Eveline S. Litscher

A profile of fertilization in mammals

Fertilization is defined as the process of union of two gametes, eggs and sperm. When mammalian eggs and sperm come into contact in the female oviduct, a series of steps is set in motion that can lead to fertilization and ultimately to development of new individuals. The pathway begins with species-specific binding of sperm to eggs and ends a relatively short time later with fusion of a single sperm with each egg. Although this process has been investigated extensively, only recently have the molecular components of egg and sperm that participate in the mammalian fertilization pathway been identified. Some of these components may participate in gamete adhesion and exocytosis, whereas others may be involved in gamete fusion. Here we describe selected aspects of mammalian fertilization and address some of the latest experimental evidence that bears on this important area of research.

The ZP domain is a conserved module for polymerization of extracellular proteins

Many eukaryotic extracellular proteins share a sequence of unknown function, called the zona pellucida (ZP) domain. Among these proteins are the mammalian sperm receptors ZP2 and ZP3, non-mammalian egg coat proteins, Tamm-Horsfall protein (THP), glycoprotein-2 (GP-2), α- and β-tectorins, transforming growth factor (TGF)-β receptor III and endoglin, DMBT-1 (deletd in malignant brain tumour-1), NompA (no-mechanoreceptor-potential-A), Dumpy and cuticlin-1 (refs 1,2). Here, we report that the ZP domain of ZP2, ZP3 and THP is responsible for polymerization of these proteins into filaments of similar supramolecular structure. Most ZP domain proteins are synthesized as precursors with carboxy-terminal transmembrane domains or glycosyl phosphatidylinositol (GPI) anchors. Our results demonstrate that the C-terminal transmembrane domain and short cytoplasmic tail of ZP2 and ZP3 are not required for secretion, but are essential for assembly. Finally, we suggest a molecular basis for dominant human hearing disorders caused by point mutations within the ZP domain of α-tectorin.

Mammalian fertilization: the egg's multifunctional zona pellucida.

The zona pellucida (ZP) is a specialized extracellular coat that surrounds the plasma membrane of mammalian eggs. Its presence is essential for successful completion of oogenesis, fertilization and preimplantation development. The ZP is composed of only a few glycoproteins which are organized into long crosslinked fibrils that constitute the extracellular coat. A hallmark of ZP glycoproteins is the presence of a ZP domain, a region of polypeptide responsible for polymerization of the glycoproteins into a network of interconnected fibrils. The mouse egg ZP consists of only three glycoproteins, called ZP1, ZP2, and ZP3, that are synthesized and secreted exclusively by growing oocytes. One of the glycoproteins, ZP3, serves as both a binding partner for sperm and inducer of sperm exocytosis, the acrosome reaction. Female mice lacking ZP3 fail to assemble a ZP around growing oocytes and are completely infertile. Sperm bind to the carboxy-terminal region of ZP3 polypeptide encoded by ZP3 exon-7 and binding is sufficient to induce sperm to complete the acrosome reaction. Whether sperm recognize and bind to ZP3 polypeptide, oligosaccharide, or both remains an unresolved issue. Purified ZP3 self-assembles into long homomeric fibrils under non-denaturing conditions. Apparently, sperm added to ZP3 bind to the fibrils and are prevented from binding to ovulated eggs in vitro. These, as well as other aspects of ZP structure and function are addressed in this article.

Mouse zona pellucida genes and glycoproteins

The zona pellucida (ZP) is a thick extracellular coat that surrounds all mammalian eggs. The ZP plays important roles during oogenesis, fertilization, and preimplantation development. The mouse ZP consists of only three glycoproteins, called ZP1, ZP2, and ZP3. All three glycoproteins are essential structural components of the ZP. Additionally, ZP3 serves as a primary sperm receptor and acrosome reaction-inducer, and ZP2 serves as a secondary sperm receptor during fertilization. ZP1, ZP2, and ZP3 are encoded by single-copy genes present on three different chromosomes. The genes are expressed exclusively by mouse oocytes as they grow and the cellular specificity can be ascribed to cis-acting sequences close to the site of transcription initiation and to certain trans-acting factors. Concomitantly, ZP polypeptides are synthesized, modified with N- and O-linked oligosaccharides, secreted, and assembled into crosslinked filaments that exhibit a structural repeat. Nascent ZP glycoproteins are incorporated into large secretory vesicles that fuse with the oocyte plasma membrane and deposit nascent ZP glycoproteins into the innermost layer of the thickening ZP. Each ZP polypeptide possesses several characteristic features, including an N-terminal signal sequence, a ZP domain, a consensus furin cleavage site, and a C-terminal transmembrane domain. The latter is required for assembly of nascent ZP polypeptides into a ZP, cleavage at the consensus furin cleavage site is required for secretion, and the ZP domain supports protein:protein interactions during ZP assembly. At ovulation, when meiotic maturation of oocytes occurs and chromosomes condense into bivalents, expression of the three ZP genes ceases. Using “knockout mice”, in the absence of either ZP2 or ZP3 expression, a ZP fails to assemble around growing oocytes and females are infertile. There is no effect on males. In the absence of ZP1 expression, a disorganized ZP assembles around growing oocytes and females exhibit reduced fertility. These observations are consistent with the current model for ZP structure in which ZP2 and ZP3 form long Z filaments crosslinked by ZP1.

Structure and function of the mammalian egg zona pellucida

The zona pellucida is a thick extracellular coat that surrounds all mammalian eggs and preimplantation embryos. The zona pellucida supports communication between oocytes and follicle cells during oogenesis; protects oocytes, eggs, and embryos during development, and regulates interactions between ovulated eggs and free-swimming sperm during and following fertilization. Mutant females that produce eggs that lack a zona pellucida are infertile. The functions of the zona pellucida during fertilization now can be ascribed to certain of its glycoproteins. Here we describe some aspects of zona pellucida structure and function as they relate to mammalian fertilization. J. Exp. Zool. (Mol. Dev. Evol.) 285:251-258, 1999.

Towards the Molecular Basis of Sperm and Egg Interaction during Mammalian Fertilization

During the past 2 decades, a number of genes have been cloned from mammals which encode polypeptides that participate in the process of fertilization. Among these are glycoproteins ZP1–3 that constitute the zona pellucida of eggs from mice to human beings. In mice, one of these glycoproteins, mZP3, acts as a primary sperm receptor and acrosome reaction-inducer. The evidence suggests that acrosome-intact sperm recognize and bind to a specific class of mZP3 oligosaccharides present on two serine residues (O-linked) located near the carboxy-terminus of the polypeptide. Mutagenesis of either of these residues results in the synthesis of an inactive form of the receptor. Therefore, mammalian fertilization is a carbohydrate-mediated event. It is possible that changes in the structure of these oligosaccharides (e.g., composition, sequence, linkages, modifications, etc.) could account for species-specific binding of sperm to eggs. Stably transfected somatic cells, null mutant animals, and DNA constructs are now available to test this possibility both in vivo and in vitro.

Recent aspects of mammalian fertilization research.

Mammalian fertilization has been the subject of intensified research in recent times. Application of recombinant DNA, transgenic and gene targeting technology, in particular, to issues in mammalian fertilization has revolutionized the field. Here, we present some of the latest results coming from application of these and other technologies to four aspects of mammalian fertilization: 1. formation of the egg zona pellucida (ZP) during oocyte growth; 2. species-specific binding of sperm to the egg zona pellucida; 3. induction of the sperm acrosome reaction (AR) by the egg zona pellucida 4. binding of sperm to and fusion with egg plasma membrane. In virtually every instance, new information and new insights have come from relatively recent investigations.

The PLAC1-homology region of the ZP domain is sufficient for protein polymerisation

BackgroundHundreds of extracellular proteins polymerise into filaments and matrices by using zona pellucida (ZP) domains. ZP domain proteins perform highly diverse functions, ranging from structural to receptorial, and mutations in their genes are responsible for a number of severe human diseases. Recently, PLAC1, Oosp1-3, Papillote and CG16798 proteins were identified that share sequence homology with the N-terminal half of the ZP domain (ZP-N), but not with its C-terminal half (ZP-C). The functional significance of this partial conservation is unknown.ResultsBy exploiting a highly engineered bacterial strain, we expressed in soluble form the PLAC1-homology region of mammalian sperm receptor ZP3 as a fusion to maltose binding protein. Mass spectrometry showed that the 4 conserved Cys residues within the ZP-N moiety of the fusion protein adopt the same disulfide bond connectivity as in full-length native ZP3, indicating that it is correctly folded, and electron microscopy and biochemical analyses revealed that it assembles into filaments.ConclusionThese findings provide a function for PLAC1-like proteins and, by showing that ZP-N is a biologically active folding unit, prompt a re-evaluation of the architecture of the ZP domain and its polymers. Furthermore, they suggest that ZP-C might play a regulatory role in the assembly of ZP domain protein complexes.

Mass Spectrometric Evidence That Proteolytic Processing of Rainbow Trout Egg Vitelline Envelope Proteins Takes Place on the Egg

The rainbow trout egg vitelline envelope (VE) is constructed of three proteins, called VEα,VEβ, and VEγ, that are synthesized and secreted by the liver and transported in the bloodstream to the ovary, the site of VE assembly around eggs. All three proteins possess an N-terminal signal peptide, a zona pellucida domain, a consensus furin-like cleavage site (CFLCS) close to the C terminus, and a short propeptide downstream of the CFLCS. Proteolytic processing at the CFLCS results in loss of the short C-terminal propeptide from precursor proteins and enables incorporation of mature proteins into the VE. Here mass spectrometry (matrix-assisted laser desorption ionization time-of-flight-mass spectrometry and liquid chromatography-mass spectrometry with a micromass-quadrupole TOF hybrid mass and a QSTAR Pulsar i mass spectrometer) was employed with VE proteins isolated from rainbow trout eggs in a peptidomics-based approach to determine the following: 1) the C-terminal amino acid of mature, proteolytically processed VE proteins; 2) the cellular site of proteolytic processing at the CFLCS of VE precursor proteins; and 3) the relationship between proteolytic processing and limited covalent cross-linking of VE proteins. Peptides derived from the C-terminal region were found for all three VE proteins isolated from eggs, indicating that processing at the CFLCS occurs after the arrival of VE precursor proteins at the egg. Consistent with this conclusion, peptides containing an intact CFLCS were also found for all three VE proteins isolated from eggs. Furthermore, peptides derived from the C-terminal propeptides of VE protein heterodimers VEα-VEγ and VEβ-VEγ were found, suggesting that a small amount of VE protein can be covalently cross-linked on eggs prior to proteolytic processing at the CFLCS. Collectively, these results provide important evidence about the process of VE formation in rainbow trout and other non-cyprinoid fish and allow comparisons to be made with the process of zona pellucida formation in mammals.

Zona pellucida glycoprotein ZP3 and fertilization in mammals

An early step in mammalian fertilization is species‐restricted binding of sperm to the eggs zona pellucida (ZP), a thick extracellular coat that surrounds eggs. Sperm bind to the ZP of unfertilized eggs, but not to the ZP of fertilized eggs. Shortly after binding to the unfertilized egg ZP, sperm undergo the acrosome reaction, a form of cellular exocytosis that enables sperm to penetrate the ZP. Three glycoproteins, mZP1‐3, constitute the mouse eggs ZP and participate in the process of fertilization. For example, sperm exposed to unfertilized egg mZP3 at nanomolar concentrations are inhibited from binding to eggs and undergo the acrosome reaction. Neither mZP1 nor mZP2 has an effect on sperm binding or the acrosome reaction. Furthermore, mZP3 from fertilized eggs has no effect on sperm binding and is unable to induce the acrosome reaction. These and other properties of mZP3 suggest that it is a receptor for sperm and inducer of the acrosome reaction. Mapping of the mZP3 combining‐site for sperm suggests that it is located near the C‐terminus of the polypeptide, just downstream of the ZP domain, in a region encoded by exon‐7 of the mZP3 gene. This region of mZP3 is a site of positive Darwinian selection. When mZP3 exon‐7 is fused to the Fc fragment of human IgG and sperm exposed to the chimeric protein, sperm are inhibited from binding to eggs. However, the chimeric protein does not induce the acrosome reaction. Therefore, polypeptide encoded by mZP3 exon‐7 is necessary and sufficient for binding of mouse sperm. Mol. Reprod. Dev. 76: 933–941, 2009.