Normal Fertilization Occurs with Eggs Lacking the Integrin α6β1 and Is Cd9-Dependent

Miller, Brent; Georges-Labouesse, Elisabeth; Primàkoff, Paul; Myles, Diana G.

doi:10.1083/jcb.149.6.1289

Cited by 178 publications

(153 citation statements)

References 31 publications

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“…After an incubation period of 50 min, eggs were washed in M199*ϩ0.4% BSA and fixed in 2% formaldehyde-PBS-PVP, and the number of bound sperm per egg was determined at the widest egg diameter. The in vitro sperm-egg fusion assay was performed as described previously (28). Isolated eggs were treated with hyaluronidase in M199*ϩ0.3% BSA to remove cumulus cells and then treated with 10 g of chymotrypsin/ml in M199*ϩ0.3% BSA for 3 min to loosen the ZP.…”

Section: Methodsmentioning

confidence: 99%

Loss of Zona Pellucida Binding Proteins in the Acrosomal Matrix Disrupts Acrosome Biogenesis and Sperm Morphogenesis

Lin

Roy

Yan³

et al. 2007

Molecular and Cellular Biology

202

135

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Zona pellucida binding protein 1 (ZPBP1), a spermatid and spermatozoon protein that localizes to the acrosome, was originally identified in pigs and named for its binding to the oocyte zona pellucida. In an in silico search for germ cell-specific genes, Zpbp1 and its novel paralog, Zpbp2, were discovered and confirmed to be expressed only in the testes in both mice and humans. To study the in vivo functions of both ZPBP proteins, we disrupted Zpbp1 and Zpbp2 in mice. Males lacking ZPBP1 were sterile, with abnormal round-headed sperm morphology and no forward sperm motility. Ultrastructural studies demonstrated that absence of ZPBP1 prevents proper acrosome compaction, resulting in acrosome fragmentation and disruption of the Sertoli-spermatid junctions. Males null for ZPBP2 were subfertile, demonstrated aberrant acrosomal membrane invaginations, and produced dysmorphic sperm with reduced ability to penetrate zona pellucida. Molecular phylogenetic analysis of ZPBPs from amphibians, birds, and mammals suggests that these paralogous genes coevolved to play cooperative roles during spermiogenesis. Whereas ZPBP1 was discovered for an in vitro role in sperm-egg interactions, we have shown that both ZPBP proteins play an earlier structural role during spermiogenesis.Studies on sperm-egg interactions in model organisms have provided conceptual understandings for how spermatozoa bind, penetrate, and fertilize the egg (15, 49). In placental mammals (Eutheria), the egg investment, called the zona pellucida (ZP), is a reticular meshwork assembled from three groups of sulfated glycoproteins, ZP2, ZP3, and ZP1/ZP4 that completely encircles mammalian eggs (12, 15). The ZP is responsible for the initial sperm binding and the subsequent induction of the acrosome reaction that allows sperm penetration. The ZP also functions as a physical barrier to select for functional spermatozoa capable of successful penetration, to prevent polyspermy, and to protect early embryos. However, the molecular details of sperm binding and zona penetration are mostly unresolved (36). Therefore, much effort has been focused on identifying sperm proteins involved in these processes.The acrosome is a cap-shaped, Golgi-derived organelle located over the anterior part of the sperm nucleus and highly conserved throughout evolution (2, 13). One exception in vertebrates is the teleost (bony fish) lineage, in which acrosomeless sperm can fertilize the egg by swimming through a specialized opening on the egg investment known as the micropyle (10, 32). During the acrosome reaction, the vesiculization and removal of the sperm plasma membrane and the outer acrosomal membrane expose the inner acrosomal membrane and the overlaying acrosomal matrix materials for subsequent sperm-egg interactions, including zona penetration and spermegg fusion (15,16,49). Although acrosome biogenesis is important for sperm during gamete interaction, recent studies have also revealed its involvement in sperm morphogenesis (21). During spermiogenesis, close association of the acrosome ...

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Section: Methodsmentioning

confidence: 99%

Loss of Zona Pellucida Binding Proteins in the Acrosomal Matrix Disrupts Acrosome Biogenesis and Sperm Morphogenesis

Lin

Roy

Yan³

et al. 2007

Molecular and Cellular Biology

202

135

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“…In contrast, when the sperm were incubated with Tg ϩ CD9 Ϫ/Ϫ eggs in the medium containing anti-CD9 mAb, no increase in intensity of CD9-EGFP on the sperm heads was detected. Anti-CD9 mAbs have been reported to inhibit sperm-egg fusion (4,15,16). Our findings demonstrate that the anti-CD9 mAb inhibited the association of sperm with CD9-containing vesicles in parallel to inhibition of sperm-egg fusion.…”

Section: Resultsmentioning

confidence: 99%

The fusing ability of sperm is bestowed by CD9-containing vesicles released from eggs in mice

Miyado

Yoshida

Yamagata

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

172

189

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Membrane fusion is an essential step in the encounter of two nuclei from sex cells-sperm and egg-in fertilization. However, aside from the involvement of two molecules, CD9 and Izumo, the mechanism of fusion remains unclear. Here, we show that spermegg fusion is mediated by vesicles containing CD9 that are released from the egg and interact with sperm. We demonstrate that the CD9 ؊/؊ eggs, which have a defective sperm-fusing ability, have impaired release of CD9-containing vesicles. We investigate the fusion-facilitating activity of CD9-containing vesicles by examining the fusion of sperm to CD9 ؊/؊ eggs with the aid of exogenous CD9-containing vesicles. Moreover, we show, by examining the fusion of sperm to CD9 ؊/؊ eggs, that hamster eggs have a similar fusing ability as mouse eggs. The CD9-containing vesicle release from unfertilized eggs provides insight into the mechanism required for fusion with sperm.

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“…Mouse CD9, a member of the tetraspanin family of proteins, is an egg protein that is essential for fertility. The protein is also found in several nonreproductive cell types in the mouse and is proposed to play a scaffold-like role during gamete fusion (Le Naour et al, 2000;Miller et al, 2000;Miyado et al, 2000). Another mouse protein essential for fertility is the endoplasmic reticulum resident chaperone calmegin, which likely is involved in proper folding of key sperm proteins (Watanabe et al, 1995;Ikawa et al, 2001;Yamagata et al, 2002).…”

Section: Introductionmentioning

confidence: 99%

TheChlamydomonasFus1 Protein Is Present on the Mating TypeplusFusion Organelle and Required for a Critical Membrane Adhesion Event during Fusion withminusGametes

Misamore

Gupta

Snell³

2003

MBoC

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The molecular mechanisms of the defining event in fertilization, gamete fusion, remain poorly understood. The FUS1 gene in the unicellular, biflagellated green alga Chlamydomonas is one of the few sex-specific eukaryotic genes shown by genetic analysis to be essential for gamete fusion during fertilization. In Chlamydomonas, adhesion and fusion of the plasma membranes of activated mtϩ and mtϪ gametes is accomplished via specialized fusion organelles called mating structures. Herein, we identify the endogenous Fus1 protein, test the idea that Fus1 is at the site of fusion, and identify the step in fusion that requires Fus1. Our results show that Fus1 is a ϳ95-kDa protein present on the external surface of both unactivated and activated mtϩ gametes. Bioassays indicate that adhesion between mating type plus and mating type minus fusion organelles requires Fus1 and that Fus1 is functional only after gamete activation. Finally, immunofluorescence demonstrates that the Fus1 protein is present as an apical patch on unactivated gametes and redistributes during gamete activation over the entire surface of the microvillous-like activated plus mating structure, the fertilization tubule. Thus, Fus1 is present on mtϩ gametes at the site of cell-cell fusion and essential for an early step in the fusion process. INTRODUCTIONGamete fusion defines the beginning of a new organism in all sexual species. In most organisms, the events that precede fusion have been well characterized at the cellular level (Snell and White, 1996;Wassarman et al., 2001;Primakoff and Myles, 2002). Initial cell-cell interactions between sperm surface proteins and extracellular matrix molecules on the egg trigger activation of the sperm and exposure of previously cryptic regions of the plasma membrane proposed to be involved in gamete fusion. Once the sperm has made its way to the egg plasma membrane, the membranes of the two gametes interact more intimately, finally bringing about gamete fusion. Several sperm and egg molecules implicated in activation of the sperm have been identified, and we know much about the morphological events that accompany gamete fusion (Yanagimachi, 1994). On the other hand, little is known about the molecular mechanisms that underlie the late steps in fertilization, when the plasma membranes of the interacting gametes adhere and fuse (for review, see Primakoff and Myles, 2002). In the absence of any purely eukaryotic systems in which bona fide fusion proteins have been identified, the best models for fusion of the external plasma membrane of cells have come from studies of fusion of viruses with their eukaryotic target cells (White, 1996;Eckert and Kim, 2001). In these systems, a viral transmembrane surface protein interacts with a receptor in the host cell plasma membrane, leading to docking of the virus particle on the cell surface. Subsequent conformational changes in the interacting proteins finally lead to complete fusion (Dimitrov, 2000;Malashkevich et al., 2001;Mayer, 2001).A small number of eukaryotic genes has been sho...

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Normal Fertilization Occurs with Eggs Lacking the Integrin α6β1 and Is Cd9-Dependent

Cited by 178 publications

References 31 publications

Loss of Zona Pellucida Binding Proteins in the Acrosomal Matrix Disrupts Acrosome Biogenesis and Sperm Morphogenesis

Loss of Zona Pellucida Binding Proteins in the Acrosomal Matrix Disrupts Acrosome Biogenesis and Sperm Morphogenesis

The fusing ability of sperm is bestowed by CD9-containing vesicles released from eggs in mice

TheChlamydomonasFus1 Protein Is Present on the Mating TypeplusFusion Organelle and Required for a Critical Membrane Adhesion Event during Fusion withminusGametes

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