Luca Jovine

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.

Structure, function and evolution of the signal recognition particle

The signal recognition particle (SRP) is a ribonucleoprotein particle essential for the targeting of signal peptide‐bearing proteins to the prokaryotic plasma membrane or the eukaryotic endoplasmic reticulum membrane for secretion or membrane insertion. SRP binds to the signal peptide emerging from the exit site of the ribosome and forms a ribosome nascent chain (RNC)–SRP complex. The RNC–SRP complex then docks in a GTP‐dependent manner with a membrane‐anchored SRP receptor and the protein is translocated across or integrated into the membrane through a channel called the translocon. Recently considerable progress has been made in understanding the architecture and function of SRP.

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.

Crystal Structure of SRP19 in Complex with the S Domain of SRP RNA and Its Implication for the Assembly of the Signal Recognition Particle

The signal recognition particle (SRP) is a ribonucleoprotein particle involved in GTP-dependent translocation of secretory proteins across membranes. In Archaea and Eukarya, SRP19 binds to 7SL RNA and promotes the incorporation of SRP54, which contains the binding sites for GTP, the signal peptide, and the membrane-bound SRP receptor. We have determined the crystal structure of Methanococcus jannaschii SRP19 bound to the S domain of human 7SL RNA at 2.9 A resolution. SRP19 clamps the tetraloops of two branched helices (helices 6 and 8) and allows them to interact side by side. Helix 6 acts as a splint for helix 8 and partially preorganizes the binding site for SRP54 in helix 8, thereby facilitating the binding of SRP54 in assembly.

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.

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.

Crystal structure of the Ffh and EF-G binding sites in the conserved domain IV of Escherichia coli 4.5S RNA

BACKGROUND Bacterial signal recognition particle (SRP), consisting of 4.5S RNA and Ffh protein, plays an essential role in targeting signal-peptide-containing proteins to the secretory apparatus in the cell membrane. The 4.5S RNA increases the affinity of Ffh for signal peptides and is essential for the interaction between SRP and its receptor, protein FtsY. The 4.5S RNA also interacts with elongation factor G (EF-G) in the ribosome and this interaction is required for efficient translation. RESULTS We have determined by multiple anomalous dispersion (MAD) with Lu(3+) the 2.7 A crystal structure of a 4.5S RNA fragment containing binding sites for both Ffh and EF-G. This fragment consists of three helices connected by a symmetric and an asymmetric internal loop. In contrast to NMR-derived structures reported previously, the symmetric loop is entirely constituted by non-canonical base pairs. These pairs continuously stack and project unusual sets of hydrogen-bond donors and acceptors into the shallow minor groove. The structure can therefore be regarded as two double helical rods hinged by the asymmetric loop that protrudes from one strand. CONCLUSIONS Based on our crystal structure and results of chemical protection experiments reported previously, we predicted that Ffh binds to the minor groove of the symmetric loop. An identical decanucleotide sequence is found in the EF-G binding sites of both 4.5S RNA and 23S rRNA. The decanucleotide structure in the 4.5S RNA and the ribosomal protein L11-RNA complex crystals suggests how 4.5S RNA and 23S rRNA might interact with EF-G and function in translating ribosomes.

Polypeptide encoded by mouse ZP3 exon-7 is necessary and sufficient for binding of mouse sperm in vitro.

Fertilization in mice is initiated by species‐specific binding of sperm to mZP3, one of three mouse zona pellucida (ZP) glycoproteins. At nanomolar concentrations, purified egg mZP3 binds to acrosome‐intact sperm heads and inhibits binding of sperm to eggs in vitro. Although several reports suggest that sperm recognize and bind to a region of mZP3 encoded by mZP3 exon‐7 (so‐called, sperm combining‐site), this issue remains controversial. Here, exon‐swapping and an IgG(Fc) fusion construct were used to further evaluate whether mZP3 exon‐7 is essential for binding of sperm to mZP3. In one set of experiments, hamster ZP3 (hZP3) exon‐6, ‐7, and ‐8 were individually replaced with the corresponding exon of mZP3. Stably transfected embryonal carcinoma (EC) cell lines carrying the recombinant genes were produced and secreted recombinant glycoprotein was purified and assayed for the ability to inhibit binding of sperm to eggs. While EC‐hZP3, a recombinant form of hZP3 made by EC cells, is unable to inhibit binding of mouse sperm to eggs in vitro, the results suggest that substitution of mZP3 exon‐7 for hZP3 exon‐7, but not mZP3 exon‐6 or ‐8, can impart inhibitory activity to EC‐hZP3. In this context, a fusion construct consisting of human IgG(Fc) and mZP3 exon‐7 and ‐8 was prepared, an EC cell line carrying the recombinant gene was produced, and secreted chimeric glycoprotein, called EC‐huIgG(Fc)/mZP3(7), was purified and assayed. It was found that the chimeric glycoprotein binds specifically to plasma membrane overlying sperm heads to a similar extent as egg mZP3 and, at nanomolar concentrations, inhibits binding of mouse sperm to eggs in vitro. Collectively, these observations provide new evidence that sperm recognize and bind to a region of mZP3 polypeptide immediately downstream of its ZP domain that is encoded by mZP3 exon‐7. The implications of these findings are discussed. J. Cell. Physiol. 207: 30–39, 2006.

Egg zona pellucida, egg vitelline envelope, and related extracellular glycoproteins.

Abstract Vertebrate eggs are surrounded by an extracellular coat that performs vital functions during development, especially during fertilization. In mammalian and non-mammalian animals the egg coat is called a zona pellucida (ZP) and vitelline envelope (VE), respectively. The ZP of mammalian eggs, from mice to humans, and VE of fish, bird, and amphibian eggs, consists of only a few glycoproteins that are related to each other and often possess several recognizable elements (e.g., a signal sequence, ZP domain, consensus furin cleavage-site, and transmembrane domain). In most instances, ZP and VE glycoproteins are synthesized in the ovary by growing oocytes and surrounding follicle cells, but in some cases (e.g., in fish and birds) certain VE glycoproteins are synthesized in the liver and transported to the ovary. Many other families of extracellular proteins, found in vertebrates and invertebrates, share a conserved ZP domain (∼260 amino acids and 8 conserved Cys residues) with ZP and VE glycoproteins. The ZP domain enables these proteins to multimerize and perform their various functions, for example, as receptors and mechanotransducers. It is likely that the list of proteins containing a ZP domain will grow considerably in coming years and that our understanding of structural-functional relationships for such proteins will be clarified.