Chapter Ten - The Mouse Egg's Zona Pellucida

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Abstract

All mammalian eggs are surrounded by a highly specialized extracellular matrix (ECM), called the zona pellucida (ZP), that functions before, during, and after fertilization. Unlike somatic cell ECM the mouse ZP is composed of three different proteins, ZP1–3, that are synthesized and secreted by growing oocytes and assembled into long interconnected fibrils. ECM or vitelline envelope (VE) that surrounds fish, reptilian, amphibian, and avian eggs also consists of a limited number of proteins all closely related to ZP1–3. Messenger RNAs encoding ZP1–3 are expressed only by growing oocytes at very high levels from single-copy genes present on different chromosomes. Processing at the amino- and carboxy-termini of nascent ZP1–3 permits secretion of mature proteins into the extracellular space and assembly into fibrils and matrix. Structural features of nascent ZP proteins prevent assembly within secretory vesicles of growing oocytes. Homozygous knockout female mice that fail to synthesize either ZP2 or ZP3 are unable to construct a ZP, ovulate few if any eggs, and are infertile. ZP1–3 have a common structural feature, the ZP domain (ZPD), that has been conserved through 600 million years of evolution and is essential for ZP protein assembly into fibrils. The ZPD consists of two subdomains, each with four conserved cysteine residues present as two intramolecular disulfides, and resembles an immunoglobulin (Ig) domain found in a wide variety of proteins that have diverse functions, from receptors to mechanical transducers. ZP2 and ZP3 function as receptors for acrosome-reacted and acrosome-intact sperm, respectively, during fertilization of ovulated eggs, but are inactivated as sperm receptors as a result of fertilization.

Section snippets

Introduction to the Zona Pellucida

Nearly all animal cells are surrounded by an acellular component called the extracellular matrix (ECM) that participates in a myriad of biological activities (Alberts et al., 2014). ECM affects gene expression, differentiation, morphogenesis, cellular adhesion, cellular migration, and intercellular communication. Integrins are transmembrane receptors that mediate interactions between ECM and intracellular actin cytoskeleton, permitting outside-in and inside-out signal transduction. Most cells

Oogenesis in Mice

The ZP is laid down during the process of oogenesis in females that results in production of unfertilized eggs (Gosden, 2013; Rodrigues, Limback, McGinnis, Plancha, & Albertini, 2008; Wassarman & Albertini, 1994; Zuccotti, Merico, Cecconi, Redi, & Garagna, 2011) (Fig. 1A). In mice oogenesis begins early in fetal development with appearance of 15–100 primordial germ cells (PGCs) in yolk sac endoderm and in the region of the allantois arising from the primitive streak. PGCs migrate into the

Zona Pellucida Proteins

The mouse egg's ZP is a gel-like structure that is composed of three proteins, called ZP1–3, that have apparent MWs of about 200, 120, and 83 kDa, respectively, and account for virtually all proteins (3.5 ng) in the ZP (Bleil & Wassarman, 1980a; Litscher & Wassarman, 2015; Wassarman, 1988a) (Fig. 3). ZP2 and ZP3 are present in roughly equimolar amounts in the ZP and are monomers, whereas ZP1 is the least abundant protein and is a dimer of identical polypeptide chains held together by

Zona Pellucida Domain Proteins

All three mouse ZP proteins possess a zona pellucida domain (ZPD)—ZP1, amino acids (aa) 271–540; ZP2, aa 364–628; and ZP3, aa 45–302. ZP1–3 are prototypical ZPD proteins (Bork & Sander, 1992) that are present in virtually all vertebrate egg ZP and VE, as well as in a wide variety of tissues and organs in both vertebrates and invertebrates (Jovine, Darie, Litscher, & Wassarman, 2005; Litscher & Wassarman, 2015; Plaza, Chanut-Delalande, Fernandes, Wassarman, & Payre, 2010). The ZPD arose more

Zona Pellucida Protein Synthesis

Are oocytes, follicle cells, or both the site of synthesis of mouse ZP proteins? This question was resolved in the early 1980s using growing oocytes, with or without surrounding follicle cells, cultured in vitro in the presence of radiolabeled aa or sugars. It was concluded that ZP proteins are synthesized only by growing mouse oocytes, not by follicle cells (Bleil & Wassarman, 1980b; Greve, Salzmann, Roller, & Wassarman, 1982; Salzmann, Greve, Roller, & Wassarman, 1983; Shimizu, Tsuji, & Dean,

Zona Pellucida-Knockout Mice

With the advent of gene targeting procedures mouse lines have been established in which ZP genes have been inactivated by homologous recombination and insertional mutagenesis. Homozygous null males are unaffected by the mutations. Oocytes and eggs from mutant females carrying a single ZP3 allele (heterozygous nulls; ZP3+/−) possess a ZP and the females reproduce normally (Table 1). However, the ZP is less than one-half the thickness (2.7 ± 1.2 μm) of wild-type ZP (6.2 ± 1.9 μm) and contains about

Zona Pellucida Protein Secretion and Assembly

Certain elements involved in secretion of ZP proteins by growing oocytes are located in the CTP. One of these elements is the CFCS that must be cleaved by a proprotein convertase for secretion of ZP proteins to take place (Litscher & Wassarman, 1999; Williams & Wassarman, 2001). When the CFCS is mutated, it abolishes cleavage and results in accumulation of unprocessed ZP proteins in the ER. Under these conditions, secretion of ZP proteins is reduced as much as 20-fold, but the low level of

Zona Pellucida and Fertilization

For sperm to fertilize ovulated eggs they must bind to and penetrate the ZP, reach the egg's plasma membrane, and fuse with it. To penetrate the ZP, acrosome-intact (AI) sperm must first bind to the ZP and undergo the acrosome reaction (ARx) (Buffone, Hirohashi, & Gerton, 2014), a process that involves multiple membrane fusions (Wassarman & Litscher, 2008). The ARx is a Ca2 +-dependent event that leads to signaling through sperm G proteins to produce intracellular ionic changes and second

Summary

(1) Mouse and all other mammalian eggs are surrounded by a relatively thick, gel-like ECM called the ZP. (2) The mouse egg's ZP is composed of three unique proteins, ZP1–3, encoded by single-copy genes on different chromosomes. (3) ZP genes are expressed solely by growing mouse oocytes and, therefore, only by female mice. (4) ZP1–3 are prototype ZPD proteins that are synthesized and processed only by growing oocytes and secreted for self-assembly into fibrils and matrix. (5) ZP2 and ZP3

Acknowledgments

Many thanks to the postdoctoral fellows, graduate students, and research assistants who contributed to our research on ZP proteins and mammalian fertilization at Harvard Medical School, Roche Institute of Molecular Biology, and Icahn School of Medicine at Mount Sinai. Our research was supported in part by the National Institutes of Health (NICHD) and by Hoffmann-La Roche Inc.

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