Novel insights into the molecular mechanism of sperm–egg fusion via IZUMO1

When a spermatozoon fertilizes an oocyte in mammals, there must be an extremely precise regulation system for successful gamete fusion to occur, which is the final step of fertilization. Using gene-modified animals, IZUMO1 on the sperm side and its receptor, JUNO, on the ovum side, have been unveiled as indispensable factors for triggering membrane fusion. We recently analyzed the detailed molecular machinery of the IZUMO1–JUNO recognition system and clarified the tertiary architecture of the IZUMO1–JUNO complex based on the crystal structure. Over the past 2 years, important discoveries have successively emerged, presenting a new perspective on fertilization. In this mini-review, I will initially explain the historical background of the molecular mechanism study of gamete fusion, and go on to describe our latest study data.     

Monitoring dimeric status of IZUMO1 during the acrosome reaction in living spermatozoon

The acrosome reaction (AR) is indispensable for successful spermatozoon-oocyte fusion. Recent studies have indicated that sperm IZUMO1 gradually gathers in the equatorial segment (EQ), which is the initiation site of sperm-egg fusion, after the AR. In addition, by examining the binding process of oocytes and Izumo1-expressing cultured cells to reconstitute the early steps of fertilization, we previously demonstrated that robust IZUMO1-dependent adhesion specifically occurs at the contact site along with the dimerization of IZUMO1. However, when IZUMO1 dimerizes after the AR in living spermatozoon is unknown. Here, we report dynamics of IZUMO1 dimerization during the AR in spermatozoa by combining transgenic mice and time-lapse imaging using a set of bimolecular fluorescence complementation (BiFC) probes. Surprisingly, dimeric IZUMO1 was already formed at the acrosomal cap region before the AR and redistributed into the EQ after the AR. We categorized the translocation of the dimer into two types: Type 1, the near-simultaneous appearance of BiFC signals with IZUMO1-mCherry; and Type 2, the delayed formation of dimer in the EQ. Those findings suggest that, before encountering oocytes, spermatozoa are prepared to boost their affinity with JUNO.     

Lack of tyrosylprotein sulfotransferase-2 activity results in altered sperm-egg interactions and loss of ADAM3 and ADAM6 in epididymal sperm

Tyrosine O-sulfation is a post-translational modification catalyzed by two tyrosylprotein sulfotransferases (TPST-1 and TPST-2) in the trans-Golgi network. Tpst2-deficient mice have male infertility, sperm motility defects, and possible abnormalities in sperm-egg membrane interactions. Studies here show that compared with wild-type sperm, fewer Tpst2-null sperm bind to the egg membrane, but more of these bound sperm progress to membrane fusion. Similar outcomes were observed with wild-type sperm treated with the anti-sulfotyrosine antibody PSG2. The increased extent of sperm-egg fusion is not due to a failure of Tpst2-null sperm to trigger establishment of the egg membrane block to polyspermy. Anti-sulfotyrosine staining of sperm showed localization similar to that of IZUMO1, a sperm protein that is essential for gamete fusion, but we detected little to no tyrosine sulfation of IZUMO1 and found that IZUMO1 expression and localization were normal in Tpst2-null sperm. Turning to a discovery-driven approach, we used mass spectrometry to characterize sperm proteins that associated with PSG2. This identified ADAM6, a member of the A disintegrin and A metalloprotease (ADAM) family; members of this protein family are associated with multiple sperm functions. Subsequent studies revealed that Tpst2-null sperm lack ADAM6 and ADAM3. Loss of ADAM3 is strongly associated with male infertility and is observed in knockouts of male germ line-specific endoplasmic reticulum-resident chaperones, raising the possibility that TPST-2 may function in quality control in the secretory pathway. These data suggest that TPST-2-mediated tyrosine O-sulfation participates in regulating the sperm surface proteome or membrane order, ultimately affecting male fertility.     

The cell biology of fertilization: Gamete attachment and fusion

Fertilization is defined as the union of two gametes. During fertilization, sperm and egg fuse to form a diploid zygote to initiate prenatal development. In mammals, fertilization involves multiple ordered steps, including the acrosome reaction, zona pellucida penetration, sperm–egg attachment, and membrane fusion. Given the success of in vitro fertilization, one would think that the mechanisms of fertilization are understood; however, the precise details for many of the steps in fertilization remain a mystery. Recent studies using genetic knockout mouse models and structural biology are providing valuable insight into the molecular basis of sperm–egg attachment and fusion. Here, we review the cell biology of fertilization, specifically summarizing data from recent structural and functional studies that provide insights into the interactions involved in human gamete attachment and fusion.

IntroductionDuring sexual reproduction, the oocyte and sperm fuse to generate a new and unique embryo. The journey of a sperm to an egg ends in the ampulla of the female oviduct. From there, the sperm must overcome a number of physical and biochemical barriers. After undergoing the acrosome reaction and binding the ova, the sperm penetrates through the cumulus oophorus cells and the zona pellucida (ZP) to reach the perivitelline space (PVS) and oocyte membrane. Upon fusion of the sperm and egg membranes, the sperm nucleus and organelles are incorporated into the egg cytoplasm.An understanding of the mechanisms of mammalian fertilization is crucial to treat infertility and develop new methods of birth control. Infertility affects 15% of couples globally, and in one third of these cases, the underlying cause is unknown (Gelbaya et al., 2014). Developments in assisted reproductive technologies have provided couples with new options to conceive but may have epigenetic side effects (Mani et al., 2020). Furthermore, only 40% of couples manage to have a child despite 2 yr of treatment. Safety, efficacy, and acceptability of contraceptives are also critically important, but many current female contraceptive methods have side effects that limit long-term use (Aitken et al., 2008), while male contraceptives are limited to condoms or vasectomy (Kanakis and Goulis, 2015). A better understanding of the molecular players involved in fertilization is necessary to drive innovation in both assisted reproductive technologies and contraception.In this review, we will first briefly review the events that prepare the gametes for fertilization. We will then discuss how recent studies of genetically altered mice and structural biology efforts have shed light on the molecular mechanisms of sperm–egg attachment and fusion. We will also discuss the gaps in current knowledge and suggest new perspectives and future directions in the search for other protein factors involved at the gamete fusion synapse.Cell biology of gametesFertilization requires proper gametogenesis (oogenesis in the female and spermatogenesis in the male), which produces haploid cells and introduces diversity. Primordial germ cells (PGC) are the embryonic precursors to spermatocytes and ova. The cells produced by the first few divisions of the fertilized egg are totipotent and capable of differentiating into any cell type, including germ cells. PGCs originate within the primary ectoderm of the embryo and then migrate into the yolk sac. Between weeks 4 and 6, the PGCs migrate back into the posterior body wall of the embryo, where they stimulate cells of the adjacent coelomic epithelium and mesonephros to form primitive sex cords and induce the formation of the genital ridges and gonads. The sex (gonadal) cords surround the PGCs and give rise to the tissue that will nourish and regulate the development of the maturing sex cells (ovarian follicles in the female and Sertoli cells in the male).EggOogenesis is a complex differentiation process by which mature functional ova develop from germ cells (Fig. 1 A; Edson et al., 2009). In humans, oogenesis begins in the ovary at 6–8 wk of fetus development, when PGCs differentiate into oogonia. By the 12th week, several million oogonia enter prophase, the first meiotic division and become dormant until shortly before ovulation (Hayashi et al., 2020). Due to their large and watery nuclei, these cells are referred to as germinal vesicles (Pan and Li, 2019). These primary oocytes become enclosed by follicle cells to form primordial follicles. The number of primordial follicles peaks at ∼7 million by the fifth month of fetal life, with ∼700,000 left at birth and 400,000 by puberty (Marcozzi et al., 2018). All of the egg cells that the ovaries will release are already present at birth.Open in a separate windowFigure 1.Gametogenesis and fertilization. (A–C) Illustration of oogenesis and follicle development (A), spermatogenesis (B), and the major steps in fertilization (C): (1) initial contact, (2) acrosome reaction, (3) ZP penetration, (4) sperm–egg fusion, (5) entry of sperm nucleus, (6) cortical reaction, and (7) fusion of the sperm and egg nuclei. The oocyte with its ZP measures 130 μm in diameter. Created with BioRender.During each menstrual cycle, hormones from the hypothalamic–pituitary–gonadal axis restart the division of the primary oocytes in meiosis I and follicular development (Atwood and Vadakkadath Meethal, 2016). Primary follicles develop into secondary follicles, containing each growing oocyte surrounded by two or more layers of proliferating follicle cells. ZP glycoproteins are secreted by the oocyte of the primary follicle and possibly the follicular cells (Törmälä et al., 2008). Although these glycoproteins form a physical barrier between the follicle cells and the oocyte, follicle cells and the oocyte remain connected through transzonal cytoplasmic projections from the follicle cells until fertilization (Makabe et al., 2006). A reciprocal dialog between the oocyte and its surrounding follicular cells coordinates the different phases of follicular development and the maintenance of meiotic arrest (Dalbies-Tran et al., 2020). Oocyte-derived microvilli control female fertility by optimizing ovarian follicle selection in mice (Zhang et al., 2021). The epithelium of 5–12 primary follicles proliferates to form a multilayered capsule around the oocyte. A few of these growing follicles continue to enlarge in response to follicle-stimulating hormone (FSH; Visser and Themmen, 2014). A single follicle becomes dominant, and the others degenerate by atresia (Atwood and Vadakkadath Meethal, 2016). Meiosis of the oocyte in the mature preovulatory follicle is blocked until a surge in levels of FSH and luteinizing hormone that occurs midway through the menstrual cycle. The membrane of the germinal vesicle nucleus breaks down, the chromosomes align in metaphase, and the oocyte expels its first polar body. The secondary oocyte then begins the second meiotic division, which is arrested at the meiotic metaphase II stage until ovulation (Gougeon, 1996). Ovulation depends on the breakdown of the follicle wall and occurs ∼38 h after the increase in levels of FSH and luteinizing hormone (Holesh et al., 2021). The disruption of the follicle wall expulses the oocyte, which is captured by the fimbriated mouth of the oviduct and moved into the ampulla. The oocyte retains its ability to be fertilized for ∼24 h and completes meiosis only if it is fertilized.SpermIn contrast to oogenesis, which is complete before birth, spermatogenesis is a continuous process that begins at puberty (Fig. 1 B). In humans, spermatogenesis takes 74 d to complete; thus, multiple spermatogenesis events occur simultaneously to allow for continual sperm production. Spermatogenesis occurs in the testis in a stepwise manner, beginning with diploid spermatogonia at the basal surface of seminiferous tubules and ending with mature elongated spermatozoa that are released in tubule lumens in a process called spermiation (Clermont, 1972; Yang and Oatley, 2014). During spermatogenesis, mitosis results in gene amplification, meiosis results in genome reduction, and finally maturation occurs (Hess and Renato de Franca, 2008). At this stage, sperm are not motile and are fertilization incompetent. Two additional sperm maturational processes are required outside the testis. First, sperm undergo a maturation process during epididymal transit (Bedford et al., 1973) involving posttranslational modifications of previously synthesized proteins and acquisition of proteins from the epididymal epithelium (James et al., 2020; see text box). After ejaculation into the female reproductive tract, dilution triggers additional changes in sperm, collectively termed capacitation (see text box), that prepare the sperm for the acrosome reaction.Epididymal maturationSperm exchange with the epididymal epithelium occurs by direct interaction with epithelial cells, by interaction with soluble proteins in the epididymal fluid or via extracellular exosome-like vesicles released by epithelial cells called epididymosomes (James et al., 2020). The purposes of this exchange are to redistribute sperm proteins and change the composition and lipid balance of the sperm membrane. These changes take place during the transit from the epididymis initial segment, through the caput and the corpus, to the cauda where sperm are stored (Cornwall, 2009). Epididymal transit lasts 10–12 d in mammals, but storage is dependent on sexual activity. Since fertilization is not immediate, fertilizing capacities of the spermatozoa are preserved by decapacitation factors that are active in the epididymis. An example of a decapacitation factor is SPINK3, which is secreted by seminal vesicles; it impairs sperm membrane hyperpolarization and calcium influx through CatSper (Zalazar et al., 2020). Epididymal plasma and sperm represent only a small fraction (5%) of semen in men (Batruch et al., 2011). Two thirds of the volume of semen comes from the seminal vesicles and the other third from the prostate. These secretions protect the sperm and prevent early maturation.Sperm capacitationMore than 70 yr ago, Austin and Chang described capacitation as the changes required for sperm to fertilize oocytes in vivo (Austin, 1952; Chang, 1951). Once sperm enter the female reproductive tract, they undergo capacitation. Capacitation results in hyperactivation of sperm movement and initiation of the acrosome reaction (Saling et al., 1979; Florman and First, 1988). During capacitation, stabilizing or decapacitation factors that are adsorbed on the sperm plasma membrane are removed (Bedford and Chang, 1962). These agents that initiate removal of decapacitation factors are electrolytes, energy substrates, and proteins such as seminal plasma protein or albumin. Removal of decapacitation factors increases sperm plasma membrane fluidity, allowing an increase in the permeability to calcium, chloride, and bicarbonate ions (Gangwar and Atreja, 2015). Sperm motility depends on the membrane potential, intracellular pH, and balance of intracellular ions (reviewed in Nowicka-Bauer and Szymczak-Cendlak, 2021). The most important ion for this function is Ca²⁺ (Hwang et al., 2019). This secondary messenger is an important signaling pathways activator that regulates sperm motility (Finkelstein et al., 2020). The activation of soluble adenyl cyclases generates cyclic adenosine monophosphate that in turn activates serine/threonine protein kinase A, which induces a cascade of protein phosphorylation initiating the induction of sperm motility (Chen et al., 2000). Protein phosphorylation, sperm hyperactivation, and the acrosome reaction are used in vitro to evaluate capacitation. Capacitation can be induced in vitro by incubation in medium containing calcium, bicarbonate ions, and serum albumin (Touré, 2019).Mammalian sperm capacitation occurs during sperm migration in the female tract. Mammalian males ejaculate millions of sperm cells into the female reproductive tract, but only a few hundred sperm at most reach the oocytes. This massive elimination process likely prevents polyspermy (reviewed in Kölle, 2015). Selection of human sperm during the journey begins in the acidic environment of the vagina. In the cervix, only morphologically normal sperm can migrate. Some sperm immediately pass into the cervical mucus, whereas the remaining sperm becomes a part of the coagulum. The next selection occurs at the uterus–tubal junction, the connection between the uterus and the oviduct that represents a major obstacle for sperm migration (Kölle, 2015). Experiments in mice indicate that sperm motility alone is insufficient for sperm migration through the uterus–tubal junction (Fujihara et al., 2018). Uterine contractions facilitate sperm transport as do molecular interactions. Several proteins, such as ADAM3 and other ADAM family members, are known to be involved in this step in mice (Yamaguchi et al., 2009; Xiong et al., 2019); most ADAM proteins have human orthologues.Spermatogenesis takes place in a species-specific cycle called the seminiferous epithelial cycle and is regulated in particular through the hypothalamic–pituitary–testicular axis. Indeed, at puberty, the testes (interstitial steroidogenic Leydig cells) secrete an increased amount of testosterone, which triggers growth of the testes, maturation of the seminiferous tubules, and the commencement of spermatogenesis. The Sertoli cells are the major somatic cells present in the seminiferous tubules and are considered to be the main regulators of spermatogenesis. They orchestrate spermatogenesis by supporting spermatogonial stem cells, determining the testis size, organizing meiotic and postmeiotic development and sperm output, supporting androgen production by maintaining the development and function of Leydig cells, and regulating other aspects of testis function like peritubular myoid cells, immune cells, and the vasculature, which participate in the maintenance of the spermatogonial stem cell niche.Acrosome reactionThe acrosome is a secretory vesicle located on the anterior region of sperm that originates from the spermatid Golgi apparatus. An acrosomal granule is formed by the fusion of proacrosomal vesicles in the vicinity of the nucleus. The region increases in size and spreads over the anterior part of the nucleus. The acrosome reaction is driven by SNARE complexes and results in the exocytosis of the contents of the acrosome upon fusion of the plasma membrane with the outer acrosomal membrane (Fig. 1 C; reviewed in Okabe, 2016; De Blas et al., 2005). The timing of the acrosome reaction is critical. Only sperm that have undergone this reaction are fertilization competent, but when a high proportion of sperm undergo the acrosome reaction prematurely, success of in vitro fertilization is low (Wiser et al., 2014). Several studies indicate that only a fraction of sperm is capable of undergoing spontaneous acrosome reaction. In human and mice sperm samples, 15–20% of cells undergo spontaneous acrosome reaction (Nakanishi et al., 2001), whereas only 20–30% undergo progesterone-induced acrosome reaction (Stival et al., 2016), suggesting physiological heterogeneity of sperm population. In addition, Inoue et al. demonstrated that acrosome-reacted mouse spermatozoa recovered from the PVS can fertilize other eggs (Inoue et al., 2011).Based on in vitro data, it was thought that the acrosome reaction occurs when the sperm contacts the ZP, particularly the ZP3 protein (Litscher and Wassarman, 1996). Using transgenic mice that express fluorescent markers in the acrosome (Nakanishi et al., 1999) and the midpiece mitochondria (Hasuwa et al., 2010), real-time observation of acrosomal exocytosis was possible. These experiments showed that most mouse spermatozoa capable of fertilization had undergone the acrosome reaction before contact with the oocyte ZP (Jin et al., 2011). Most spermatozoa begin to react in the isthmus of the oviduct before reaching the ampulla (Hino et al., 2016; La Spina et al., 2016). Contact with the ZP in vitro probably makes it possible to complete a partial acrosome reaction. The most important function of the acrosome reaction is to induce changes in the sperm membrane (Okabe, 2016). The relocations of IZUMO1 and SPACA6, proteins essential for sperm–egg fusion, that occur after the acrosome reaction are illustrative examples of these changes (Sosnik et al., 2009; Barbaux et al., 2020; Satouh et al., 2012). The presence of these proteins on the sperm membrane, in addition to the classic markers Pisum sativum agglutinin, Peanut agglutinin lectins, or CD46, can be used as markers for the acrosome reaction (Ito and Toshimori, 2016). The acrosome and its disruption are both crucial for effective fertilization, as low fertilization rates are observed upon intracytoplasmic sperm injection of acrosome-intact sperm (Morozumi and Yanagimachi, 2005) or round spermatozoa lacking acrosomes (Dávila Garza and Patrizio, 2013).ZP penetrationThe ZP is a physical barrier between the oocyte and the follicular cells that forms from glycoproteins secreted from the primary follicles (Fig. 1 C). The human ZP consists of four glycoproteins (hZP1–hZP4; Harris et al., 1994). Mice, which have been used for most of the ZP studies in mammals, express only three ZP glycoproteins (mZP1–mZP3; Litscher and Wassarman, 2007). Analysis of mouse lines expressing human ZP proteins demonstrated that only hZP2 is important in human sperm–egg binding (Gupta, 2021). Experiments using purified native or recombinant human ZP proteins have shown that hZP1, hZP3, and hZP4 bind to the capacitated human spermatozoa and induce the acrosome reaction (Gupta, 2021). ZP1 is required for the structural integrity of the ZP (Chakravarty et al., 2008). To better understand the roles of ZP glycoproteins, further studies, particularly on ZP protein glycosylation, are needed. The species-specific binding of the ZP to sperm is presumably related to these carbohydrate moieties (Clark, 2014). The sialyl-Lewis(x) sequence is the major carbohydrate ligand for human sperm–egg binding (Pang et al., 2011). The current hypothesis that hZP1, hZP3, and hZP4 bind to capacitated sperm and hZP2 binds to sperm with intact acrosomes will need to be revisited due to the recent demonstration that the acrosome reaction takes place before ZP contact. Regardless, the role of the ZP in preventing polyspermy is clear. Indeed, ZP hardening is due to ZP2 cleavage by ovastacin, a protease released into the PVS by cortical granules after the first sperm–egg fusion (Burkart et al., 2012).Sperm–egg attachment and membrane fusionAfter penetration of the ZP, the sperm enters the PVS and can attach and fuse with the egg plasma membrane. The development of genetic knockout animal models has proven critical in determining the importance of various sperm and egg proteins in sperm–egg attachment and fusion. Surprisingly, genetic knockout studies revealed that many factors originally thought to be important for fertilization were in fact not necessary (reviewed in Okabe, 2018, 2015). The proteins from sperm and egg that are essential for sperm–egg membrane interaction and fusion are listed in

Analysis of CAPZA3 localization reveals temporally discrete events during the acrosome reaction

In mammals, the starting point of development is the fusion between sperm and egg. It is well established that sperm fuse with the egg through the equatorial/post‐acrosomal region. Apart from this observation and the requirement of two proteins (CD9 in the egg and IZUMO1 in the sperm) very little is known about this fundamental process. Actin polymerization correlates with sperm capacitation in different mammalian species and it has been proposed that F‐actin breakdown is needed during the acrosome reaction. Recently, we have presented evidence that actin polymerization inhibitors block the movement of IZUMO1 that accompany the acrosome reaction. These results suggest that actin dynamics play a role in the observed changes in IZUMO1 localization. This finding is significant because IZUMO1 localization in acrosome‐intact sperm is not compatible with the known location of the initiation of the fusion between the sperm and the egg. To further understand the actin‐mediated changes in protein localization during the acrosome reaction, the distribution of the sperm‐specific plus‐end actin capping protein CAPZA3 was analyzed. Like IZUMO1, CAPZA3 shows a dynamic pattern of localization; however, these movements follow a different temporal pattern than the changes observed with IZUMO1. In addition, the actin polymerization inhibitor latrunculin A was unable to alter CAPZA3 movement. J. Cell. Physiol. 224: 575–580, 2010. © 2010 Wiley‐Liss, Inc.     

Identification and Disruption of Sperm-Specific Angiotensin Converting Enzyme-3 (ACE3) in Mouse

Background

IZUMO1 is the only sperm protein which is proven to be essential for sperm-egg fusion. However, the IZUMO1 is a structurally simple protein with single Ig domain and seems not to include either a “fusogenic peptide” or a fusion machinery domain. This led us to assume the existence of an IZUMO1-interacting protein(s) which makes a functional fusion machine interacting with IZUMO1.

Methodology/Principal Findings

We produced a transgenic mouse line which expresses His-tagged IZUMO1 in the Izumo1 ^−/− genetic background. After solubilization of sperm membranes, we purified His-tagged IZUMO1 using anti-His affinity chromatography and found a protein that interacts with IZUMO1. After being separated on SDS-PAGE gel, the IZUMO1-interacting protein was subjected to LC-MS/MS analysis and from the partial fragments, we identified the protein as ACE3. We raised the antibody against ACE3 and found that ACE3 is localized on the acrosomal cap area as in the case of IZUMO1. However, ACE3 disappeared from sperm after acrosome reaction while IZUMO1 remained on sperm. In order to investigate the role of ACE3 in vivo, we generated Ace3-deficient mice by homologous recombination and examined the fertilizing ability of the males. Unexpectedly, the male mice showed no defect in fertilizing ability in in vivo or in an in vitro fertilization system.

Conclusions/Significance

We identified an IZUMO1-interacting protein in sperm, which we identified as testis specific ACE homologue ACE3. We produced an Ace3 disrupted mouse line, and found the localization of IZUMO1 spread in a little wider area on sperm, but the elimination of ACE3 did not result in a loss of sperm fertilizing ability, differing from the case of ACE disruption.     

The functional domain of GCS1-based gamete fusion resides in the amino terminus in plant and parasite species

Fertilization is one of the most important processes in all organisms utilizing sexual reproduction. In a previous study, we succeeded in identifying a novel male gametic transmembrane protein GCS1 (GENERATIVE CELL SPECIFIC 1), also called HAP2 (HAPLESS 2) in the male-sterile Arabidopsis thaliana mutants, as a factor critical to gamete fusion in flowering plants. Interestingly, GCS1 is highly conserved among various eukaryotes covering plants, protists and invertebrates. Of these organisms, Chlamydomonas (green alga) and Plasmodium (malaria parasite) GCS1s similarly show male gametic expression and gamete fusion function. Since it is generally believed that protein factors controlling gamete fusion have rapidly evolved and different organisms utilize species-specific gamete fusion factors, GCS1 may be an ancient fertilization factor derived from the common ancestor of those organisms above. And therefore, its molecular structure and function are important to understanding the common molecular mechanics of eukaryotic fertilization. In this study, we tried to detect the central functional domain(s) of GCS1, using complementation assay of Arabidopsis GCS1 mutant lines expressing modified GCS1. As a result, the positively-charged C-terminal sequence of this protein is dispensable for gamete fusion, while the highly conserved N-terminal domain is critical to GCS1 function. In addition, in vitro fertilization assay of Plasmodium berghei (mouse malaria parasite) knock-in lines expressing partly truncated GCS1 showed similar results. Those findings above indicate that the extracellular N-terminus alone is sufficient for GCS1-based gamete fusion.     

Gamete fusion site on the egg cell and autonomous establishment of cell polarity in the zygote

Gamete fusion activates the egg in animals and plants, and the gamete fusion site on the zygote might provide a possible cue for zygotic development and/or embryonic patterning. In angiosperms, a zygote generally divides into a two-celled proembryo consisting of an apical and a basal cell with different cell fates. This is a putative step in the formation of the apical-basal axis of the proembryo. We observed the positional relationship between the gamete fusion site and the division plane formed by zygotic cleavage using an in vitro fertilization system with rice gametes. There was no relationship between the gamete fusion site and the division plane leading to the two-celled proembryo. Thus, the gamete fusion site on the rice zygote does not appear to function as a determinant for positioning the zygote division plane, and the zygote apparently possesses autonomous potential to establish cell polarity along the apical-basal axis for its first cleavage.Key words: asymmetric division, egg cell, fertilization, gamete fusion, rice, sperm cell, two-celled proembryo, zygote     

HAP2(GCS1)-Dependent Gamete Fusion Requires a Positively Charged Carboxy-Terminal Domain

HAP2(GCS1) is a deeply conserved sperm protein that is essential for gamete fusion. Here we use complementation assays to define major functional regions of the Arabidopsis thaliana ortholog using HAP2(GCS1) variants with modifications to regions amino(N) and carboxy(C) to its single transmembrane domain. These quantitative in vivo complementation studies show that the N-terminal region tolerates exchange with a closely related sequence, but not with a more distantly related plant sequence. In contrast, a distantly related C-terminus is functional in Arabidopsis, indicating that the primary sequence of the C-terminus is not critical. However, mutations that neutralized the charge of the C-terminus impair HAP2(GCS1)-dependent gamete fusion. Our results provide data identifying the essential functional features of this highly conserved sperm fusion protein. They suggest that the N-terminus functions by interacting with female gamete-expressed proteins and that the positively charged C-terminus may function through electrostatic interactions with the sperm plasma membrane.     

Evolutionary History of the HAP2/GCS1 Gene and Sexual Reproduction in Metazoans

The HAP2/GCS1 gene first appeared in the common ancestor of plants, animals, and protists, and is required in the male gamete for fusion to the female gamete in the unicellular organisms Chlamydomonas and Plasmodium. We have identified a HAP2/GCS1 gene in the genome sequence of the sponge Amphimedon queenslandica. This finding provides a continuous evolutionary history of HAP2/GCS1 from unicellular organisms into the metazoan lineage. Divergent versions of the HAP2/GCS1 gene are also present in the genomes of some but not all arthropods. By examining the expression of the HAP2/GCS1 gene in the cnidarian Hydra, we have found the first evidence supporting the hypothesis that HAP2/GCS1 was used for male gamete fusion in the ancestor of extant metazoans and that it retains that function in modern cnidarians.     

Expression and structure-function analysis of de,a sperm cysteine-rich secretory protein that mediates gamete fusion

Rat sperm epididymal glycoprotein DE belongs to the cysteine-rich secretory protein (CRISP) family and participates in sperm-egg fusion through its binding to complementary sites on the egg surface. To investigate the molecular mechanisms underlying the role of DE in gamete fusion, in the present work we expressed DE in a prokaryotic system, and examined the relevance of carbohydrates and disulfide bonds for the biological activity of the protein. Immunofluorescence and sperm-egg fusion assays carried out in the presence of recombinant DE (recDE) revealed that this protein exhibits the ability to bind to the DE-egg binding sites and to inhibit gamete fusion, as does native DE (nDE). Comparison of the proteins indicated, however, that the inhibitory ability of recDE was significantly lower than that of nDE. This difference would not be due to the lack of carbohydrates in the bacterially expressed protein because enzymatically deglycosylated nDE was as able as the untreated protein to inhibit gamete fusion. To examine whether disulfide bridges are involved in DE activity, the presence of sulfhydryls in nDE and recDE was evaluated by the biotin-maleimide technique. Results indicated that, unlike nDE, in which all cysteines are involved in disulfide bonds, recDE contains free thiol groups. Subsequent experiments showed that reduction of nDE with dithiothreitol significantly decreased the ability of the protein to inhibit gamete fusion. Together, these results indicate that whereas carbohydrates do not have a role in DE-mediated gamete fusion, disulfide bridges are required for full biological activity of the protein. To our knowledge, this is the first study reporting the relevance of structural components for the function of a CRISP member.     

The Chlamydomonas Fus1 protein is present on the mating type plus fusion organelle and required for a critical membrane adhesion event during fusion with minus gametes

The molecular mechanisms of the defining event in fertilization, gamete fusion, remain poorly understood. The FUS1 gene in the unicellular, biflagellated green alga Chlamydomonas is one of the few sex-specific eukaryotic genes shown by genetic analysis to be essential for gamete fusion during fertilization. In Chlamydomonas, adhesion and fusion of the plasma membranes of activated mt+ and mt- gametes is accomplished via specialized fusion organelles called mating structures. Herein, we identify the endogenous Fus1 protein, test the idea that Fus1 is at the site of fusion, and identify the step in fusion that requires Fus1. Our results show that Fus1 is a approximately 95-kDa protein present on the external surface of both unactivated and activated mt+ gametes. Bioassays indicate that adhesion between mating type plus and mating type minus fusion organelles requires Fus1 and that Fus1 is functional only after gamete activation. Finally, immunofluorescence demonstrates that the Fus1 protein is present as an apical patch on unactivated gametes and redistributes during gamete activation over the entire surface of the microvillous-like activated plus mating structure, the fertilization tubule. Thus, Fus1 is present on mt+ gametes at the site of cell-cell fusion and essential for an early step in the fusion process.     

PDIA3的结构与功能及其在精卵融合过程中的作用

二硫键异构酶家族的一员PDIA3(protein disulfide-isomerase A3)最初确定其位于内质网腔内,作为钙联接蛋白和钙网质蛋白的分子伴侣复合物的组成成分之一,促进新合成的糖蛋白在内质网中的正确折叠。PDIA3在内质网中的功能依赖其蛋白二硫键氧化还原异构酶活性。目前研究发现位于精子表面的PDIA3的蛋白二硫键氧化还原酶活性在精卵融合中也发挥着重要的作用。就目前关于PDIA3的结构和功能及在精卵质膜融合中的作用研究进展做一概述。     

Sperm protein "DE" mediates gamete fusion through an evolutionarily conserved site of the CRISP family

The first member of the cysteine-rich secretory protein (CRISP) family was described by our laboratory in the rat epididymis, and it is known as DE or CRISP-1. Since then, numerous CRISPs exhibiting a high amino acid sequence similarity have been identified in animals, plants and fungi, although their functions remain largely unknown. CRISP-1 proteins are candidates to mediate gamete fusion in the rat, mouse and human through their binding to complementary sites on the egg surface. To elucidate the molecular mechanisms underlying CRISP-1 function, in the present work, deletion mutants of protein DE were generated and examined for their ability to bind to the rat egg and interfere with gamete fusion. Results revealed that the egg-binding ability of DE resides within a 45-amino acid N-terminal region containing the two motifs of the CRISP family named Signature 1 and Signature 2. Subsequent assays using synthetic peptides and other CRISPs support that the egg-binding site of DE falls in the 12-amino-acid region corresponding to Signature 2. The interesting finding that the binding site of DE resides in an evolutionarily conserved region of the molecule provides novel information on the molecular mechanisms underlying CRISP-1 function in gamete fusion with important implications on the structure-function relationship of other members of the widely distributed CRISP family.     

MHC-linked susceptibility to a bacterial infection, but no MHC-linked cryptic female choice in whitefish

Non-random gamete fusion is one of several potential cryptic female choice mechanisms that have been postulated and that may enhance the survival probability of the offspring. Previous studies have found that gamete fusion in mice is influenced by genes of the major histocompatibility complex (MHC) region. Here we test (i) whether there is MHC-dependent gamete fusion in whitefish (Coregonus sp.) and (ii) whether there is a link between the MHC and embryo susceptibility to an infection by the bacterium Pseudomonas fuorescens. We experimentally bred whitefish and reared sibships in several batches that either experienced or did not experience strong selection by P. fluorescens. We then determined the MHC class II B1 genotype of 1016 surviving larvae of several full sibships. We found no evidence for MHC-linked gamete fusion. However, in one of seven sibships we found a strong connection between the MHC class II genotype and embryo susceptibility to P. fluorescens. This connection was still significant after correcting for multiple testing. Hence, the MHC class II genotype can considerably influence embryo survival in whitefish, but gamete fusion seems to be random with respect to the MHC.     

Different developmental functions for calmodulin in Dictyostelium: trifluoperazine and R24571 both inhibit cell and pronuclear fusion but enhance gamete formation

The calmodulin antagonists trifluoperazine and compound R24571 were used to study the function of calmodulin during sexual development in Dictyostelium discoideum. Calmodulin activity is required for both cell fusion and pronuclear fusion. However, cell fusion and pronuclear fusion were each maximally inhibited at different concentrations of the same inhibitor suggesting differential calmodulin activity during these events. In contrast, trifluoperazine and R24571 were both found to enhance rather than inhibit the formation of gametes. This suggests an additional role for calmodulin as a negative regulator of gamete development. These results provide evidence of a role for calmodulin as both a positive (biomembrane fusion) and a negative (gamete development) regulator of developmental events in Dictyostelium. They also reveal calmodulin as a mediator of pronuclear fusion for zygote development in this eukaryote.     

Gamete membrane dynamics during double fertilization in Arabidopsis

In the double fertilization of angiosperms, one sperm cell fertilizes an egg cell to produce a zygote, whereas the other sperm cell fertilizes a central cell to give rise to an endosperm. There is little information on gamete membrane dynamics during double fertilization even though the cell surface structure is critical for male and female gamete interactions. In a recent study, we analyzed gamete membrane behavior during double fertilization by live-cell imaging with Arabidopsis gamete membrane marker lines. We observed that the sperm membrane signals occasionally remained at the boundary of the female gametes after gamete fusion. In addition, sperm membrane signals entering the fertilized female gametes were detected. These findings suggested that plasma membrane fusion between male and female gametes occurred with the sperm internal membrane components entering the female gametes, and this was followed by plasmogamy.     

Downregulation of egg cell-secreted EC1 is accompanied with delayed gamete fusion and polytubey

One major player known to be essential for successful gamete interactions during double fertilization in Arabidopsis thaliana is the recently identified family of egg cell-secreted EC1 proteins. Both gamete fusion events are affected in EC1-deficient female gametophytes. Here, we show that the number of ovules with unfused sperm cells is considerably higher than the number of undeveloped seeds in the same ec1-RNAi knockdown lines. We found that some sperm cells are able to fuse with the female gametes even 2 to 3 days after pollination, as reflected by delayed embryo and endosperm development, and by polytubey. We propose that the egg cell secretes EC1 proteins upon sperm arrival to promote rapid sperm activation, thereby accelerating gamete fusion and preventing polytubey.     

Novel species-specific glycoprotein on the surface of Mytilus edulis and M. trossulus eggs

Protein–protein interactions play a central role in the gamete attraction, binding, and fusion stages of gamete interactions and fertilization for broadcast spawning species, such as marine mussels in the Mytilus edulis species complex. Although assortative gamete interaction has been implicated in the level of reproductive isolation among the three species in this complex, the molecular basis of these interactions has not been elucidated. Using mass spectrometry peptide sequencing, cDNA sequencing, and bioinformatics approaches, we have investigated species-level variation in the proteins expressed on the surface of mussel eggs. We herein describe an extracellular protein, MESP-1, from the surface of the eggs of M. edulis and M. trossulus that has a unique domain structure when compared to protein structures that have heretofore been identified. Given variation in the size of MESP-1 predicted from cDNA sequences versus those estimated from SDS-PAGE gels, we conclude this protein is subject to significant species-specific post-translation modifications. Further, bioinformatic analysis of the novel structure of MESP-1 suggests that this protein may be an integral membrane protein involved in sperm–egg fusion, and/or released to the vitelline envelope.     

Analysis of gamete membrane dynamics during double fertilization of Arabidopsis

Angiosperms have a unique sexual reproduction system called “double fertilization.” One sperm cell fertilizes the egg and another sperm cell fertilizes the central cell. To date, plant gamete membrane dynamics during fertilization has been poorly understood. To analyze this unrevealed gamete subcellular behavior, live cell imaging analyses of Arabidopsis double fertilization were performed. We produced female gamete membrane marker lines in which fluorescent proteins conjugated with PIP2a finely visualized egg cell and central cell surfaces. Using those lines together with a sperm cell membrane marker line expressing GCS1-GFP, the double fertilization process was observed. As a result, after gamete fusion, putative sperm plasma membrane GFP signals were occasionally detected on the egg cell surface adjacent to the central cell. In addition, time-lapse imaging revealed that GCS1-GFP signals entered both the egg cell and the central cell in parallel with the sperm cell movement toward the female gametes during double fertilization. These findings suggested that the gamete fusion process based on membrane dynamics was composed of (1) plasma membrane fusion on male and female gamete surfaces, (2) entry of sperm internal membrane components into the female gametes, and (3) plasmogamy.