamily may have originated ∼750 million years ago in sponges as a primitive sandwich-like
fold to be used in vertebrate extracellular recognition systems (54–56).
In ZP3 crystals, two ZP3 molecules are arranged as homodimers in antiparallel orientation
to form an asymmetric structure (52). The two ZPDs are bonded by electrostatic interactions
between the ZP-N and ZP-C of opposing molecules: ZP-N(1):ZP-C(2) and ZP-N(2):ZP-C(1).
In contrast, there are no ZP-N(1):ZP-N(2) or ZP-C(1):ZP-C(2) contacts within the homodimer.
The ZP-N and ZP-C of uromodulin, another ZPD protein, are similar to those of ZP3, having
the same fold, disulfide connectivities, and a conserved tyrosine (Tyr) residue in the ZP-N (47).
Mutation of this Tyr residue in tectorin, a ZPD protein associated with the tectorial membrane,
results in hearing loss (57).
In general, the ZPD is a conserved evolutionary unit essential for polymerization of proteins,
whereas adjoining regions contribute functional diversification that may be caused by selective
pressure related to species-specific functions. The strong structural similarity between the ZP-N
and ZP-C subdomains suggests that the ZPD may have arisen by duplication of an ancestral gene
encoding a protein containing a single ZP-N subdomain. Certain molecular features must regulate
whether ZPD proteins assemble into homopolymers (e.g., uromodulin fibrils) or heteropolymers
(e.g., ZP fibrils). For different ZPD proteins, the protease-sensitive linker between ZPD subdo-
mains can be either flexible or rigid (short), and differences in the flexibility or plasticity of the
linker region may modulate polymerization of structurally similar ZPD proteins (52).

6. STRUCTURE OF ZONA PELLUCIDA FIBRILS AND MATRIX

6.1. Structural Features
The mouse ZP consists of cross-linked fibrils 7–8 nm in width and several micrometers in length
with a ZP2–ZP3 dimer located every 14–15 nm along the fibrils (4, 21, 58). The structural pe-
riodicity of fibrils can be visualized as protuberances (bumps or knobs) along fibrils in electron
micrographs of solubilized ZP (Figure 7), as well as in micrographs of solubilized ZP prepara-
tions decorated with monoclonal antibodies against ZP2 or ZP3.
The surface of the ZP is covered with many fenestrations or pores (∼50 pores per mouse
ZP), giving it a laminated, spongelike appearance “somewhat reminiscent of layers of sliced Swiss
cheese put down irregularly on top of one another” (59, p. 10). Scanning electron microscopy
clearly revealed the porous and fibrous nature of the ZP of mouse and human eggs (Figure 8) (60).
Furthermore, polarized light microscopy examinations of hamster, mouse, and human ZP revealed
that the ZP is a multilayered structure consisting of inner, outer, and intervening layers (61, 62).
Fibrils in the inner and outer layers are oriented perpendicular and parallel, respectively, to the
oolemma; fibrils in the intervening layer are oriented randomly. In addition to asymmetric fibril
orientation in the inner and outer layers of the ZP, fibrils in the inner layer are more densely packed
than those in the outer layer. Such packing is consistent with the observation that nascent ZP
proteins are deposited solely into the innermost layer of the ZP, so that nascent matrix deposited

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200 nm

Figure 7
Transmission electron micrograph of mouse zona pellucida (ZP) fibrils. ZP fibrils prepared from isolated
ZP of fully grown oocytes were solubilized in the presence of elastase and sprayed as a fine mist onto
glow-discharged, carbon-coated Formvar grids and negatively stained with uranyl acetate (4, 58). The fibrils
are approximately 10 nm wide, and a structural repeat is visible along the fibrils every 14–15 nm (arrows).
The same structural repeat is seen in micrographs of freeze-dried and unidirectionally shadowed ZP fibrils.

around small oocytes ends up near the surface of the ZP of fully grown oocytes (9). The layer of
matrix near the surface of the ZP must undergo considerable stretching in order to accommodate
the severalfold increase in oocyte circumference during growth. For example, the circumference
of mouse oocytes increases from approximately 38 to 250 μm, a six- to sevenfold change, during
oocyte growth. Stretching of fibrous matrix closest to the surface of the ZP (outer layer) may
lead to reorientation of fibrils from a perpendicular to a parallel orientation with respect to the
oolemma.

6.2. Changes Postfertilization

The physical and biological properties of the oocyte, egg, and embryo ZP undergo changes as a
result of conversion of oocytes to eggs (meiotic maturation) and eggs to embryos (fertilization).
Following fertilization by a single sperm, (a) additional sperm can no longer fuse with the oolemma
(fast membrane block), (b) the ZP undergoes a significant reduction in its solubility (hardening
reaction), (c) sperm that had partially penetrated the ZP prior to fertilization can penetrate no
further, and (d) free-swimming sperm are no longer able to bind to the ZP of fertilized eggs (loss
of receptor activity).
Cortical granules (CGs; 0.2–0.6 μm in diameter; ∼4,500 per mouse egg) appear during oocyte
growth as a product of the Golgi apparatus (63). Changes in the properties of the ZP following

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a b c

Human ZP Human ZP Mouse ZP

Figure 8
Scanning electron micrographs of the surface of human and mouse oocytes. (a) Outer surface of the zona pellucida (ZP) of a human
oocyte showing the presence of many pores (yellow arrows) (9,000× magnification). (b) Higher magnification of the outer surface of the
ZP of a human oocyte showing its fibrillar organization (50,000× magnification). The fibrils are 0.1–0.4 μm long and 10–14 nm wide.
(c) Outer surface of the ZP of a mouse oocyte showing its fibrillar organization (50,000× magnification). These micrographs were
obtained with samples treated with saponin–ruthenium red–osmium–thiocarbohydrazide to reveal ZP fibrils. Figure adapted with
permission from G. Familiari (60, figure 3).

fertilization are largely attributable to fusion of CGs with the egg’s oolemma (cortical reaction)
by a SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein–
mediated pathway (64). As a consequence of fusion, egg ZP2 is cleaved by a protease, but this
process does not result in the release of a ZP2 peptide (or peptides), and the ZP undergoes a
hardening reaction (65, 66). Recent results suggest that fusion of CGs and the fertilized egg’s
oolemma leads to accumulation of Zn2+ in the ZP (100–500 μM Zn2+ /ZP), called zinc sparks, and
to release of a Zn2+ metalloendopeptidase, called ovastacin, from the CG that cleaves ZP2 near its
N terminus and inactivates it as a sperm receptor (67–70). Inactivation of ZP3 as a sperm receptor
also occurs following cortical reaction, but the molecular basis of the inactivation is unclear (7).
The use of micropipette indentation, tactile sensors, atomic force microscopy, and/or other
methodology has shown that the viscosity and elasticity of the ZP change markedly following
fertilization (71–74). For example, the stiffness and viscosity of the ZP of embryos increase by
approximately 2.6- and 4.4-fold, respectively, compared with the ZP of oocytes (75). The increase
in stiffness is reflected in increased resistance of the ZP to dissolution by various proteases, heat,
and reducing agents. The molecular basis of the mechanical changes is unclear; however, it is likely
that it involves cleavage of ZP2 and concomitant changes in ZP fibril and matrix conformation,
as well as a marked increase in noncovalent interactions between ZP fibrils.

7. ZONA PELLUCIDA PROTEINS AS AMYLOIDS

7.1. Nature
Traditionally, amyloids have been thought to cause diseases but have recently been found to be
widespread in many organisms and to have functional roles (76, 77). The term amyloid refers
to proteins that aggregate and form long, unbranched fibrils that display a cross-β-fiber X-ray
diffraction pattern and other properties, for instance, a range of physical characteristics that can

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be assessed by dye binding and morphological techniques (78, 79). The fibrils share some char-
acteristic structural features: (a) They are unbranched, 5–15 nm in width, many micrometers in
length, and composed of two or more protofilaments; (b) each fibril is an aggregate of a single
protein that has a typical cross-β-fiber X-ray diffraction pattern with reflections at 4.6–4.8 Å and
at 8–10 Å (the former reflections represent spacing between strands in a β-sheet; the latter, spacing
between β-sheets); (c) the β-strands of fibrils are arranged in an orientation perpendicular to the
fibril axis, whereas the β-sheets line up horizontally across the fibrils; and (d) the typical fibril has a
diameter of ∼10 nm and is made up of several protofilaments, but monoprotofilaments also exist.
Notably, the width of ZP fibrils ranges from 5 to 10 nm, suggesting that each may be composed
of more than one protofilament (76, 80).

7.2. Amyloids
The analogy between amyloid and ZP fibrils comes from biophysical descriptions of the fibrils.
Amyloidogenic properties can be associated with relatively short peptide sequences that stack atop
one another as individual β-strands and form an extended β-sheet. The length of a protofilament
consists of a stack of β-strands that form hydrogen bonds with neighboring β-strands, giving rise to
a single β-sheet, whereas the width of a protofilament generally consists of two single β-sheets (80).
The ZP-N subdomain of ZP proteins adopts an Ig-like β-sandwich fold arranged as two
antiparallel β-sheets composed of eight β-strands (A–G), with each sheet composed of four
β-strands and linked by two disulfide bonds (e.g., for mouse ZP3, C46→C139 and C78→C98).
The region between the A and G β-strands is a conserved, hydrophobic, and relatively exposed
surface area that could be favorable for successive monomer interactions to form polymers.
However, other contact sites, such as those between β-strands of IHP segments and between
E β-strands of adjacent ZP-N subdomains, have been noted. This observation suggests that
ZP-N subdomains of adjacent ZP proteins, such as ZP2 and ZP3, interact and contribute to
polymerization of ZP fibrils (48, 52).
Several recent studies of peptide analogs of human and fish ZP proteins have concluded
that ZP fibrils have properties analogous to amyloid fibrils. For example, a ZP1 structural
model based on the human ZP-N subdomain predicts how this subdomain could assemble
into fibrils. Short peptides with aggregation-prone properties were modeled by AMYLPRED2
(AMYLoidPREDiction2) (81), a consensus method or algorithm used for assessment of the
propensity of peptides present in proteins to form amyloids. After 1–2 weeks in distilled water at
pH 5.5, peptide A (276Ala–Thr–Val–Gln–Ala–Phe281 with Cys280 substituted by Ala) and pep-
tide G (370Phe–Gln–Leu–His–Val–Arg–Ala376 with Cys376 substituted by Ala), representing
β-strands A and G of the human ZP1 ZP-N subdomain, self-assembled into amyloid-like fibrils
(82). Peptide A self-assembled into long, unbranched fibrils 10–12 nm in diameter, with a pair of
protofibrils, each 4–5 nm in diameter, wrapped around each other to form a double-helical struc-
ture. Peptide G also self-assembled into amyloid-like fibrils that interacted laterally and formed
ribbons and gels, and a mixture of peptides A and G gave rise to two types of fibrils similar to
those just described. Experiments with peptide analogs of human ZP2, ZP3, and ZP4, whose
ZP-N subdomain has the same overall three-dimensional fold, yielded similar results. These find-
ings suggest that the interface contact between A/A and G/G β-strands of the ZP-N subdomains
could be the basis for ZP fibril formation.
AMYLPRED2 has also been used with mouse ZP1–3 to identify fibril-forming sequences in
the proteins, and predicted amyloidogenic sites were probed with specific antibodies (83). All
three ZP proteins had relatively short stretches of 4–12 aa that were identified as fibril-forming
segments, many of which mapped to β-strands in the ZP-N subdomain of the ZPD. Structural

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studies also were carried out using powder X-ray diffraction with air-dried whole ZP in quartz
capillaries. The ZP diffracted with the typical amyloid cross-β-type pattern, with reflections at 4.65
and 10 Å; however, the 4.65-Å reflection was faint or blurred, possibly due to irregular spacing of
strands in the β-sheets (84). Solubilized ZP samples examined by electron microscopy exhibited a
meshwork of packed fibrils resembling beads on a string. These observations led to the conclusion
that ZP1–3 are amyloidogenic and that the ZP is a functional amyloid.
Zipper DB is an algorithm that uses a three-dimensional profile method on tightly mated β-
sheets that result in a structural scaffold, called a steric zipper (85). Zipper DB and AMYLPRED2
were used with aa sequences from mouse ZP3, and profiles from both algorithms were compared
(E.S. Litscher, unpublished results). For example, the sequences 49Ala–Glu–Leu–Val–Val–Thr–
Val–Ser56 (Zipper DB) and 50Glu–Leu–Val–Val–Thr–Val–Ser–Arg–Asp58 (AMYLPRED2) that
correspond to β-strand F of the β-sandwich fold displayed a strong propensity to form fibrils. Sim-
ilar results were obtained with other sequences from mouse ZP1 and ZP2. However, depending
on the algorithms (Zipper DB versus AMYLPRED2) and sequences (e.g., ZP3 109Leu–Val–Tyr–
Ser–Thr–Phe114 versus 108Ala–Leu–Val–Tyr–Ser–Thr–Phe–Leu–Leu116), the results were not
always consistent with fibril formation. Furthermore, if, as suggested, ZP2 and ZP3 interact via
specific ZP-N contact sites to form fibrils, they would be heteromeric aggregates rather than the
homomeric aggregates typical of amyloids (86).

SUMMARY POINTS
1. The ZP is an ECM that surrounds mammalian oocytes, eggs, and preimplantation
embryos and plays essential roles during oogenesis, fertilization, and preimplantation
development.
2. The ZP is composed of either three or four glycosylated proteins, ZP1–4, each with a
unique polypeptide chain encoded by a single-copy gene that is highly expressed only in
females during oocyte growth.
3. ZP proteins are synthesized, processed, and secreted by growing oocytes into the extra-
cellular space, where ZP2 and ZP3 form heterodimers that polymerize into long fibrils
that exhibit a structural repeat and are cross-linked by ZP1 and/or ZP4.
4. Female mice lacking either ZP2 or ZP3 are unable to assemble a ZP during oocyte
growth and are infertile due to a scarcity of growing oocytes and ovulated eggs, whereas
mice lacking ZP1 assemble a very porous ZP and exhibit reduced fertility due to early
embryonic loss.
5. ZP1–4 have a ZPD that is ∼270 aa in length; have eight conserved Cys residues present
as four intramolecular disulfides; and consist of two subdomains, ZP-N and ZP-C, that
have an Ig-like three-dimensional structure.
6. The ZP-N subdomains of ZP2 and ZP3 are required for assembly of fibrils during oocyte
growth, and the fibril cross-linker, ZP1, has a very Pro-rich N-terminal region that may
confer flexibility or elasticity on the ZP.
7. The ZP is a multilayered ECM with fibrils oriented in different directions in its inner
and outer layers and whose physical and biological properties change after fertilization
due to the cortical reaction and modification of ZP2 and ZP3.
8. ZP proteins possess some amyloid-like structural and other physical features and are
predicted to be functional amyloids.

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FUTURE ISSUES
1. Is assembly of ZP2-ZP3 dimers and/or polymerization of dimers into fibrils mediated
by specific oocyte factors?
2. Is the ZPD linker region, PRR, and/or other regions of ZP proteins responsible for the
flexibility or elasticity of ZP fibrils?
3. Does the ZPD linker region determine or influence whether homomeric or heteromeric
polymers are produced from nascent ZP proteins?
4. What dynamic changes occur when ZP protein precursors are cleaved at the CFCS and
converted into a conformation that permits polymerization?
5. What causes the reorientation of ZP fibrils from perpendicular to parallel to the
oolemma as the ZP thickens during oocyte growth?
6. What dynamic changes occur when ZP2 is cleaved postfertilization and hardening of
the ZP takes place?
7. Are postfertilization changes in ZP matrix due to expulsion of water, analogous to
squeezing a wet sponge, accompanied by increased molecular interactions between
fibrils?
8. Are ZP proteins really authentic amyloid proteins? It is very likely that further X-ray
crystallographic, cryo-electron microscopic, advanced imaging, and other experimental
approaches will provide answers to these and other questions in the near future.

DISCLOSURE STATEMENT
The authors are not aware of any affiliations, memberships, funding, or financial holdings that
might be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS
We are grateful to the graduate students, postdoctoral fellows, and research technicians who
worked with us on various aspects of the synthesis, structure, and function of ZP proteins. We
thank Giuseppe Familiari at the University of Rome, Italy, and Luca Jovine at the Karolinska In-
stitute, Sweden, for kindly providing images from their publications for this review. In a number of
instances we have referred to reviews rather than primary papers, and we apologize to those inves-
tigators whose contributions were not cited directly. Research from our laboratory was supported
in part by the National Institutes of Health and F. Hoffmann–La Roche AG.
Our review is dedicated to the memory of Sydney Brenner (1927–2019; FRS 1965, Nobel
2002), an extraordinary scientist, renowned raconteur, and last of the 1967 six-member Govern-
ing Board of the Medical Research Council Laboratory of Molecular Biology that also included
Francis Crick (1916–2004; FRS 1959, Nobel 1962), Hugh Huxley (1924–2013; FRS 1960), John
Kendrew (1917–1997; FRS 1960, Nobel 1962), Max Perutz (1914–2002; FRS 1954, Nobel 1962),
and Fred Sanger (1918–2013; FRS 1954, Nobel 1958, 1980).

LITERATURE CITED
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2. Litscher ES, Wassarman PM. 2015. A Guide to Zona Pellucida Domain Proteins. Hoboken, NJ: Wiley
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