REVIEW

The molecular mechanisms mediating mammalian fertilizationHanisha H. Bhakta, Fares H. Refai and Matteo A. Avella*

ABSTRACTFertilization is a key biological process in which the egg and spermmust recognize one another and fuse to form a zygote. Although theprocess is a continuum, mammalian fertilization has been studied asa sequence of steps: sperm bind and penetrate through the zonapellucida of the egg, adhere to the egg plasma membrane and finallyfuse with the egg. Following fusion, effective blocks to polyspermyensure monospermic fertilization. Here, we review how recentadvances obtained using genetically modified mouse lines bringnew insights into the molecular mechanisms regulating mammalianfertilization. We discuss models for these processes and we includestudies showing that these mechanisms may be conserved acrossdifferent mammalian species.

KEYWORDS: Sperm binding, Acrosome exocytosis, Gamete fusion,Polyspermy block

IntroductionFertilization is an essential step in sexual reproduction and consistsof a carefully orchestrated series of events that culminate with thegeneration of a genetically unique zygote. Upon ejaculation in thefemale genital tract, millions of sperm ascend the uterus. However,only a few pass through the uterotubal junctions to migrate towardsthe ampulla of the oviduct. During this transit, sperm acquire theability to fertilize eggs through a process defined as capacitation(Austin, 1951, 1960; Chang, 1951), which consists of a series ofphysiological and molecular changes that sperm acquire in thefemale reproductive tract. These changes pertain to the spermmotility pattern (Chung et al., 2014) and to their ability to undergoacrosome exocytosis (the fusion of the sperm plasma membranewith the outer acrosomal membrane), which is essential forfertilization (Puga Molina et al., 2018). Capacitated spermtransverse the cumulus oophorous (or cumulus mass), a mass offollicle cells kept together by hyaluronic acid and, within hours ofovulation, only one single spermatozoon successfully fuses with theegg. Effective mechanisms ensure monospermic fertilization, whichis essential for successful embryonic development and healthypregnancy. The first of these is a species-specific gameterecognition process at the zona pellucida (zona; ZP), the envelopesurrounding mammalian oocytes, which mediates sperm bindingand subsequent penetration through the zona. In this Review, we use‘sperm binding’ to refer to the interaction between the sperm plasmamembrane and the egg zona (Fig. 1, 1), and the term ‘spermpenetration’ to refer to the passage of the sperm through the zona(Fig. 1, 2). After penetration, the sperm reach a space enclosedbetween the inner aspect of the zona and the egg plasma membrane

(the oolemma) known as the perivitelline space. Here, a secondspecies-specific gamete recognition process mediates ‘gameteadhesion’; the interaction between the acrosome-reacted spermplasma membrane and the oolemma (Fig. 1, 3). Gamete recognitionat the zona and gamete adhesion prior to fusion occur in a species-specific manner. Finally, after gamete adhesion, the spermmembrane and the oolemma fuse to generate the zygote, whichwe refer to as ‘gamete fusion’ (Fig. 1, 4). Following gamete fusion isthe exocytosis of cortical granules: membrane-bound vesiclesderived from the Golgi apparatus that contain biochemical matrix-remodelling apparatus (Ducibella et al., 1988) (Fig. 1, 5). Theresulting molecular changes in the zona and the oolemma establisheffective blocks to polyspermy to prevent polyploidy, which isembryonic lethal in mammals (Fig. 1, 6).

Fertilization provides an excellent physiological platform for thestudy of ligand-receptor interactions. Moreover, understanding themolecular mechanisms that mediate fertilization will help to resolvethe idiopathic causes of human infertility, establish novel treatmentsfor providing superior fertility care and develop effectivecontraceptive agents. Here, we provide a comprehensive review ofthe fertilization process in mammals. We describe the most recentfindings on the mechanisms mediating each of the steps describedabove. For each step, we report the most recent advances fromstudies performed on genome-edited mouse lines that describe thefunction and interactions of proteins during fertilization, focusingspecifically on the proteins that are reported to be essential forfertilization.

Composition and structure of the zona pellucidaA structurally intact zona pellucida plays major roles in mammalianfertilization: it mediates species-specific gamete recognition, itprevents polyspermy and it protects the preimplantation embryofrom being resorbed into the oviduct epithelial lining (Bronson andMcLaren, 1970; Modlinski, 1970). The zona of most mammalianspecies contains either three or four glycoproteins: ZP1, ZP2/ZPA,ZP3/ZPC and ZP4/ZPB (Boja et al., 2003; Harris et al., 1994;Lefievre et al., 2004) (Box 1, Fig. 2A). Extensive studies in micehave shed light on the structure of the zona, which is composed ofZP1, ZP2 and ZP3 (Bleil and Wassarman, 1980a).

Mutations of the genes encoding each zona protein have beenshown to affect the zona structure either partially or severely, whichleads to female subfertility or infertility (Box 2). Gene deletions intransgenic mice have shown that the absence of ZP1 results in amisshapen, thinner and more fragile zona structure compared withthe wild type. Although still capable of supporting sperm bindingand fertilization, ZP1 null mutations are associated with femalesubfertility (Rankin et al., 1999). A more severe phenotype isobserved in mutant mice lacking either ZP2 or ZP3: homozygousnull females form a very thin zona (Zp2Null) or do not form a zona atall (Zp3Null), which results in defective follicle formation, scarcity ofovulated eggs (that also lack the zona) and ultimately in femaleinfertility (Rankin et al., 1996, 2001). To date, a complete crystalstructure of a ZP protein has only been reported for chicken ZP3

Department of Biological Science, College of Engineering and Natural Sciences,The University of Tulsa, Tulsa, OK 74104, USA.

*Author for correspondence (matteo.avella@utulsa.edu)

H.H.B., 0000-0002-3017-2570; F.H.R., 0000-0003-1650-7351; M.A.A., 0000-0003-0104-3304

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(Han et al., 2010). Further characterization of the moleculararchitecture of the zona matrix will allow a better understandingof the putative interactions between zona proteins in establishingand maintaining a three-dimensional structure that guaranteessuccessful sperm-egg recognition.

Sperm bind to the zona pellucida with species-specificityUncovering the zona protein that mediates sperm binding to the egghas been a compelling and debated biological question over the pastfew decades. Results have varied based on the assays employed orthe model systems adopted. Previous studies have reported eachindividual zona protein as the ligand for gamete recognition (Bleiland Wassarman, 1980b; Ganguly et al., 2010; Yonezawa et al.,2012). Controversy still persists on whether a single zona proteinmay be sufficient to support sperm binding and which of the fourzona proteins mediate gamete recognition.To identify the zona ligand that mediates sperm binding, pioneer

studies have used soluble SDS-PAGE mouse ZP proteins in in vitrocompetitive sperm binding assays with mouse ovulated eggs. Thesestudies showed significant sperm binding inhibitory effects only inthe presence of ZP3. On the other hand, with ZP1, ZP2 or ZP3 thatwere isolated from the two-cell embryo zonae, to which sperm areunable to bind, no effect on sperm binding to ovulated eggs wasrecorded (Bleil and Wassarman, 1980b). These observationsintroduced a widely accepted model that indicate ZP3 as the ligandfor gamete recognition at the zona. This model later implicatedO-glycans attached to Ser332 and Ser334 of ZP3 as the zona ligands forsperm binding (Chen et al., 1998; Florman and Wassarman, 1985).More specifically, the α1,3 galactose and the N-acetylglucosaminewere proposed as the ligand for mouse sperm binding (Bleil andWassarman, 1988; Miller et al., 1992), and the sialyl-LewisX antigenfor human sperm binding (Pang et al., 2011). However, fertility ispreserved in genome-edited mice either lacking the α1,3 galactose orthe putative sperm receptor for N-acetylglucosamine (Thall et al.,1995). Indeed, no carbohydrates have been found on Ser332 andSer334 in normal mouse zonae (Boja et al., 2003), and mutation of the

sites to prevent glycosylation in transgenic mice has not led to femaleinfertility (Gahlay et al., 2010). Moreover, mice that lack the glycosyltransferases MGAT1 and T-synthase are still fertile (Shi et al., 2004;Williams et al., 2007).

An alternative model of recognitionAnother thought-provoking model suggests that the process ofsperm binding to the zona might not be necessary for penetration,and that no gamete recognition at the zona occurs before spermcrossing the zona. This model originates from observations inknockout mice lacking ADAM3 (a disintegrin and metalloprotease3). Although sperm from Adam3Null mice show impaired spermbinding to the zona, the sperm can fertilize the eggs in vitro(Yamaguchi et al., 2009). In addition, deletion of a number of genesthat are necessary for normal expression of ADAM3 in sperm, suchas Ace (Krege et al., 1995), Clgn (Ikawa et al., 1997), Tpst2(Marcello et al., 2011), Calr3 (Ikawa et al., 2011) and Pdilt(Tokuhiro et al., 2012), leads to defective sperm binding to the zona(Yamaguchi et al., 2009). Of note, humans have two orthologues ofAdam3 (ADAM3A and ADAM3B), and both are pseudogenes.However, male fertility is preserved in the absence of the ADAM3orthologues (Grzmil et al., 2001). Moreover, the observation thatmammalian sperm bind to the zonawith species-specificity (Bedford,1981) indicates a role of the zona in mediating gamete recognition.For example, human sperm bind to the zonae of human and hominoidprimates (gibbon, gorillas), but do not bind to the zonae of other sub-hominoid primates (baboons, rhesus monkeys, squirrel monkeys) ormouse and other rodents (Baibakov et al., 2012; Bedford, 1977;Hoodbhoy et al., 2005; Lanzendorf et al., 1992). Likewise, horsesperm can bind to the zonae of horse, but not to the zonae of pigs(Mugnier et al., 2009). Conversely, mouse and rat sperm bind withsimilar efficiency to the zonae of either rodent (Hoodbhoy et al.,2005), and pig sperm bind to equine and bovine zonae as effectivelyas to pig zonae (Mugnier et al., 2009; Takahashi et al., 2013). Thus,two main observations prompt reconsideration for alternative modelsmediating sperm binding to the zona. First, mouse sperm cannot bind

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Fig. 1. Schematic representation of mammalianfertilization. After crossing the cumulus mass,sperm bind to the zona pellucida surrounding themetaphase (M)II oocyte (1), penetrate through thezona (2) and adhere to the oolemma, the eggplasma membrane (3). Sperm-egg fusion (4) isfollowed by the exocytosis of the cortical granules,which are organelles distributed in the cortex of theunfertilized eggs (5). The release of the corticalgranule content is followed by a block topolyspermy (6), which ensures monospermicfertilization, essential for successful mammalianembryonic development. Figures representingsperm and eggs are not drawn to scale. Theshapes of the proteins are drawn for facilevisualization of putative protein interactionspurposes but are not representative of the actualfolding structure.

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to the two-cell embryos and the only characterized biochemicalmodification in the zona is a ZP2 cleavage after fertilization (Bleilet al., 1981) – remarkably, mouse sperm can bind de novo two-cellembryos when ZP2 remains uncleaved after fertilization (Baibakovet al., 2007; Gahlay et al., 2010). Second, as reported above, humansperm are selective and only bind to human and hominoid primateseggs (gibbon, gorillas), but do not bind to mouse eggs (Bedford,1977; Lanzendorf et al., 1992).

Experimental evidence for species-specific zona recognitionKeeping this species-specificity in mind, gain-of-function assaysusing mouse genetics have been established by individuallyexpressing human ZP1, ZP2 or ZP3 genes in the appropriatemouse Zp1, Zp2 or Zp3 null background (Rankin et al., 1996, 1999,2001) (Fig. 2). The three established transgenic mouse lines havebeen defined as human (hu)ZP1Rescue, huZP2Rescue and huZP3Rescue.Moreover, a fourth line expressing mouse ZP1-3 and human ZP4,

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Fig. 2. Sperm bind to the zona pellucida via the N terminus of ZP2. (A) Secreted ectodomains of ZP1-4. Yellow bars denote cysteine residues. Asterisk on ZP2represents the post-fertilization cleavage site: 166LA*DE169 in mouse, 171LA*DD174 in human. ZP2 sperm binding and trefoil domains on ZP1 and ZP4 are alsoshown. The red inverted tripod indicates the N83 glycan. (B) Composition of the zona pellucida. Human zona contains ZP1-4 (top), mouse zona contains ZP1-3(bottom). (C) Human ZP2 N terminus is necessary and sufficient to mediate sperm binding. Transgenic (hu)ZP2Rescue eggs express mouse ZP1, ZP3 and humanZP2. Chimeric human/mouse (hu/mo)Zp2 eggs express a mouse ZP2 in which the N terminus has been replaced with the human ZP2 N terminus. Mouse/human(mo/hu)ZP2 eggs express a chimeric human ZP2, in which the human ZP2 N terminus has been replaced with the mouse ZP2 N terminus. Human sperm bind to(hu)ZP2Rescue and hu/moZP2 zonae pellucidae (gain of function), but do not bind to mo/huZP2 eggs (loss of function). (D) Mouse ZP2 N terminus is necessary andsufficient to support sperm binding. Zonae lacking ZP2 (moZp2Null; huZP4) or the ZP2 N terminus [moZP2Trunc (moZP251-149)] fail to support sperm binding andfemales are infertile. Eggs producing a fusion ZP2/ZP4 protein carrying the ZP2 amino acids 35-149 (moZp235-149; huZP4) support sperm binding and are fertile,despite the absence of endogenous ZP2. (E) Human and mouse sperm binding assays. Human sperm can bind to huZP2Rescue and hu/moZp2, but not tomo/huZP2 eggs (left); immature human oocytes have been used as internal positive control for human spermbinding andmouseZp3EGFPeggs [composed ofmouseZP1-3 and expressing a recombinant ZP3 transgenically tagged with GFP (green zona)] have been used as internal negative control. Mouse sperm can bind tozonae containing normal or truncated ZP2 or to mouse zonae lacking mouse ZP2 (right). Zp3EGFP eggs have been used as internal positive control formouse sperm binding and two-cell embryos have been used as internal negative control. Sperm binding images reprinted with permission (figures 1E, 2E, 3C, 4D inAvella et al., 2014).

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defined as huZP4Transgenic, has also been generated (Baibakov et al.,2012; Rankin et al., 1998, 2003). To assess which glycoprotein issufficient to support human sperm binding, eggs from eachhuZP1Rescue, huZP2Rescue, huZP3Rescue and huZP4Transgenic linehave been inseminated with human sperm, revealing that humansperm bind avidly only to the huZP2Rescue eggs (Baibakov et al.,2012) (Fig. 2B,C,E). In addition, human sperm injected in vivo intothe oviduct ampulla of huZP2Rescue and huZP3Rescue female micehave been observed only in the perivitelline space of thehuZP2Rescue eggs (Avella et al., 2014). After penetration throughthe zona, however, human sperm cannot fuse with the mouse eggs,owing to species-specific mechanisms controlling gamete adhesionand fusion (Bianchi and Wright, 2015; Quinn, 1979; Yanagimachi,1984). These observations demonstrate that ZP2 is sufficient tosupport human sperm binding and penetration of the zona pellucidain transgenic mice (Avella et al., 2014; Baibakov et al., 2012), in theabsence of sialyl-LewisX antigen (Avella et al., 2014; Pang et al.,2011).Complementary loss-of-function assays have been performed by

crossing mouse lines to obtain zonae lacking either mouse or humanZP2. Transgenic zonae containing mouse ZP1, ZP3 and human ZP4cannot support mouse sperm binding. Likewise, transgenic zonaecontaining human ZP1, ZP3 and ZP4 cannot support mouse andhuman sperm binding, and female mice are infertile (Avella et al.,2014). These observations further support the model that ZP2 isnecessary and sufficient for mouse and human sperm binding intransgenic mice (Avella et al., 2014) (Fig. 2D). Moreover, thegamete recognition domain has been refined to the N terminus ofZP2 by deleting a region coding for the ZP2 N terminus (ZP251-149).These mutant zonae cannot support mouse sperm binding andfemale mice are infertile (Avella et al., 2014) (Fig. 2D,E). Inaddition, a recombinant fusion mouse Zp235-149/huZP4 protein-encoding gene expressed in the Zp2Null background has been shownto re-establish formation of a zona matrix, which could supportsperm binding in vitro, resulting in fertile female mice (Tokuhiro

and Dean, 2018) (Fig. 2D). Genetic ablation of the proposed glycanattachment site on the N terminus of ZP2 does not affect spermbinding or female fertility in transgenic mice (Tokuhiro and Dean,2018), which demonstrates that gamete recognition mediated by theN terminus of ZP2 is glycan independent (Boja et al., 2003; Avellaet al., 2014; Tokuhiro and Dean, 2018) (Fig. 2A). The N terminus ofZP2 accounts for the species-specificity observed for human spermbinding. Chimeric human/mouse ZP2 proteins, with the humanN-terminal domain in place of the mouse N terminus, supporthuman sperm binding analogous to the huZP2Rescue zonae matrices(Avella et al., 2014) (Fig. 2C,E). All these results are consistent withthe N-terminal region of ZP2 being necessary and sufficient forsperm binding to the zona in mice and humans, along with femalefertility in mice (Avella et al., 2014; Tokuhiro and Dean, 2018).These recent results support a model of gamete recognition in whichmouse and human sperm bind to the zona via the N terminus of ZP2and the binding appears to be independent of O- or N-linked zonaprotein glycans (Tokuhiro and Dean, 2018).

A plausible alternative interpretation for these observations is thatspermmay not directly interact with ZP2 and that the ZP2 N terminusis necessary to preserve a zona structure that guarantees normal spermbinding. Indeed, the absence of the ZP2 N terminus (Avella et al.,2014) may mimic a possibly modified zona structure occurring afterfertilization, which would impede sperm binding and lead toinfertility. Because sperm binding is necessary for penetrationthrough the zona (Avella et al., 2014; Baibakov et al., 2012; Tokuhiroand Dean, 2018) and occurs with species-specificity (Bedford, 1977),onewould expect that sperm recognize a putative zona sperm bindingsite, the structure of which is preserved by an intact ZP2 N terminus.After fertilization, the cleavage of ZP2 would disrupt the nativeconformation of this putative sperm binding site, impedingsupernumerary sperm binding to the zona. If this model is indeedcorrect, the data obtained from genetically edited mouse lines mayhelp to localize the putative sperm binding site on the zona: Zp1Null

female mice still form a zona that is capable of supporting sperm

Box 1. ZP protein structureEach ZP protein contains a signal peptide that directs it into the secretorysystem and a transmembrane domain that tethers the protein to theendomembrane system (Boja et al., 2003; Lefie vre et al., 2004; Lianget al., 1990). Upon species-specific protein glycosylation with N- and O-glycans (Boja et al., 2003), each ZP protein is transferred towards theperiphery of the oocyte and cleaved upstream of a dibasic motif(Hoodbhoy et al., 2006; Jimenez-Movilla and Dean, 2011), whichreleases the ZP from the oolemma (Jimenez-Movilla and Dean, 2011;Monné and Jovine, 2011). The proteins then establish non-covalentinteractions with other ZP proteins to form a three-dimensional zonamatrix. Both the ZP domain and the cytoplasmic tail control this finalassembly (Jimenez-Movilla and Dean, 2011; Monné and Jovine, 2011).ZP proteins also contain a ∼260 amino acid domain with eight or tenconserved cysteine residues, which form specific disulphide bonds forthe correct folding of the protein (Bork and Sander, 1992) (Fig. 2A). TheZP2 and ZP3 ectodomains contain an even number of cysteine residues,whereas ZP1 has an uneven number, which is required for ZP1homodimerization for matrix structure stabilization (Bleil andWassarman, 1980a; Epifano et al., 1995). Moreover, ZP1 and ZP4contain a trefoil domain: six cysteine residues that form disulphide bondsthat preserve the distinctive three-looped shape of the trefoil. The trefoilresides immediately upstream of the zona domain (Boja et al., 2003;Bork, 1993) and the functions of the trefoil domain for fertilization remainuncertain (Sommer et al., 1999) (Fig. 2A). Preliminary evidence in catshas shown that the ZP4 trefoil domain appears to preserve the structuralstability of the zona (Braun, 2009).

Box 2. Pathogenic variants in zona protein genes causehuman infertilityZP1, ZP2 and ZP3 are crucial for maintaining the structural integrity ofthe zona and for fertility in humans. Female infertility with autosomalrecessive inheritance, characterized by eggs that lack the zona, hasbeen associated with a homozygous frameshift deletion(c.1169_1176del) in the ZP1 gene, which results in a premature stopcodon predicted to generate a truncated protein (I390fs404*). Thetruncated form sequesters ZP3 during oocyte growth, preventing normalzona biogenesis (Huang et al., 2014). A second female patient carryingone heterozygous nonsense mutation in ZP2 (c.2092C>T) and oneheterozygous frameshift mutation in ZP3 (c.1045_1046insT) presentedwith infertility associated with the absence of zona formation. Mimickingthese mutations in transgenic mice recapitulates the phenotypeobserved in the patient (Liu et al., 2017). A third case has reported 11female patients from three different families carrying a heterozygousmissense mutation in human ZP3 (c.400G>A), which is associated withempty follicle syndrome, due to the absence of the zona formation (Chenet al., 2017). In vitro studies in CHO cells have shown that recombinantZP3 mutants with the same missense mutation fail to interact with ZP2,even when co-expressed with wild-type ZP1-4 proteins (Chen et al.,2017). Recently, two homozygous variants of ZP2 (c.1695-2A>G andc.1691_1694dup), from two consanguineous families have beenassociated with female infertility (Dai et al., 2018). Both variantsproduce a ZP2 protein that is truncated at the zona domain, whichmight prematurely interact with other zona proteins during oocyte growth,generating a thin zona (Dai et al., 2018).

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binding, so the sperm binding site should reside on ZP2 and/or ZP3.Also, we may exclude the ZP2 C terminal region, which is notrequired for sperm binding, because recent studies in transgenic miceshow that ZP251-149 is necessary (Avella et al., 2014) and sufficient(Tokuhiro and Dean, 2018) for sperm binding. In addition, humansperm, which are normally unable to bind to mouse zonae (Bedford,1977), can bind and penetrate huZP2Rescue zonae, but not huZP3Rescue

zonae, in vitro and in vivo (Avella et al., 2014; Baibakov et al., 2012).Together, this evidence indicates that the putative sperm binding siteshould not reside on human ZP3. Moreover, recombinant mouse orhuman ZP2 N termini directly interact with mouse or human sperm inpeptide-bead binding assays (Avella et al., 2014, 2016; Baibakov et al.,2012). From all these data, we hypothesize a direct interaction betweensperm and the N terminus of ZP2 and we conclude that, for successfulfertilization, sperm bind to the zona via the N terminus of ZP2.Also, this model raises the prediction of the existence of a putative

sperm receptor for the ZP2 N terminus, which would mediate spermbinding to the zona. In the past decades, numerous studies havefocused on ZP3 as a ligand to identify the sperm receptor (Bleil andWassarman, 1990; Cheng et al., 1994; Ensslin and Shur, 2003;Hanayama et al., 2004; Muro et al., 2012; Neutzner et al., 2007;Silvestre et al., 2005; Tardif et al., 2010). However, ablation of eachnovel candidate in transgenic mice has not resulted in maleinfertility and the identity of this receptor is still unknown. Here, wereport a number of examples including SED1 [secreted protein thatcontains Notch-like epidermal growth factor (EGF) repeats anddiscoidin/F5/8 type C domains] (also known as MFGE8), the ZP3receptor (ZP3R; also known as sp56), and zonadhesin. The spermsurface protein SED1 was originally identified using porcine zonaproteins from the boar orthologue, p47. Western blot analyses haveshown that SED1 interacts with ZP3 (Ensslin and Shur, 2003).However, deletion of the SED1-encoding gene, either shows noimpact on male fertility (Hanayama et al., 2004; Neutzner et al.,2007) or leads to male subfertility (Ensslin and Shur, 2003; Silvestreet al., 2005). Thus, SED1 is not necessary for sperm binding. A

second candidate, ZP3R, localizes on the sperm plasma membraneand shows binding affinity for the glycans on ZP3 (Bleil andWassarman, 1990; Cheng et al., 1994). In an in vitro competitivebinding assay, ZP3R (recombinant or native) can inhibit spermbinding to ovulated mouse eggs, but not to embryos. Nonetheless,genetic ablation of the gene encoding ZP3R does not affect malefertility in transgenic mice (Muro et al., 2012). Finally, zonadhesinhas been also an appealing candidate; it is a testis-enriched proteinthat presents several cell-adhesion domains and becomes exposed inthe sperm plasma membrane during capacitation. Recombinantzonadhesin can bind to the zona, and antibodies against zonadhesinsignificantly inhibit sperm binding and in vitro fertilization (Tardifet al., 2010). Nevertheless, mutant null males are fertile, which leadsto the conclusion that zonadhesin is not necessary for sperm binding(Tardif et al., 2010).

Today, the definition that the ZP2 N terminus is the ligandnecessary and sufficient for gamete recognition at the zona in miceand humans may offer a new path toward the identification of novelsperm receptor candidates. Moreover, recent studies reported thatthe folding of the N terminus of ZP2 shows structural similaritieswith a specific region of the egg coat protein VITELLINEENVELOPE RECEPTOR for LYSIN (VERL) from the marinemollusc, abalone. During abalone fertilization, the egg VERLdirectly interacts with the sperm lysin to guarantee species-specificgamete recognition (Raj et al., 2017). Even though it appearsplausible that ZP2 may not interact with a mammalian homolog oflysin, the functional (Avella et al., 2014; Baibakov et al., 2012;Tokuhiro and Dean, 2018) and structural (Raj et al., 2017) definitionof the oocyte ligand open new perspectives for the identification ofthe putative receptor for mammalian sperm binding to the zona.

Acrosome exocytosis is necessary for fertilizationThe acrosome is a Golgi-derived subcellular organelle that underliesthe anterior plasma membrane of mammalian sperm heads. Inner andouter acrosomal membranes delimit this vesicle (Fig. 3A), which

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Fig. 3. Acrosome-reacted sperm fuse with theegg plasma membrane. (A) During acrosomeexocytosis, the sperm plasma membrane fuseswith the outer acrosomal membrane (OAM) torelease acrosomal contents and acrosome-reactedsperm expose the inner acrosomal membrane(IAM). Acrosome exocytosis is necessary forgamete fusion, which occurs between theoolemma and the equatorial segment, a residualfragment of the plasma membrane (left). (B) Site ofacrosome exocytosis during fertilization. Anongoing scientific debate has been entertained inthe past decades on the location of acrosomeexocytosis. Current models envision exocytosis tooccur in the oviduct before encountering the egg orits surrounding layers, while sperm crosses thecumulus oophorus surrounding the egg, uponsperm binding to the zona pellucida or during initialpenetration through the zona matrix of themetaphase (M)II oocyte.(C) Schematic of putative protein interactionsmediating gamete fusion. As the GPI-anchoredprotein JUNO binds IZUMO1, CD9, through theEC2 domain (larger loop) binds in cis a number ofstill-undefined proteins, which may include theoocyte fusogens (asterisk). Three tetraspaninspartners, CD9P-1, IGSF8 and α6β1 integrin havebeen reported to interact with CD9 in the oocyte.

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includes a mixture of digestive enzymes – although none of thedigestive enzymes is necessary for binding or penetration through thezona pellucida (Buffone et al., 2014). To gain fusion competencewith the egg, capacitated mammalian sperm must undergo theacrosome reaction, an exocytotic event, which involves the fusion ofthe sperm plasma membrane with the outer acrosomal membrane.This membrane fusion event lets the acrosome-reacted sperm exposethe inner acrosomal membrane, together with the equatorial segment.The sperm equatorial segment is defined by two regions, one inwhich the inner and outer acrosomal membrane are tightly apposed,and another one in which the inner and outer acrosomal membranesseparate to include the acrosomal matrix (Yanagimachi and Noda,1970). Acrosome-reacted sperm can fuse with the oolemma, the eggplasma membrane, via a remnant of the sperm plasma membrane,overlying the equatorial segment (Fig. 3A).Only acrosome-reacted sperm are found in the perivitelline space

of eggs from guinea-pig, mouse and rabbit (Fleming andYanagimachi, 1982; Inoue et al., 2011; Kuzan et al., 1984), whichindicates that, at some point before fusion, sperm must undergoacrosome exocytosis. The site of acrosome reaction has been anintriguing matter of debate for decades and remains highlycontroversial (Fig. 3B). Based on functional studies reporting theability of solubilized mouse zonae or mouse ZP3 to trigger acrosomeexocytosis in vitro (Bleil and Wassarman, 1990), acrosome reactionhas been thought to be induced during gamete recognition at the zonapellucida (Saling et al., 1979). However, binding to the zona has beenreported to be insufficient to induce acrosome exocytosis of AcrGFP

sperm, even several hours after binding (Baibakov et al., 2007). Theseobservations prompted consideration of alternative models ofacrosome exocytosis induction.Early studies in rabbit show that acrosome-reacted sperm are able

to bind and fertilize de novo rabbit ovulated eggs (Kuzan et al., 1984).This observation could be repeated in mouse using supernumeraryperivitelline sperm recovered from inseminated Cd9Null eggs, whichare unable to fuse with mouse sperm (discussed below) (Inoue et al.,2011). In addition, time-lapse imaging ofAcrGFPmouse sperm showsthat acrosome-reacted sperm penetrate the zonawith higher efficiencythan acrosome-intact sperm (Jin et al., 2011). These observations areconsistent with recent findings showing that both mouse and humansperm can remain bound to the N terminus of ZP2 after acrosomeexocytosis (Avella et al., 2016). However, this model does not fullyexplain the acrosomal shrouds (residues of the vesiculated acrosomalcaps) that are found attached to the zonae surrounding fertilized eggsin different mammalian species (VandeVoort et al., 1997;Wakayamaet al., 1996; Yanagimachi and Phillips, 1984). Indeed, if spermundergo acrosome exocytosis before binding to the egg, no shroudshould be observed on the zona. Moreover, species-specificity hasbeen observed in the induction of acrosome exocytosis during zonapellucida penetration; field vole sperm penetrate mouse and hamsterzonae, and the acrosome remains in the perivitelline space, but theyare unable to fuse with the eggs (Wakayama et al., 1996).To clarify these diverse sets of observations, it is conceivable that

different mechanisms could induce acrosome exocytosis, whichwould depend on the status of the egg investments encountered bythe fertilizing sperm. A tight cumulus mass surrounding freshlyovulated eggs could induce acrosome exocytosis before spermpenetration through the zona matrix. Conversely, a more dispersedor absent cumulus mass, as it is the case for some marsupials(Rodger and Bedford, 1982; Talbot and DiCarlantonio, 1984),would fail to induce acrosome exocytosis and would therefore bedependent on zona penetration (Baibakov et al., 2007; Wakayamaet al., 1996). Adding more complexity to the model, recent studies

have indicated that the majority of sperm have undergone acrosomeexocytosis before encountering the cumulus mass (Hino et al.,2016; Muro et al., 2016), and only ∼5% of the sperm traversing theampulla are acrosome-intact (La Spina et al., 2016). Thereforeacrosome exocytosis of the fertilizing sperm might be occurringduring sperm migration through the female oviduct and theacrosome-reacted sperm cross the cumulus before binding to thezona (Fig. 3B). This model raises some intriguing predictions to betested in future assays: as sperm migrate through the female oviductor as they cross the cumulus mass, what are the molecularmechanisms that mediate the induction of acrosome exocytosis?Also, both acrosome-intact and acrosome-reacted sperm have theability to bind to the zona, so is there only one putative spermplasma membrane receptor for binding to the zona pellucida, whichis relocated to the equatorial segment upon acrosome reaction? Orare there two or more different sperm receptors, located in differentsites of the sperm head? A clear understanding of the mechanismsand sites of the induction of acrosome exocytosis are necessary for abetter understanding of the processes mediating gamete recognitionand fusion in mammals.

Species-specific gamete adhesion is required for gametefusionGamete adhesion between the oolemma and the sperm plasmamembrane overlying the equatorial segment is a precursor stepnecessary for gamete fusion and shows extensive species-specificityin mammals (Bedford, 1977; Bianchi and Wright, 2015; Quinn,1979; Yanagimachi, 1984). Gamete adhesion begins with known orputative molecular interactions between proteins of sperm and theoolemma. Upon gamete adhesion, a closer apposition of themembranes establishes mixing of lipid bilayers, followed by theformation of fusion pores, which generates cytoplasmic continuityand gamete fusion (Yanagimachi, 1984). Gamete adhesion andfusion have been intensively investigated over the past decades.Pioneering in vitro fertilization (IVF) studies have reported howdefective or uncontrolled mammalian gamete adhesion and fusioncauses failed fertilization, including eggs remaining unfertilized(Chang, 1959; Edwards et al., 1970) or polyspermy (Wentz et al.,1983), both of which lead to infertility.

Gamete adhesionDespite decades of investigation, only a few proteins have beenfound to be necessary for the adhesion of the sperm plasmamembrane to the oolemma. Using loss-of-function assays intransgenic or mutant mice, two sperm proteins have been found tomediate the sperm adhesion to the oolemma that is necessary forfertilization: IZUMO1 (Inoue et al., 2005) and SPACA6 (Lorenzettiet al., 2014). IZUMO1 is a testis-specific cell-surface protein andpart of the immunoglobulin type-I cell superfamily, characterizedby a cytoplasmic C-terminal tail, a transmembrane region and aconserved ‘Izumo domain’, which is linked to an extracellularimmunoglobulin-like (Ig-like) C2-type domain by a centralβ-hairpin region. Each of these domains plays an important role inmediating gamete adhesion (Aydin et al., 2016; Inoue et al., 2005;Ohto et al., 2016). Upon acrosome exocytosis, IZUMO1, which isnot detectable on the plasma membrane of acrosome-intact sperm,localizes to the equatorial segment (Satouh et al., 2012) to mediategamete adhesion with the egg plasma membrane. Absence ofIZUMO1 leads to impairment of gamete adhesion and to anaccumulation of sperm in the perivitelline space (Inoue et al., 2005).SPACA6 is a testis-specific protein that includes an Ig-like domain.Random integration of a transgene has been reported to disrupt the

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open reading frame of Spaca6 (intron 2-exon 6), which results inloss of SPACA6 expression and sperm-egg adhesion impairment,with an accumulation of supernumerary sperm in the perivitellinespace (Lorenzetti et al., 2014).On the oolemma, two proteins are necessary for gamete adhesion.

CD9 was the first protein identified to mediate gamete adhesion andbelongs to the family of tetraspanins. Females lacking CD9 showseverely reduced fertility, or infertility, due to the inability of theoolemma to support sperm adhesion (Le Naour et al., 2000). Asecond tetraspanin, CD81, has also been found to play a role inmediating gamete adhesion. Cd81Null female mice accumulatesupernumerary sperm in the perivitelline space, although they arestill fertile – albeit with reduced fertility (Rubinstein et al., 2006).More recently, an oocyte glycosylphosphatidylinositol (GPI)-

anchored protein, folate receptor 4 (FOLR4, also known asIZUMO1R), has been identified as the oolemma-bound receptorfor IZUMO1 (Bianchi et al., 2014). Although this protein presentsstructural similarity with other folate receptors, it is unable to bind tofolates and therefore it is also called JUNO, after the Romangoddess of fertility and marriage. Female mice lacking JUNO areinfertile, owing to the inability of the egg to adhere to sperm(Bianchi et al., 2014). Structural studies using X-ray crystallographyhave confirmed direct interaction between human IZUMO122-254

and JUNO20-228, and show that the two proteins form a stablecomplex that is necessary for gamete adhesion (Aydin et al., 2016;Ohto et al., 2016). These structural studies have identified 19residues in JUNO that interact with 20 amino acids in IZUMO1(Aydin et al., 2016; Ohto et al., 2016). In addition, this IZUMO1-JUNO interaction (Jean et al., 2019) regulates the species-specificgamete adhesion observed between human sperm and zona-freehamster oocytes (discussed in detail below) (Bianchi and Wright,2015; Han et al., 2016; Yanagimachi, 1984). Additional evidencesuggests that after gamete adhesion, a transmembrane region ofIZUMO1 is involved in protein dimerization, which allows dimericIZUMO1 to interact with a putative oocyte receptor (Inoue et al.,2015). To date, JUNO and IZUMO1 are the only egg receptor andsperm ligand known to interact directly to guarantee sperm-eggadhesion and this binding ability is conserved in several mammalianspecies (Bianchi and Wright, 2015; Grayson, 2015).Themechanismsmediating the transition between gamete adhesion

and fusion are still uncertain. Nevertheless, it is conceivable thatduring gamete adhesion, certain proteins may act in cis to recruit othermolecular players which would mediate gamete fusion.

Gamete fusionFusogen proteins mediate fusion of membranes during cellinteractions. Examples of fusogens include the mammaliansyncytins, which are necessary for cytotrophoblast fusion duringplacentation, the Caenorhabditis elegans Epithelial Fusion Failure-1(EFF-1) and Anchor-cell Fusion Failure-1 (AFF-1) proteins, whichmediate cell-fusion mechanisms during embryo/larval development(reviewed by Hernández and Podbilewicz, 2017). Despite intensiveinvestigation, the existence and identity of gamete fusogens remainuncertain and our understanding of the mechanisms mediatinggamete fusion are incomplete.One model for cell-cell fusion that might be applied to fertilization

arises from studies on myoblast fusion. Upon cell adhesion,myoblasts generate actin-propelled membrane protrusions thatinvade the other cell, which responds in turn through a myosin II-mediated increase of cortical tension to the myoblast invasion. Thecontrast between invasive and resistance forces disrupt the lipidbilayers of each cell, leading to the formation of fusion pores (Kim

et al., 2015). This compelling model is reminiscent of earlyobservations documenting species-specificity in gamete fusionbetween human sperm and hamster oocytes (Yanagimachi andNoda, 1970). In these IVF assays, the zona was removed fromovulated hamster oocytes and denuded eggs were inseminated withhuman sperm. Soon after gamete adhesion, protruding microvillifrom the egg surrounded the human sperm, leading to gamete fusion(Yanagimachi, 1984). Asmentioned above, this cross-species gametefusion event is dependent on the IZUMO1-JUNO interaction(Bianchi and Wright, 2015; Han et al., 2016). The ‘myoblastfusion’ model raises the prediction that interaction between JUNOand IZUMO1 should be sufficient to induce gamete fusion. To testthis hypothesis, IZUMO1 and JUNO were ectopically expressed ineukaryotic COS-7 cells, and despite the occurrence of cell-adhesion,no cell fusion was recorded. These data indicate that the IZUMO1-JUNO interaction is not sufficient to induce cell fusion (Inoue et al.,2015), which is consistent with the fact that neither IZUMO1 norJUNO show sequence similarities to currently identified membranefusogens (Bianchi and Wright, 2015).

One final model envisions a pre-fusion step (in between gameteadhesion and fusion), during which the direct bond betweenIZUMO1 and JUNO generates an accumulation of CD9 in theadhesion area (Chalbi et al., 2014). By acting independently(Ohnami et al., 2012), both CD9 and CD81 directly interact withthree oocyte tetraspanin partners, CD9P-1 (PTGFRN), α6β1integrin and IGSF8 (also known as EWI-2). In addition, CD9 andCD81 may recruit in cis other proteins (although still undefined) tomediate gamete fusion (Jegou et al., 2011; Ohnami et al., 2012;Rubinstein et al., 2006). In particular, studies in vitro have shownthat gamete fusion might be mediated by a three-residue epitope(SFQ) in the EC2 domain of CD9, which organizes a tetraspaninprotein network. More specifically, wild-type CD9 mRNA partiallyrescues the ability of Cd9Null eggs to adhere with sperm, whereasmutant CD9 mRNA that lacks the SFQ epitope fails to rescue thefusion competence in Cd9Null eggs (Zhu et al., 2002). Moreover,incubation of wild-type eggs with monoclonal antibodies againstthe SFQ epitope can inhibit sperm-egg fusion in mice, sheep andgoats (Xing et al., 2010), but not gamete adhesion in mice (Milleret al., 2000; Zhu et al., 2002). Incubation of the recombinant EC2domain with sperm does not preclude gamete adhesion and fusion,which implicates that sperm may lack a receptor acting as a transpartner for CD9 (Miller et al., 2000; Zhu et al., 2002). Thus, uponIZUMO1-JUNO interaction, it is conceivable that the SFQ epitopein the EC2 domain may play a part in mediating gamete fusion byrecruiting still-unidentified fusogens. Then, the sperm and eggfusogen interactions would mediate fertilization (Fig. 3C).

Effective block to polyspermy ensures monospermicfertilizationMammalian eggs cannot tolerate the physiological polyspermyobserved in other vertebrate species (such as urodeles,elasmobranchs, reptiles and birds) owing to the absence of amechanism that selects one single male pronucleus for interdigitatingwith the female pronucleus. Hence, polyploidy in mammals isembryonic-lethal: early studies in humans show ∼20% ofspontaneously aborted concepti to be polyploid (Hassold et al.,1980), of which 66-69% are diandric – the result of the fertilization byone diploid sperm or by two sperm (Jacobs et al., 1978; Zaragozaet al., 2000). Therefore, promptly after fertilization cortical granuleexocytosis releases zinc (Box 3) and a cortical granule-specificprotease that biochemically modifies the zona to preventsupernumerary sperm binding with and penetrating the zona matrix

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(known as the ‘zona block’) (Stewart-Savage and Bavister, 1988).Moreover, molecular changes at the oolemma prevent other spermadhering to and fusing with the oolemma (referred to as the‘membrane block to polyspermy’) (Sato, 1979). These eventstogether are defined as the block to polyspermy, which ensuresmonospermic fertilization (Fig. 4). These distinct biologicalprocesses are independent of each other and work with differentefficiencies in different mammalian models.

Membrane block to polyspermyIn mammals, the membrane block does not depend on the rapidmembrane depolarization (Jaffe et al., 1983) that occurs rapidly (afew seconds) after fertilization in species such as the starfish (Tyleret al., 1956) and the sea urchin (Jaffe, 1976). Studies in mice haveshown that zona-free, unfertilized oocytes remain competent forfusion for several minutes before a membrane block is established(Wolf and Hamada, 1977). One important aspect to note is that thismembrane block is independent of sperm entry into the cytoplasm(Wolf and Hamada, 1977): when one-cell zygotes or two-cellembryos were generated by intracytoplasmic sperm injection (ICSI)(Wolf and Hamada, 1977), which consists of the injection of asingle sperm in the oocyte cytoplasm, bypassing gamete fusion(Horvath et al., 1993; Maleszewski et al., 1996), their cell plasmamembranes still presented the ability to fuse de novo with othersperm.The molecular mechanism that regulates the membrane block in

mammals remains incompletely understood. In mice, a twofolddecrease in CD9 protein content and a reorganization of microvillidistribution have been reported to play a putative role in themembrane block (Zyłkiewicz et al., 2010). Moreover, shedding ofJUNO upon fertilization impedes further sperm adhesion to thefertilized egg oolemma (Bianchi et al., 2014) (Fig. 4, 2). However,as JUNO appears to persist for ∼40 min after fertilization, othermechanisms may account for the effective membrane block topolyspermy at the oolemma. Unfortunately, to date, no fusionmolecule(s) have been identified on either the egg or sperm plasmamembrane, and what the exact mechanism mediating the membrane

block is remains a compelling, but still unanswered, biologicalquestion.

A better understanding of the molecular basis of the membraneblock will be beneficial to elucidate the complexity of the differenteffective membrane blocks observed among mammalian species. Infact, in vitro and in vivo observations appear to indicate that thismembrane block works with different efficiencies in differentmammalian species. For example, tens to hundreds of sperm arefound in the perivitelline space of one-cell zygotes recovered fromrabbit, pocket gopher and mole females, whereas in other speciessuch as mice, rats, cats, pigs, dogs, sheep and humans perivitellinesperm are rarely observed (Gardner and Evans, 2006). Suchobservations raise the prediction that the former group may present amore effective membrane block, whereas the latter possess a moreeffective zona block.

Zona blockIn the last few years, the molecular mechanisms mediating the zonablock have been extensively characterized in transgenic mice. Earlystudies report that, within hours of fertilization, the zona fromfertilized eggs loses the ability to support sperm binding (Bleil et al.,1981). Thirty minutes after fertilization, the initiation of apost-fertilization cleavage of ZP2 at a diacidic residue in theN-terminal region is the only biochemical modification documented(Bleil et al., 1981) (Fig. 2A), and mutation of this ZP2 cleavage sitepermits sperm to bind to the zona surrounding fertilized eggs

Box 3. Zinc releaseZinc release is initiated byan increase in calcium transients (Duncan et al.,2016) (Fig. 4, 1). The reduction in intracellular zinc in eggs following thesecalcium oscillations is necessary to resume the cell cycle and enableproper development (Kim et al., 2011). Zinc is present in both the corticalgranules (Tokuhiro and Dean, 2018) and in defined cortical zinc vesicles(Que et al., 2015) of mouse oocytes. Within the cortical granules, aproportion of the zinc available appears to be bound to the active site ofovastacin, whereas the second pool of zinc that is responsible for the zincsparks observed after gamete fusion (Kim et al., 2011; Que et al., 2015) isbelieved to reside in the interstices between ovastacin molecules.Consistent with this hypothesis, zinc cannot be detected in the mouseoocytes in the absence of ovastacin (Tokuhiro and Dean, 2018). The zincreleased upon gamete fusion appears to play a role in regulating thetransient block to zona penetration. One possibility is that zinc perturbs thezona structure density (as observed by transmission electron microscopy)and, as a consequence, the zonamay no longer be able to support spermbinding (Que et al., 2017). Alternatively, zinc may prevent spermpenetration by affecting sperm motility of the bound and/or penetratingsperm during interaction with the egg: studies have shown that thepresence of increasing concentration of zinc in the media (25-50 µmZnSO4) partially or almost completely inhibits sperm penetration throughthe zona by affecting sperm motility as recorded by computer-assistedsperm analysis (CASA) (Tokuhiro and Dean, 2018).

Zincrelease

Zona penetration~minutes post fertilization

1

Membrane block ~minutes

post fertilization2

IZUMO1

e

Zona block~4 hr post fertilization

3

Zona pellucida

ZP4ZP3

ZP1

*ZP2

I

MII Oocyte

JUNO(vesicles)

Ovastacin

Perivitellinespace

Oolemma

Fig. 4. Effective post-fertilization block to polyspermy guaranteesmonospermic fertilization. After fertilization and cortical granule exocytosis,two immediate blocks and one delayed block occur to prevent polyspermicfertilization, which is embryonic-lethal. (1) Zinc release: zinc released within afewminutes of fertilization affects the forward motility of sperm that are bindingor have initiated penetration of the zona, thus possibly providing a block topenetration to hyperactive sperm in vivo. (2) Membrane block: ∼40 min afterfertilization, JUNO is shed from the oolemma within vesicles (green) into theperivitelline space to act as a decoy that blocks any supernumerary sperm fromadhering and fusing with the oolemma. (3) Zona block: within 4 h post-fertilization, ovastacin cleaves ZP2 at the N terminal region (asterisk) and thecleavage of ZP2 provides a definitive block by preventing further sperm bindingto the zona pellucida.

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(Gahlay et al., 2010). This post-fertilization cleavage of ZP2 occursfollowing cortical granules exocytosis and a precise translocation ofthe cortical granules to the cortex of the oocytes is necessary topreserve the zona block. This translocation is guaranteed by aRAB27a-regulated cytoplasmic actin network (Cheeseman et al.,2016; Bhuin and Roy, 2014). In ashen mice, a splice site mutation inRab27a generates a nonfunctional RAB27a protein (Wilson et al.,2000), which is associated with the disruption of cortical granulestranslocation and results in the accumulation of supernumeraryperivitelline sperm in fertilized homozygous Rab27a mutant eggs(Cheeseman et al., 2016). In addition, recent studies used high- andsuper-resolution imaging to document cortical granule dynamics atsingle granule resolution in transgenic mice (Vogt et al., 2019). Inthese studies, MATER, a component of the subcortical maternalcomplex (SCMC) of the oocyte has been shown to be necessary inanchoring the cortical granules at the oocyte cortex. Conditionalgenetic ablation of MATER in the oocyte has been associated with aspread distribution of the cortical granules throughout thecytoplasm, which delays cortical granule exocytosis afterfertilization. This prevents the post-fertilization cleavage of ZP2,which results in supernumerary sperm in the perivitelline space(Vogt et al., 2019). Upon gamete fusion, the oocyte internal calciumconcentration increases, which prompts the cortical granules to fusewith the oolemma and exocytose their contents of trypsin-likeproteases, ovoperoxidase, N-acetylglycosaminidase and zinc(Ducibella et al., 2002) (Box 3) (Fig. 1, 5).The cortical granules also release an oocyte-specific astacin-like

metallo-endoprotease encoded by the Astl gene, named ovastacin,which recognizes the cleavage site on ZP2 (Burkart et al., 2012)(Fig. 4, 3). Ablating the gene encoding ovastacin prevents cleavageof ZP2, and as a consequence, zona from these mutant eggs stillsupport sperm binding after fertilization (Burkart et al., 2012).Ovastacin cleaves ZP2 with species-specificity; mouse ovastacindoes not cleave human ZP2 in transgenic mice, which allowstransgenic zonae surrounding two-cell embryos to support de novomouse sperm binding (Baibakov et al., 2007). To ensuremonospermic fertilization, ovastacin must act at the right time andin the right place. A precocious activity of ovastacin on the zonabefore fertilization would be detrimental, as it would induce apremature block to sperm binding that would, in turn, cause femaleinfertility. The ovastacin signal peptide localizes the proteinectodomain into the endomembrane system, in which it is thensequestered into the cortical granules of the oocyte cortical area.During oocyte maturation, the precise localization of ovastacin inthe cortical granules is ensured by a seven amino acid motif (Xionget al., 2017). Indeed, deletion of this motif affects ovastacinlocalization because of the mutant protein being retained in theendomembrane system and leads to a premature and partial cleavageof ZP2. With the zona of MII eggs unable to support normal spermbinding, female fertility is severely affected (Xiong et al., 2017).These observations are puzzling; it is conceivable to expect that theretention of mutant ovastacin in the endomembrane system couldinhibit ZP2 cleavage, thus leading to a faulty polyspermy block.However, it could be hypothesized that the mutant ovastacin isreleased at higher concentration compared with the normalphysiological release of native ovastacin observed shortly beforeovulation (Ducibella et al., 1988). This continuous release would bethe reason for the partial premature ZP2 cleavage observed (Xionget al., 2017) and would be consistent with previously reportedphenotypes associated with premature cleavage of ZP2. FETUB, acystatin superfamily protein (thiol protease inhibitors), plays animportant role by inhibiting the proteolytic action of the premature

physiological release of ovastacin that occurs before ovulation(Ducibella et al., 1988). The effects of FETUB last until corticalgranule exocytosis when the overcoming amount of ovastacin actson the zona by cleaving ZP2 (Dietzel et al., 2013). Female micelacking FETUB are infertile, owing to a premature cleavage of ZP2by an early release of ovastacin from the cortical granules (Dietzelet al., 2013). In addition, a block to sperm penetration through thezona, dependent on cortical granule exocytosis (Inoue and Wolf,1975), acts transiently. This has been recently shown using eggsexpressing a mutant ZP2 that encodes a protein that remainsuncleaved after fertilization and cortical granule exocytosis (Gahlayet al., 2010). The study showed that after artificial induction ofcortical granule exocytosis by exposure to strontium chloride, thezona of eggs inseminated de novo with mouse sperm are capable ofsupporting sperm binding, although it remains refractory to spermpenetration (Tokuhiro and Dean, 2018). Nine hours post-corticalgranule exocytosis this block disappears, and the mutant zonabecomes permissive for penetration (Tokuhiro and Dean, 2018).This transient block appears to be mediated by zinc released uponfertilization (Que et al., 2017) (Box 3) that, together with a block tofusion and block to zona binding, provides an effective block topolyspermy in mammals that is imperative for the successful onsetof development.

Conclusions and perspectivesIn conclusion, a number of important recent discoveries usinggenetically modifiedmice have led to a novel model for fertilization,but fundamental questions remain answered. The current modelenvisions sperm binding to the zona via the N terminus of ZP2,which predicts the presence of a putative sperm receptor yet to bediscovered (Avella et al., 2014; Raj et al., 2017; Tokuhiro and Dean,2018) (Fig. 2). Indeed, this putative sperm receptor might also beinvolved in the induction of sperm acrosome exocytosis duringsperm penetration through the zona, however, the location andregulation of sperm acrosome exocytosis remains poorly understood(Inoue et al., 2011; Jin et al., 2011). After zona penetration, directinteraction between IZUMO1 and JUNO (Aydin et al., 2016; Katoet al., 2016; Ohto et al., 2016) mediates gamete adhesion (Bianchiet al., 2014) and drives accumulation of CD9 in the adhesion area(Chalbi et al., 2014). CD9 recruits in cis other oocyte proteins,which need to be identified (Fig. 3C). Similarly, whether otherundefined sperm proteins form complexes upon sperm adhesionwith the oolemma to mediate gamete fusion remains to be shown(Ellerman et al., 2009). Following fertilization, the cortical granulesat the periphery of the oocyte undergo exocytosis to release zinc andovastacin that cleaves ZP2 at the N terminus. Zinc release affects themotility of supernumerary sperm penetrating the zona, whereascleavage of ZP2 and shedding of JUNO from the oolemma impedefurther sperm binding and fusing with the egg (Bianchi et al., 2014;Burkart et al., 2012; Tokuhiro and Dean, 2018; Xiong et al., 2017)(Fig. 4).

It is remarkable that, despite decades of intensive investigation, westill know little about the molecular interactions mediatingfertilization, potentially because of the nature of the extracellularinteractions that regulate gamete recognition and fusion. Interactionsthat involve transmembrane proteins can be challenging to identify,because some receptor-ligand interactions present low affinity, aretransient or require local protein clustering to increase bindingefficiency. This can be circumvented by using recombinantoligomerized ectodomains as probes for the identification of cis/trans-acting factors controlling gamete recognition and fertilization(Bianchi et al., 2014). Finally, recent advances in chemoproteomic

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and genome editing technologies currently offer outstanding toolswith which to address these and other long-standing issues inreproductive biology.

AcknowledgementsWe apologize to colleagues whose work could not be cited owing to spacelimitations. We thank Drs Bonett and Toomey for critical comments on themanuscript.

Competing interestsThe authors declare no competing or financial interests.

FundingThis research was supported by the Department of Biological Science and by theOffice of Research and Sponsored Programs at the University of Tulsa (FacultyResearch Grant and Faculty Research Summer Fellowship) to M.A.A.

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