sperm meets egg: the genetics of mammalian fertilization

GE50CH05-Wright ARI 24 October 2016 11:35

Sperm Meets Egg: TheGenetics of MammalianFertilizationEnrica Bianchi and Gavin J. WrightCell Surface Signalling Laboratory, Wellcome Trust Sanger Institute, Cambridge CB10 1SA,United Kingdom; email: [email protected], [email protected]

Annu. Rev. Genet. 2016. 50:93–111

First published online as a Review in Advance onSeptember 2, 2016

The Annual Review of Genetics is online atgenet.annualreviews.org

This article’s doi:10.1146/annurev-genet-121415-121834

Copyright c© 2016 by Annual Reviews.All rights reserved

Keywords

fertilization, sperm, egg, fusion, reproduction, gametes

Abstract

Fertilization is the culminating event of sexual reproduction, which involvesthe union of the sperm and egg to form a single, genetically distinct organism.Despite the fundamental role of fertilization, the basic mechanisms involvedhave remained poorly understood. However, these mechanisms must involvean ordered schedule of cellular recognition events between the sperm and eggto ensure successful fusion. In this article, we review recent progress in ourmolecular understanding of mammalian fertilization, highlighting the areasin which genetic approaches have been particularly informative and focusingespecially on the roles of secreted and cell surface proteins, expressed in asex-specific manner, that mediate sperm-egg interactions. We discuss howthe sperm interacts with the female reproductive tract, zona pellucida, andthe oolemma. Finally, we review recent progress made in elucidating themechanisms that reduce polyspermy and ensure that eggs normally fusewith only a single sperm.

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ANNUAL REVIEWS Further

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http://www.annualreviews.org/doi/full/10.1146/annurev-genet-121415-121834


INTRODUCTION

Sexual reproduction is so successful in generating and propagating genetic diversity that the vastmajority of eukaryotic organisms reproduce in this way. Two of the most important principlesof sexual reproduction are, first, meiosis in which reductive cell divisions in the germline resultin specialized gametes that contain half the normal number of chromosomes, and, second, fer-tilization, in which two gametes recreate a genetically distinct organism and restore the normalnumber of chromosomes. These basic genetic principles are remarkably well conserved through-out the eukaryotic kingdoms of life. Despite these conserved principles and the fundamentalnature of fertilization, we know remarkably little about the molecular details involved. This maybe due to the many challenges in studying fertilization in mammals (123), particularly the rela-tionship between fertilization and the use of genetics; after all, infertility is not a heritable trait.However, genetics, and in particular the use of targeted gene-deficient mice, has made several im-portant contributions to our current understanding of fertilization. These are highlighted in thisreview.

Fertilization requires recognition and fusion between one spermatozoon and one egg. Bothsperm and eggs are fertilization competent for just a few days in most species, so strict timing isnecessary to ensure success (21, 130). In mammals, ovulation is orchestrated by regulated hormonesecretion by the gonads and pituitary gland throughout the estrous cycle. The surge of luteinizinghormone causes mature eggs to burst out of the follicles within the ovary; they are then captured bythe tubal fimbriae and propelled into the uterine tube. Eggs are not released as discrete cells; rather,they are surrounded by a glycoprotein matrix called the zona pellucida (ZP) and embedded withina hyaluronic acid–rich jelly containing somatic cumulus cells collectively termed the cumulus-oocyte complex (COC). The periodicity of the ovulation cycle and the number of eggs releasedare highly species dependent. In contrast with females, males produce sperm continuously andin much larger amounts, most likely because sperm must navigate the highly selective femalereproductive tract and penetrate the COC. As the sperm nears the egg, a vesicle in the spermhead called the acrosome fuses with the sperm membrane to release its contents: a mixture ofdigestive enzymes that help the sperm penetrate the COC and ZP. Finally, the cell membrane ofan acrosome-reacted sperm adheres and fuses with the egg membrane (oolemma), the paternalgenetic material is transferred into the egg cytoplasm to recreate a diploid cell, and the life of a neworganism begins. Once fertilized, the new zygote must rapidly protect itself from additional spermfusion events, which would lead to the formation of a polyspermic, polyploid embryo that wouldnot be viable. In this review, we discuss our current molecular understanding of these processesand how genetics in particular has helped shape this understanding.

CELLULAR AND MOLECULAR RECOGNITION EVENTS DURINGTHE SPERM’S JOURNEY TO THE EGG

In mammals, the site of fertilization is normally within the female body, which requires malegametes to navigate toward the egg after being deposited in the female reproductive tract(Figure 1). In eutherians, the ovulated eggs are located in the oviduct (Fallopian tube), whichis over a hundred to a thousand times longer (approximately 2.5 cm in the mouse and approxi-mately 11 cm in humans) than the average length of a spermatozoon (approximately 73 µm) (24).During their time in the female body, the sperm come into contact with several cell types andexperience different environmental conditions. The oviductal morphology (structure, length, anddegree of coiling) varies in different species and has been correlated with the number of spermreleased during mating, with sperm competition, and other sperm selection mechanisms known

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AmpullaUtero-tubal junction Isthmus

Infundibulum a

Ace, Calr3, Clgn, Pdilt, Pmis2Rnase10, Tpst2, Adam1a, Adam2Tex101, Prss37, Adam3, Ly6k Spam1, Hyal5

b c d

Figure 1Sperm interact with somatic cells at many anatomical sites along the female reproductive tract. (a) Image of adissected mouse oviduct, the different anatomical regions indicated: utero-tubal junction (UTJ), isthmus, andampulla. At ovulation, the cumulus-oocyte complexes are captured by the funnel-shaped infundibulum andhalt briefly in the ampulla, the dilated region where fertilization normally occurs. (b) Sperm migrate from theuterus through the oviduct and undergo strong selection at the UTJ; currently, 13 genes have been shown tobe required for this step. (c) Sperm that pass the UTJ attach to and congregate along the epithelial cells of theisthmus until the time of ovulation. Little is known about the signals that trigger sperm detachment from theisthmus. This detachment allows the resumption of the sperm’s journey toward the ampulla, where spermmay eventually encounter ovulated cumulus-oocyte complexes. (d ) Finally, sperm-derived enzymes such asSPAM1 and HAYAL5 facilitate the sperm’s penetration of the protective viscous hyaluronan matrix in orderto reach the egg. Once fertilized, zygotes navigate the isthmus to reach the implantation sites in the uterus.

collectively as cryptic female choice. Notably, the number of sperm that reach the site of fertiliza-tion is fairly constant across different species, averaging a few hundred, which is only a tiny fractionof the hundreds of millions of sperm that are typically released in a single mating (28). This sug-gests that the female reproductive tract acts as an effective barrier, if not an active selector, of thefittest sperm for fertilization. However, the female environment is not completely hostile; indeed,sperm must reside within the female reproductive tract to undergo the structural and molecularchanges, known as capacitation, that bestow the ability to fertilize an egg. Sperm that leave thetestes are morphologically mature but functionally incompetent: They are immotile, and theirability to swim is activated only after they have passed through the epididymis. Upon activation,the now motile sperm generate symmetrical flagellar beats and have an almost linear trajectory.

www.annualreviews.org • Genetics of Mammalian Fertilization 95

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The intersection of the uterine cavity with the oviduct, known as the utero-tubal junction (UTJ),is a major obstacle for sperm. So far, 13 genes have been shown to be important for male fertilityand all are necessary for successfully passing the UTJ (Figure 1b). Eleven of these [Ace, Calr3,Clgn, Pdilt, Pmis2, Rnase10, Tpst2, a disintegrin and metalloproteinase (Adam)1a, Adam2, Tex101, andPrss37] are required for the presence and proper localization of a cell surface receptor belongingto the ADAM family, ADAM3, on the surface of sperm (48). In addition to their reduced abilityto pass through the UTJ, Adam3-deficient sperm have impaired binding to the ZP, suggestingthe existence of a mechanism common to both processes (107, 125). Ly6k, whose deletion affectssperm migration but whose mechanism of action does not appear to involve ADAM3, has recentlybeen identified (35). Despite the large number of genes involved and their general convergenceon ADAM3, the molecular mechanism of UTJ crossing is currently unknown.

Once the sperm have passed the UTJ, they congregate by attaching to the mucosal epitheliumin the isthmus of the oviduct; they can wait in the epithelium for several days for ovulation to occurwhile maintaining their ability to fertilize the egg (Figure 1c). During this time, the sperm interactwith the female epithelial cells and also with the molecules in the oviductal fluid (reviewed in 45,111). There is evidence that molecules within the oviductal secretions modulate sperm function,their ability to interact with the ZP, and their ability to fertilize the egg (100). Coincubation ofsperm with oviduct epithelial cells or their conditioned media maintained sperm viability andmotility for a longer time relative to a control in vitro (85). Recent findings have also shownthat sperm can bind to the epithelium in the ampulla as well as the epithelium in the isthmus,suggesting that molecular interactions between the sperm and the female reproductive tract aremore widespread than initially thought (19).

As ovulation approaches, a yet unknown physiological signal triggers a significant change inthe pattern of sperm flagellar beating. The curvature increases in amplitude on one side of theflagellum only and produces an asymmetrical beat that might help release the sperm from theoviductal reservoir. Sperm in this state are said to be hyperactivated (110). Hyperactivation andthe capacity to undergo the acrosome reaction are the two features acquired by sperm duringcapacitation that are essential for fertilization; these two features are only partially dependent onone another, and both are triggered by largely unknown stimuli. In mice, only hyperactivated spermhave been observed detaching from the mucosal epithelium, suggesting that hyperactivation is therelease mechanism (26). However, other factors, such as a reduction of the molecular bindingaffinity between the sperm and the epithelium, may play a role in sperm release. Researchersbelieve sperm use hyperactivation to enter the oviductal lumen and swim toward the COC (for anextended review of the sperm-oviduct interaction, see 23). Sperm take a few minutes to cross theviscous hyaluronic acid matrix of the COC, but this passage is not thought to play a major role inpreparing either the sperm or the egg for fertilization because eggs isolated from the cumulus canbe efficiently fertilized in vitro. The proper formation of the COC is essential for ovulation, and thepresence of the cumulus is beneficial for fertilization (62, 103). Possibly because in vitro fertilization(IVF) is normally performed after the removal of the cumulus cells, the interaction between thesperm and the COC has not been investigated in further detail. Hyaluronidases, including SPAM1and HYAL5 (65, 80), are displayed on the surface of sperm, and their activity presumably easesthe sperm’s passage through the cumulus matrix (Figure 1d ), but the importance of any mutualinfluence between sperm and cumulus cells, as well as the role of chemotaxis, remains unclear.Although a great deal of progress has been made in understanding the recognition events betweenthe female reproductive tract and sperm, further investigations are likely to reveal interesting newbiological discoveries relating to the mechanisms of sperm competition and cryptic female choice,which influence the probabilities that individual sperm will be successful in fertilizing the egg.

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THE INTERACTION OF SPERM WITH THE ZONA PELLUCIDA

All mammalian eggs are surrounded by an extracellular coat, the ZP, that shields the egg and per-forms important functions during oocyte maturation, fertilization, and embryo preimplantation.The ZP consists of a rubbery extracellular matrix of variable size depending on the species; theZP of a mature mouse egg is ∼7 µm thick. Scanning electron microscopy reveals that the ZP isformed from a sponge-like mesh consisting of interwoven microfilaments, resulting in pores thatcentripetally narrow and bifurcate so that the network is more tightly packed (33, 76, 87). Themicrofilaments are arranged in regular arrays and held together by noncovalent bonds such thatthe ZP is permeable to antibodies, enzymes, and small viruses. The mouse (m)ZP is composed ofthree glycoproteins, mZP1 (∼200 kDa), mZP2 (∼120 kDa), and mZP3 (∼83 kDa); in humans, afourth glycoprotein, ZP4 (∼60 kDa), is present. Interestingly, a gene orthologous to ZP4 is presentin the mouse genome, but it is clearly a pseudogene because it contains multiple in-frame stopcodons (70). mZP2 and mZP3 are present in equimolar amounts and are arrayed in an alternaterepeating pattern, creating fibrils that are cross-linked by mZP1. ZP proteins are glycosylated withN- (asparagine) and O- (serine/threonine) linked oligosaccharides and show varying degrees ofsialylation and sulfation. In mice and humans, the ZP is present only on maturing and fully matureoocytes, and the synthesis of ZP proteins ceases only during the development of the metaphase IIegg (for an extended review, see 116).

Despite a great number of investigations into the molecular basis of the cellular recognitionevents between the sperm and egg that lead to fertilization, many important details remain poorlyunderstood. To convey the current state of knowledge, we sum up the models proposed over theyears, highlighting the evidence that supports them while emphasizing their incongruities, for thepurpose of stimulating further discussion and new investigations.

Two main features of the egg extracellular matrix have attracted the attention of researchers.First, the ZP confers some level of species specificity to mammalian fertilization, as sperm fromheterologous species do not bind or, if binding occurs, do not penetrate the ZP to reach the egg(128). Several examples show the ZP acting as a physical barrier impeding fertilization, such as theinability of human sperm to bind to the mouse ZP; however, in some mammals, such as the rabbit,the sperm-ZP interaction exhibits much less species specificity (8). In general, the specificity ofcross-species mammalian gamete interactions is variable (9). Second, in many mammalian species,sperm do not bind to the ZP of fertilized eggs, thereby reducing the possibility of polyspermy.The hypothesis that eggs possess specific surface receptors for corresponding molecules on thesurface of sperm is based mainly on these observations, which can be traced back to the beginningof the last century (115). Experimental support for this hypothesis was obtained when solubilizedZP glycoproteins were added to IVF assays and were shown to block sperm binding to the eggextracellular matrix. Moreover, several investigators reported that only acrosome-reacted spermwere found in the perivitelline space (34, 129), leading to the following proposed schedule ofevents: (a) molecules displayed on the plasma membrane of acrosome-intact sperm bind to areceptor displayed on the ZP; (b) this binding event triggers the acrosome reaction, leading tothe release of proteolytic enzymes to loosen the egg extracellular matrix and helping the spermpenetrate the ZP; and (c) after fertilization, the molecular modification of the sperm receptorprevents binding of subsequent sperm to the ZP. However, this model failed to explain someexperimental observations and has been questioned by some recent findings. Nonetheless, thismodel provided the impetus to determine the identity of the sperm receptor. Bleil & Wassarman(14) showed that, of the three ZP proteins, mZP3 could inhibit sperm binding to the ZP andinduce the acrosome reaction. Because the protein used for these experiments was obtained fromsolubilized native ZP, the experiment did not determine whether the protein itself or a glycan


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was responsible for the observed effects (14). Evidence then emerged that ZP2 bound acrosome-reacted sperm, which supports the idea that, following the acrosome reaction, sperm maintaintheir contact with the ZP through another receptor (13). Unfortunately, the generation of gene-deficient mice failed to shed light on which ZP protein is the primary receptor for sperm. Infact, both ZP2 and ZP3 are essential for the synthesis of the ZP, which is also required forproper oocyte development during folliculogenesis (73, 97, 98). The hypothesis that O-glycanson mZP3—specifically on Ser332 and Ser334—might be involved in sperm binding is challengedby the genetic ablation of glycosyltransferases, as well as by the fact that genetic mutation of thespecific residues (Ser332 and Ser334) does not prevent fertility (74, 118). The role of ZP3 as theprimary sperm receptor has also been questioned by Dean and colleagues (99), who generatedtransgenic mice expressing human ZP in place of the orthologous mouse protein and surprisinglyshowed that substitution of mouse ZP3 with human ZP3 did not alter the fertilizing ability ofmouse sperm or rescue the inability of human sperm to bind the mouse ZP. Using an analogousgain-of-function approach, i.e., replacing endogenous mouse proteins with homologous humanones, the same group has gathered more evidence indicating that ZP2 mediates gamete binding inmice and humans (2, 6). The role played by individual ZP proteins in sperm recognition remainsan active area of debate, arguably because there is no strong candidate for the sperm ligand. So far,all suggested candidates (ACROSIN, BETA-1,4-GALACTOSYLTRANSFERASE 1, MFGE8)have been shown to be dispensable for fertilization in vivo using gene-deficient mice (for examples,see 5, 32, 75; for an extended review, see 48).

The central assumption that the binding of sperm to the ZP triggers the acrosome reactionhas been challenged by an elegant experiment from Okabe’s group (55) in which they recoveredacrosome-reacted sperm from the perivitelline space of Cd9-knockout eggs (which do not fusewith sperm) and showed that these sperm retain their ability to traverse the cumulus of ovulatedeggs, pass through a second ZP, and fertilize the egg (Figure 2a). An earlier paper reported thesame findings in rabbits (67). Further evidence that the acrosome reaction occurs before the spermcontacts the ZP was obtained using live imaging of acrosome reporter sperm; the majority offertilizing sperm underwent the acrosome reaction before contacting the ZP (62). These findingschallenge the idea that a specific sperm surface receptor interacts with the ZP to initiate theacrosome reaction and so it therefore remains to be determined what then triggers the acrosomereaction as the sperm nears the egg. Finally, there is good evidence to suggest that sperm hyper-activation is critical for ZP penetration because sperm that undergo the acrosome reaction but donot hyperactivate fail to penetrate the ZP (109). This is in agreement with the basic notion thatfertilization is a race to reach the egg. It is therefore reasonable to think that passing through theZP quickly is important for sperm. This would logically necessitate that the sperm-ZP molecularbinding events should be transient and labile to prevent the sperm adhering within the ZP mesh;consequently, these interactions may be difficult to experimentally detect. In fact, approachesthat have focused on sperm that are firmly bound to the ZP may have counterintuitively biasedtheir experiments against those sperm that are the fittest to fertilize the egg.

THE SPERM-OOLEMMA INTERACTION

The search for the molecules and mechanisms involved in sperm-egg recognition began as soonas IVF became feasible. Extracellular calcium was quickly demonstrated to be required for thefusion event (127), as were controlled pH and temperature (44, 131). To determine which classesof molecule on the surface of the egg were required for fertilization, enzymes with broad sub-strate specificities were used to remove proteins, lipids, and carbohydrates. These experiments,however, were not especially helpful because they resulted in varying conclusions depending on

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a

2'

2

1

b Oolemma

Sper

m c

ytop

lasm

Spermmembrane

NH

3+

COO–JUNO

IZUMO1

S

S

GPI-anchor

Ig-likedomain

Zona pellucida

Metaphase IIchromosomes

Acrosomereaction

Acrosome-intactsperm

Perivitelline space

3

Egg

cyto

plas

m

Izumodomain

Figure 2Sperm–egg interactions leading to fertilization. (a) To fuse with eggs, sperm must undergo the acrosomereaction (2 and 2′). The initial idea that this reaction occurred when sperm attached to the zona pellucida(ZP) has been challenged by evidence showing that the acrosome reaction occurs before sperm reach the ZPand that the acrosome reaction is not necessary for passage through the ZP (2′). The site and timing of theacrosome reaction remains a debated subject that requires further investigation. (b) Once inside theperivitelline space, the sperm membrane can bind and fuse with the oolemma. The N-terminal domain ofthe sperm protein IZUMO1 binds the GPI-anchored protein JUNO on the surface of the egg. Thisinteraction is essential for fertilization, but other molecules are almost certainly required for sperm-eggfusion because IZUMO1 and JUNO are not sufficient to trigger membrane fusion in heterologous systems.Abbreviations: GPI, glycosylphosphatidylinositol; Ig, immunoglobulin.

the precise conditions and species used (16, 96). Similarly, carbohydrates (15, 95), sperm extracts,and antibodies elicited against sperm extracts were added, but many promising candidates result-ing from these approaches were eventually shown not to be essential because mice deficient inthese genes were fully fertile or at most only subfertile (for extended reviews, see 49, 56, 92).Coonrod et al. (22) demonstrated that treating eggs with phosphoinositide phospholipase C, anenzyme that removes the membrane-anchoring glycosylphosphatidylinositol (GPI) moiety froma specific class of surface protein molecules, rendered them infertile. This observation was sup-ported by a transgenic mouse model, in which an enzyme required for GPI anchor biosynthesiswas specifically deleted in eggs, confirming that one or more GPI-anchored proteins expressed onthe egg are essential for fertilization (1). These experiments suggested that gene-deficient micewould be a valuable tool for assessing the in vivo importance of candidate molecules. To date,three genes that encode cell surface receptors have been identified as having sex-specific sterilitywhen deleted in vivo. Two, Juno and Cd9, are expressed on the egg membrane, and one, Izumo1,is displayed on the surface of sperm.

CD9

Cd9 was the first gene encoding a cell surface protein to be identified in mammals as havinga strong sex-specific fertility defect; as is often the case in science, its discovery was entirelyserendipitous. Three independent groups generated Cd9-knockout mice, and all reported stronginfertility phenotypes in female, but not male, mice. Two groups reported a severe reductionof fertility (63, 68), and the third reported complete infertility (82). It is likely that the dif-ferent gene-targeting strategies used by each group resulted in the variations observed. Cd9-deficient female mice ovulated morphologically normal-looking eggs that were unable to fuse with


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wild-type sperm. CD9 belongs to the tetraspanin family of multipass receptors, so-called becausethe members of this family contain four transmembrane-spanning regions, with both the N and Ctermini located in the cytoplasm and with both small and large extracellular loops. The larger loopcontains two disulfide bonds and can be further subdivided into a constant and a variable region.The variable region mediates protein-protein interactions with laterally associated proteins withinthe membrane, and the constant region is thought to mediate homodimerization (43, 77).

A structure-function study highlighted the role of three residues, SFQ (173–175), that arerequired for fertilization in the variable domain of murine CD9, possibly because they facilitateessential interactions with other proteins on the egg membrane (133). The mammalian tetraspaninfamily contains more than 30 members, which are involved in the regulation of many processesincluding cell proliferation, adhesion, motility, and signaling. Tetraspanins are associated withother cell surface proteins, including other tetraspanins, thereby forming tetraspanin-enrichedmicrodomains, which are thought to be dynamic assemblies within the plasma membrane that arecharacteristic of certain differentiated cell types (43, 71). However, the mechanism by which theabsence of CD9 on the egg membrane impairs fertilization is unclear. The aberrant morphology ofmicrovilli observed on the surface of knockout eggs suggests that CD9 is required to establish andmaintain the proper shape and distribution of these structures (102). In addition, CD9-containingvesicles have been found in the egg perivitelline space. Miyado and colleagues (83) demonstratedthat transfer of CD9-containing vesicles to acrosome-reacted sperm facilitated sperm-egg fusionand restored fusion with Cd9-deficient eggs; however, others could not replicate these observations(39, 66). The situation is complicated by a related tetraspanin, CD81, which also has a role infertilization, as demonstrated by the fact that Cd81-deficient female mice are subfertile (89, 101,113). It is likely that the tetraspanins play indirect roles in fertilization by organizing other proteinsembedded within the egg membrane. CD9, for example, has been shown to organize effectivesperm adhesion sites on the egg membrane that are permissible for sperm fusion. Using cell-basedbiophysical measurements, Jegou et al. (60) demonstrated that the sperm adherence sites on Cd9-deficient eggs, although higher in number, were qualitatively different and of a lower affinity thanthose of wild-type eggs. Although the identities of the molecules associated with tetrapanins arenot yet clear, some candidates have been proposed, including IGSF8 (also known as EWI-2), amember of the immunoglobulin subfamily, which directly associates with CD9 (104).

IZUMO1

IZUMO1 is the only cell surface protein expressed on sperm that is known to be essential forsperm–egg interaction in vivo. Okabe and colleagues selected a monoclonal antibody namedOBF13 that was able to bind acrosome-reacted mouse sperm (91) and potently block fertiliza-tion in IVF assays (93). Because OBF13 is an immunoglobulin M, an antibody isotype that isoften of low affinity, it was not until several years later that the antigen recognized by OBF13was identified as a type I cell surface protein belonging to the immunoglobulin superfamily; thisprotein was named Izumo, after a Japanese marriage shrine. Importantly, Izumo1-deficient malemice produced morphologically normal sperm but were completely infertile because their spermwere unable to bind and fuse with normal eggs (53). IZUMO1 is a typical type I cell surfaceprotein containing a sequence that folds into a distinct domain, followed by an immunoglobulin-like (Ig-like) C2-type domain, a transmembrane-spanning region, and a short cytoplasmic tail(Figure 2b). The IZUMO1 Ig-like domain contains an N-glycosylation site conserved acrossmost mammalian species, including humans; given the suggested role of glycans in gamete recog-nition, this N-glycosylation site has been investigated in more detail (54, 124). The fertility ofIzumo1-deficient male mice could be rescued by introducing a transgene encoding a form of Izumo1

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that could not be glycosylated, demonstrating that the glycans played no role in egg recognition.Some degradation of IZUMO1 was observed in sperm isolated from the cauda epididymis, sug-gesting that the role of the glycans was to protect the sperm from degradation (54). Using asequence-based homology search, three IZUMO1 paralogs were identified, each containing thenow-characteristic N-terminal Izumo domain and numbered from 2 to 4 (31). Izumo3 and Izumo4are thought to be expressed in sperm. Furthermore, IZUMO4 is reported to be a secreted proteinlacking the transmembrane domain, and thus it does not affect the reproductive phenotype whendisrupted in mice (112). The addition of antibodies to human IZUMO1 blocks the fusion of humansperm with hamster eggs, indicating that IZUMO1 plays a role in human fertility; however, nosequence variants in Izumo1 have yet been identified that explain male infertility in human patients(38, 42).

IZUMO1 is undetectable on the surface of acrosome-intact sperm, but it is redistributedover the sperm head following the acrosome reaction, preferentially localizing to the equatorialregion, which is the future site of fusion with the egg membrane (105). This redistribution is likelyregulated through modification of the cytoplasmic tail of IZUMO1, and this can be posttransla-tionally modified by phosphorylation during epididymal transit (7). The failure of testis-specificserine/threonine-protein kinase 6 (tssk6)–deficient male mice to redistribute IZUMO1 providesa plausible mechanism to explain the infertility of these mice. The redistribution of IZUMO1onto the surface of sperm just at the moment of reaching the egg reinforced early hypothesesthat IZUMO1 acts as a virus-like unilateral cellular fusogen; however, despite the appeal ofthis model, functional and structural data that might provide further evidence was lacking. Theidentification of a specific receptor for IZUMO1—a GPI-anchored protein present on the eggsurface, named JUNO—revealed that IZUMO1 likely plays an essential role in recognition oradhesion between the sperm and egg.

JUNO (FOLR4)

Prior to its identification as the egg receptor for IZUMO1, most of the research on JUNO involvedits basic characterization. It was initially discovered using a sequence-based data mining strategyto detect novel folate receptors. The gene was named Folate Receptor 4 (Folr4, FR4, or Folbp3) andclassified as a member of the folic acid receptor family (108). A tissue transcript analysis in miceshowed a wide expression pattern with enrichment in the spleen and thymus, suggesting that Folr4is expressed in leukocytes, most likely T lymphocytes (108). The FOLR4 protein was subsequentlycharacterized as a marker of a subset of Cd4+ Cd25+ regulatory mouse T cells (126). The importantrole played by FOLR4 in fertilization was revealed by a search for an IZUMO1 receptor on the egg.Using a highly avid soluble recombinant mouse IZUMO1 binding probe, a specific interaction wasdetected on mouse egg membranes (10). To determine the identity of the molecule responsiblefor IZUMO1 binding, an egg cDNA library was iteratively screened until a single plasmid clonewas selected and found to encode Folr4. FOLR4, similar to other folate receptors, attaches to thecell membrane through a GPI anchor (61) (Figure 2b); this was confirmed on mouse eggs (10),thereby providing a specific molecular identity for the GPI molecule that was previously known tobe important for egg fertility. The identification, using structural studies, of the residues importantfor folate binding by folate receptors (20, 117) suggested that FOLR4 may not be able to bindfolate, a hypothesis that was confirmed experimentally (10). Because of its inability to bind folateand its newly discovered role in fertilization, it was suggested that the gene be renamed Juno, afterthe Roman goddess of marriage and fertility. Juno-deficient mice are phenotypically normal, butthe females ovulate eggs that, although morphologically normal, are unable to bind and fuse withsperm, rendering the female mice completely infertile (10). This identified the IZUMO1-JUNO


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interaction as the first essential receptor-ligand interaction required for gamete recognition in anyorganism.

THE IZUMO1-JUNO INTERACTION IS REQUIRED FOR GAMETERECOGNITION AND ADHESION

The necessity of the IZUMO1-JUNO interaction for mammalian fertilization has prompted fur-ther studies on this pair of receptors, including investigations into their mode of recognition, theirrole in species specificity of fertilization, how they evolved, and more detailed mechanistic ques-tions on the function of their interaction in fertilization. The initial identification of Juno showedthat it is the N-terminal IZUMO1 domain, rather than the Ig-like domain, that is responsible forthe interaction with JUNO (10) (Figure 2b). This is consistent with prior and subsequent studiesshowing that the isolated IZUMO1 domain can bind the egg membrane and that monoclonal an-tibodies to this region potently block fertilization in vitro (52, 93). Crystal structures of unboundIZUMO1 and JUNO as well as those in complex with each other confirmed this, identified thebinding interfaces on the two proteins, and provided a structural explanation for the inability ofJUNO to bind folate (4, 40, 86, 90).

Clear orthologs of both Juno and Izumo1 can be identified only in mammalian genomes, sug-gesting that the IZUMO1-JUNO interaction is a mammalian innovation. This conclusion issupported by the demonstration that the orthologs from pigs, opossums, mice, and humans candirectly interact (10). Normally, when isolated gametes are used in vitro, the main barrier tocross-species fertilization is the ZP; however, some cross-species gamete fusions can occur whenZP-free eggs are used (41). The ability of IZUMO1 and JUNO proteins from different species tointeract, and of their gametes to cross fertilize each other in vitro, has been demonstrated (11). Ofparticular note is the fact that human IZUMO1 can bind hamster JUNO, providing a molecularexplanation for the unusual ability of hamster eggs to fuse with human sperm, a phenomenon thathas been exploited to assay sperm function in assisted fertility treatments (132).

How might the IZUMO1-JUNO interaction function together with CD9 to mediatesperm-egg membrane fusion? A model is emerging from research in other cell fusion systems,such as skeletal muscle development, indicating that, following cellular recognition, the negativelycharged lipid bilayers are brought into close proximity through a pair of adhesion receptors tocreate fusion synapses (64). Molecules that perturb the lipid bilayer, most likely through the induc-tion of extreme membrane curvature, are recruited to these synapses to reduce the energy barriersfor membrane fusion (79). Preliminary studies suggest that the IZUMO1-JUNO interactionperforms the necessary adhesion step, but it is not sufficient to mediate fusion when IZUMO1 andJUNO are ectopically expressed on neighboring cells that do not normally fuse. The insufficiencyof the IZUMO1-JUNO interaction to mediate fusion has been demonstrated using heterologouscell lines (10), as well as in a mixed assay using cells and eggs (18, 51). What could be the roleof CD9 in this model? As discussed above, there is evidence that CD9 regulates sperm adhesionsites on the egg membrane (60). Given the discovery that JUNO is an essential GPI-linked eggreceptor, and the fact that tetraspanins and GPI-linked proteins are known to be enriched withinmembrane microdomains (69), one possibility is that CD9 organizes JUNO within localizedregions of the egg membrane. This model would be consistent with differences in IZUMO1mobility within membranes when binding to wild-type or Cd9-deficient eggs (18), as well as withour observations that both the presence of cell surface JUNO and the binding of an avid IZUMO1probe are decreased on Cd9-deficient eggs compared to wild-type eggs (E. Bianchi & G.J. Wright,unpublished observations). A refinement of this model was recently presented by Inoue and col-leagues (51), who were following up on the observation that the extracellular region of IZUMO1


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contains a region that mediates dimerization. They provided evidence that monomeric IZUMO1is recruited to sperm-egg adhesion sites through JUNO-mediated interactions and is then trans-ferred to another, as yet unidentified, egg receptor, which induces IZUMO1 dimerization andforces membranes into close proximity (51). Although our understanding of sperm-egg recogni-tion has recently advanced, many questions remain unanswered; perhaps the most pressing is theidentification of a molecule—a fusogen—that triggers the union of the sperm and egg membranes.

POLYSPERMY

Eggs are presented with a delicately balanced problem: Unless they fuse with a sperm, they will die;however, if they fuse with more than one sperm, they form a nonviable polyploid embryo. Howcan unfertilized eggs be highly receptive to sperm and yet rapidly become refractory to additionalsperm fusion events once fertilized? We have had satisfactory molecular explanations for theregulation of this phenomenon in organisms such as sea urchins and amphibians for more thana decade, but only within the past two years have we begun to understand how mammalian eggsprevent additional sperm fusion once fertilized. In mammals, the conflicting evolutionary pressureson the egg to be highly receptive to sperm and yet to avoid polyspermy have resulted in a systemthat tolerates an in vivo polyspermy incidence of between 1% and 2% (37). When fertilization isperformed in vitro, the frequency of polyspermy increases with sperm concentration (81), whichsuggests that the physical barriers presented by the female reproductive tract are major factorsreducing polyspermy in mammals. The observation that fertilized eggs of marine invertebratesbecame refractory to fusion, despite high sperm concentrations, suggests the existence of molecularmechanisms to prevent polyspermy.

MOLECULAR MECHANISMS TO REDUCE POLYSPERMY

The eggs of different species have evolved a range of mechanisms to prevent polyspermy, which cangenerally be characterized as operating at three spatially distinct sites: the ZP, the egg membrane(oolemma), and the egg cytoplasm itself. The block at the ZP is probably the best studied andis used by a wide range of organisms, including seas urchins, amphibians, and mammals. Theimportance of this mechanism was first established when it was observed that the ZPs of fertilizedeggs were no longer able to bind sperm, suggesting that a biochemical change had occurred withinthe ZP. The block at the egg membrane in mammals was discovered by observing the presence ofmany unfused sperm within the perivitelline space of rabbit embryos that had been flushed fromthe reproductive tract (72, 88). Clearly, these sperm had penetrated the ZP, but the oolemma hadbecome unreceptive to additional sperm fusion events. Finally, the eggs of some animals, includingsome insect species, urodeles, elasmobranchs, reptiles, and notably birds, fuse with several sperm.This physiological polyspermy does not lead to the formation of polyploid embryos because theselection of a single sperm pronucleus is made within the cytoplasm of the egg. Little is knownof the molecular mechanisms involved in this cytoplasmic selection process, and because it is notthought to be a major mechanism used by mammals, we do not discuss it further in this review.

The degree to which mammals rely on a block at either the ZP or the oolemma varies by species.For example, fertilized eggs of rabbits, pocket gophers, and moles have been observed to containtens to hundreds of unfused sperm in the perivitelline space, which suggests a very inefficient blockat the ZP but a highly effective membrane block. In other species, such as dogs, sheep, and voles,unfused sperm in the perivitelline space are extremely rare, indicating that these species probablyrely more on the ZP block to prevent polyspermy. Other species, including mice, rats, cats, pigs,cattle, and humans, appear to use a combination of both mechanisms; in these species, as few asone or two but as many as ten sperm can be observed within the perivitelline space.


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The Zona Pellucida Block to Polyspermy

The ZP block to polyspermy has been observed and studied in several different organisms. Fol-lowing fertilization, the egg is activated, triggering calcium oscillations. This, in turn, leads to theexocytosis of the cortical granules, which are secretory organelles located at the periphery of theoocyte, close to the oolemma. The contents of the cortical granules are thought to be primarilyenzymes whose substrates are the ZP proteins, which, when processed, become hardened (i.e.,more resistant to protease digestion) (50), rendering them unable to bind sperm and making itimpossible for sperm to traverse them.

Upon egg activation, the secretory granules fuse with the oocyte plasma membrane. The firstmeasurable activity is the cleavage of ZP2 from a large 120-kDa precursor to form the smaller23-kDa ZP2-NTF (N-terminal fragment) and 90-kDa ZP2f, which remain covalently linked bydisulfide bonds (12). This processing event is associated with the loss of sperm binding sites onthe ZP. In mice, the site of ZP2 cleavage has been mapped on the N terminus to the diacidicmotif 166LA-DE169 (36). This diacidic motif corresponds to the known cleavage sites of theastacin family of metalloproteases, and a candidate protease that was localized to cortical granuleshas been identified as ovastacin (17). Although ovastacin-null mice were fertile, eggs deficientin OVASTACIN, or with a mutated cleavage site, had two important differences compared withwild-type eggs: They were unable to proteolytically process ZP2 when fertilized, and they retainedthe ability to bind capacitated sperm at the two-cell stage (17, 36). These findings resulted in amodel in which the initial sperm receptor corresponds to the N-terminal (∼30 kDa) region ofZP2, which is then cleaved at one of three diacidic residues within the ZP2-NTF to destroythe sperm-binding region within ZP2 and render eggs refractory to further sperm adhesion andfertilization (3, 17).

The role of ovastacin was further supported by the serendipitous discovery that female, butnot male, mice with a targeted deletion in the gene corresponding to a plasma protein calledFETUIN-B are sterile. Plasma proteins are known to inhibit ZP hardening (29, 30); this hasbeen shown to be due to the major serum protease inhibitor fetuin (106). These results wereinitially confusing because inhibition of human fetuin using antibodies did not relieve hardeninginhibition. This paradox was resolved when a paralogous fetuin member was identified (94).Comparing knockout mice of both paralogs (FETUIN-A and -B) identified a role for FETUIN-Bin the inhibition of premature ovastacin-based hardening (27). Fetuin-B–deficient female micewere completely infertile but ovulated morphologically normal eggs, an effect that was rescuedby transplantation of fetuin-B–deficient ovaries into wild-type animals or by mechanical removalof the ZP on FETUIN-B–deficient eggs. FETUIN-B was shown to directly inhibit ovastacinactivity, and it was concluded that FETUIN-B is normally required to prevent the prematureand inappropriate hardening of the ZP caused by the constant but low-level leakage of ovastacinfrom the oocyte. Once the egg is fertilized, the explosive release of ovastacin overwhelms anyFETUIN-B activity, leading to ZP2 processing and the loss of sperm binding sites (27). AlthoughZP2 processing is sometimes referred to as the definitive block to polyspermy because sperm arethus unable to access the egg, transgenic mice containing a mutant ZP2 that cannot be cleavedhave only reduced fertility (36) and ovastacin-null mice are fully fertile (17), which suggests thepresence of additional mechanisms to prevent polyspermy.

The Membrane Block to Polyspermy

The observation that unfused sperm are sometimes detected in the perivitelline space of fertilizedeggs suggests that the egg membrane itself is modified after fertilization to become unreceptive toadditional sperm. This change in receptivity is sometimes referred to as the membrane block to


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polyspermy. One well-characterized mechanism is an electrical depolarization of the egg mem-brane, which was first discovered in starfish eggs (114). Since then, an electrical depolarizationof the oocyte membrane upon fertilization has been discovered in many other species, includingsea urchins, mollusks, annelids, and vertebrates such as amphibians (57, 58). In these species,upon sperm fusion the potential difference across the oocyte membrane changes rapidly (withina few seconds) from a negative to a positive potential, reducing the ability of subsequent spermto fertilize the egg; however, these findings have been challenged (25). In mammals, however, noelectrical depolarization of the oocyte has been observed, suggesting that a completely differentmechanism is used (59, 84). In fact, the timing of the membrane block to polyspermy is muchslower, taking tens of minutes rather than the seconds required for membrane depolarization.In a classic experiment, ZP-free mouse eggs were fertilized in vitro and then, at different timesfollowing fertilization, subjected to high sperm concentrations to induce polyspermy (121). Underthese conditions, it took up to 40 min for oocytes to become refractory to subsequent sperm fusionevents (121). Identifying the mechanistic basis of the mammalian membrane block proved difficult,although it became clear that the membrane block to polyspermy in the egg was independent of theparthenogenetic activation of eggs using calcium ionophores, strontium chloride, or ethanol (46).This observation suggests that the membrane block does not depend on cortical granule exocytosis(46). Moreover, the finding that neither the injection of sperm extracts nor the fertilization of eggsby intracytoplasmic sperm injection (ICSI) (78, 122) induced the membrane block implies that theactual sperm fusion event is required. Because the sperm is so much smaller than the egg, it wasconsidered unlikely that the incorporation of sperm membrane components into the egg could bea plausible mechanism, and measuring the diffusion coefficients of fluorescently labeled lipids didnot reveal any striking differences between fertilized and unfertilized mouse eggs (119, 120).

One possible mechanistic explanation for the membrane block to polyspermy is suggestedby the finding that the JUNO receptor is shed from the egg membrane after fertilization andrelocalized within vesicles confined within the perivitelline space (10). Although not direct proof,this model is compelling because the loss of JUNO should render eggs infertile, and the timing ofJUNO shedding (∼40 min after fertilization) is in agreement with the known timing of themembrane block. It has also been shown that JUNO remains on the oolemma when eggs areparthenogenetically activated or fertilized by ICSI, and the membrane block is known to be agraded response, which is consistent with the gradual loss of a sperm receptor. Finally, theshedding of the oolemma as JUNO-positive membrane vesicles could potentiate the spermblock by creating a zone of decoy eggs that would rapidly bind and neutralize subsequentacrosome-reacted sperm. One intriguing but unanswered question is the identity of the signalingpathway that causes JUNO shedding. The fact that JUNO appears to be localized within entirevesicles suggests that it is not removed by a phospholipase, and treating eggs with calciumchelators such as BAPTA does not prevent shedding of JUNO (E. Bianchi & G.J. Wright,unpublished data), demonstrating that JUNO shedding is not associated with calcium signaling.However, the identification of a plausible candidate molecule now provides a focus for furtheringthe mechanistic understanding of the membrane block to polyspermy in mammals.

CONCLUSION

Our molecular understanding of mammalian fertilization has advanced significantly in the pastdecade, and genetic approaches, not least the use of targeted gene-deficient mice, have playeda major role in this advancement. Naturally, much work remains to be done, and several keyquestions remain to be answered. For example, we do not yet have a good understanding of howthe sperm interacts with the ZP or which proteins might act as fusogens in the last stages of


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fertilization. Answering these questions is more than just a matter of academic interest. Infertilityis an expanding problem, most likely due to the increasing average age of first-time parents (47).By contrast, we cannot support an ever-increasing population on a planet with finite resources, sothere is a growing need to develop better, longer-lasting, and reversible contraceptives. Althoughmany challenges must be overcome, we have every reason to be optimistic that the development ofnew cell-based and genetic technologies will enable progress to be made in the coming years. Afterall, fertilization is a fascinating and fundamental biological phenomenon, which both scientistsand the general public find compelling. Who would not be curious to know more details abouthow our mother’s egg and father’s sperm met and fused when we were first created?

DISCLOSURE STATEMENT

The authors are not aware of any affiliations, memberships, funding, or financial holdings thatmight be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS

The authors’ research is supported by the Wellcome Trust grant number 098051 and the UKMedical Research Council grant MR/M012468/1.

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GE50-FrontMatter ARI 21 October 2016 7:49

Annual Review ofGenetics

Volume 50, 2016

Contents

A Life Investigating Pathways that Repair Broken ChromosomesJames E. Haber � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 1

Sex-Biased Gene ExpressionSonja Grath and John Parsch � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �29

Aneuploidy in Cancer and AgingRyan M. Naylor and Jan M. van Deursen � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �45

Transition Metals and Virulence in BacteriaLauren D. Palmer and Eric P. Skaar � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �67

Sperm Meets Egg: The Genetics of Mammalian FertilizationEnrica Bianchi and Gavin J. Wright � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �93

Robust Yet Fragile: Expression Noise, Protein Misfolding, and GeneDosage in the Evolution of GenomesJ. Chris Pires and Gavin C. Conant � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 113

Evolution in the Cycles of LifeJohn L. Bowman, Keiko Sakakibara, Chihiro Furumizu, and Tom Dierschke � � � � � � � � � � 133

Functions, Regulation, and Therapeutic Implications of the ATRCheckpoint PathwayStephanie A. Yazinski and Lee Zou � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 155

Control of Meiotic Crossovers: From Double-Stand Break Formationto DesignationStephen Gray and Paula E. Cohen � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 175

The Plant Microbiota: Systems-Level Insights and PerspectivesDaniel B. Muller, Christine Vogel, Yang Bai, and Julia A. Vorholt � � � � � � � � � � � � � � � � � � � � � 211

Genome-Wide Analysis of RNA Secondary StructurePhilip C. Bevilacqua, Laura E. Ritchey, Zhao Su, and Sarah M. Assmann � � � � � � � � � � � � � 235

Single-Cell and Single-Molecule Analysis of Gene Expression RegulationMaria Vera, Jeetayu Biswas, Adrien Senecal, Robert H. Singer, and Hye Yoon Park � � 267

Conservation and Variability of Meiosis Across the EukaryotesJosef Loidl � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 293

Monoallelic Gene Expression in MammalsAndrew Chess � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 317

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GE50-FrontMatter ARI 21 October 2016 7:49

Proteopathic Strains and the Heterogeneity of Neurodegenerative DiseasesLary C. Walker � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 329

The Ecology and Evolution of Cancer: The Ultra-Microevolutionary ProcessChung-I Wu, Hurng-Yi Wang, Shaoping Ling, and Xuemei Lu � � � � � � � � � � � � � � � � � � � � � � � 347

Regulation and Role of Fungal Secondary MetabolitesJuliane Macheleidt, Derek J. Mattern, Juliane Fischer, Tina Netzker,

Jakob Weber, Volker Schroeckh, Vito Valiante, and Axel A. Brakhage � � � � � � � � � � � � � � � 371

Eukaryotic DNA Polymerases in Homologous RecombinationMitch McVey, Varandt Y. Khodaverdian, Damon Meyer,

Paula Goncalves Cerqueira, and Wolf-Dietrich Heyer � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 393

Regulated Proteolysis in Bacteria: CaulobacterKamal Kishore Joshi and Peter Chien � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 423

Mosquito Vectors and the Globalization of Plasmodium falciparum MalariaAlvaro Molina-Cruz, Martine M. Zilversmit, Daniel E. Neafsey,

Daniel L. Hartl, and Carolina Barillas-Mury � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 447

Plant Transgenerational EpigeneticsLeandro Quadrana and Vincent Colot � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 467

The Genetics of Enteropathogenic Escherichia coli VirulenceJaclyn S. Pearson, Cristina Giogha, Tania Wong Fok Lung,

and Elizabeth L. Hartland � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 493

Deciphering Combinatorial GeneticsAlan S.L. Wong, Gigi C.G. Choi, and Timothy K. Lu � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 515

Interorgan Communication Pathways in Physiology: Focus onDrosophilaIlia A. Droujinine and Norbert Perrimon � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 539

Cell-Specific Targeting of Genetically Encoded Tools forNeuroscienceLucas Sjulson, Daniela Cassataro, Shamik DasGupta, and Gero Miesenbock � � � � � � � � � � � 571

Vaccination via Chloroplast Genetics: Affordable Protein Drugs forthe Prevention and Treatment of Inherited or Infectious HumanDiseasesHenry Daniell, Hui-Ting Chan, and Elise K. Pasoreck � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 595

Errata

An online log of corrections to Annual Review of Genetics articles may be found athttp://www.annualreviews.org/errata/genet

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sperm meets egg: the genetics of mammalian fertilization

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