Molecular mechanisms leading to gamete fusion
Article ID: 24-00046
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Article ID: 24-00046
The fusion between spermatozoon and oocyte represents the final stage of fertilization in mammals. Since only one of the extremely large number of spermatozoa fertilizes the oocyte, there should be a strictly regulated molecular mechanism in gamete fusion. Oocyte CD9 was first identified as a key factor for gamete fusion, followed by sperm IZUMO1 and oocyte IZUMO1 receptor JUNO. Since 2020, with the recent emergence of genome editing technologies, new gamete fusion sperm factors, SPACA6, TMEM95, FIMP, SOF1, DCST1, and DCST2, have been reported one after another. In this review, I would like to give an overview of mammalian gamete fusion based on the latest findings on these factors.
Currently, 1 out of 5.5 couples in Japan suffer from infertility, and 1 out of 9 births in 2022 will be ART (Assisted Reproductive Technology) children. As infertility is on the rise amidst the trend toward late marriages, there is an urgent need to elucidate the molecular mechanisms of fertilization.
The continuity of life is supported by two types of gametes: spermatozoon and oocyte. After spermatozoa are produced in the testis, they gradually acquire fertilizing ability as they move into the epididymis and the female reproductive tract, and when they approach the oocyte, they pass through the cumulus cell layer, undergo acrosome reaction, bind to and pass through the zona pellucida, and finally fuse with the oocyte to complete fertilization. Sperm selection is very strict, and only one of the many spermatozoa (100-300 million in the case of humans) can fertilize an oocyte. Therefore, it is easy to anticipate that there is a tightly regulated molecular mechanism is involved in gamete fusion. However, the molecular mechanism of gamete fusion, the climax of fertilization, is not fully understood. In this context, our discovery of the sperm membrane protein Izumo sperm-egg fusion 1 (IZUMO1) is a groundbreaking achievement1), identifying for the first time in the world a sperm factor essential for gamete fusion. It is expected to be a breakthrough in the elucidation of these molecular mechanisms.
In 2005, we reported IZUMO1, named after Izumo-Taisha shrine, which is famous as a god of marriage1). IZUMO1 is a type I transmembrane glycoprotein with an extracellular immunoglobulin-like domain. Since IZUMO1-deficient mice were completely male sterile due to a failure of gamete fusion, IZUMO1 was identified for the first time in the world as a sperm-side factor essential for gamete fusion. IZUMO1 localizes within the acrosome cap and is not exposed on the sperm surface before the acrosome reaction, but after the acrosome reaction it relocalizes to the entire head, including the equatorial segment where sperm-oocyte fusion is established (Fig. 1A)1,2). This relocalization gradually assembles at the equatorial segment within about 20 min3). This is a reasonable system considering that fusion is initiated from the equatorial segment on the oocyte.
In 2014, nine years after the discovery of IZUMO1, the IZUMO1 receptor JUNO was reported4). Juno is a Roman goddess of marriage and childbirth. JUNO-deficient mice are completely female infertility, with gamete fusion defects. An additional interesting physiological function of JUNO is that it disappears from the surface of the oocyte immediately after fertilization has occurred. This property may be one of the rejection systems of polyspermy fertilization.
Both IZUMO1-deficient male and JUNO-deficient female mice can bypass the gamete fusion step with intracytoplasmic sperm injection (ICSI) to obtain progeny, indicating that these factors are required for gamete fusion only1,4).
IZUMO1, JUNO, and DCST1/2 are all essential for gamete fusion in mammals.
(A) Localization of IZUMO1 before and after acrosome reaction. When the acrosome reaction is triggered with exocytosis, soluble proteins and matrix (green) within the acrosome are released externally and transmembrane IZUMO1 (red) quickly moves to the surface of the sperm head, including the equatorial segment.
(B) Tertiary structure of the IZUMO1-JUNO complex. The monomer IZUMO1 exposed on the sperm surface recognizes the monomer JUNO, the IZUMO1 counter receptor, on the oocyte surface, and as a result, spermoocyte adhesion begins. Tertiary structure analysis based on X-ray crystallography has revealed the interaction mode of the IZUMO1-JUNO complex.
(C) DCST1/2-deficient mice are male sterile due to failure of gamete fusion. Spermatozoa from DCST1/2- deficient mice have normally undergone the acrosome reaction, a morphological change in spermatozoa (red : IZUMO1), but are unable to fertilize, multiple spermatozoa are present in the perivitelline space between the zona pellucida and the oocyte.
In 2016, several research groups, including ours, clarified the tertiary structures of IZUMO1, JUNO, and the IZUMO1-JUNO complex in humans and mice5-9). IZUMO1 has an elongated rod-like structure and JUNO has a spherical structure; IZUMO1 and JUNO form a complex in a 1:1 ratio. In the extracellular region, IZUMO1 consists of an IZUMO domain consisting of a bundle of four α-helices on the N-terminal side, a β-hairpin structure in the middle, and an immunoglobulin-like domain on the C-terminal side. The central β-hairpin structure of IZUMO1 is primarily responsible for binding to JUNO, while the surface located behind the putative folate binding pocket of JUNO is involved in IZUMO1 binding (Fig. 1B). Through the structural architecture of the IZUMO1-JUNO complex, the crucial amino acid residues for IZUMO1-JUNO recognition emerged. The biological importance of the IZUMO1-JUNO interaction was confirmed by cell-oocyte assays using COS-7 cells expressing substituted amino acid mutants of mouse IZUMO1, suggesting that amino acids W148, H157 and R160 are particularly important on the IZUMO1 side6). The kD values of purified human IZUMO1-JUNO containing each mutant showed that W52 and L81 were particularly important on the JUNO side in addition to W148 in IZUMO16).
IZUMO1-JUNO interaction is essential for mammalian fertilization to occur at a very early stage after sperm attachment to the oocyte. This has been demonstrated in that the fine steric structure of the IZUMO1-JUNO complex has been determined, but it is difficult to explain the complex fertilization phenomenon using only the two IZUMO1/JUNO factors. Although IZUMO1-expressing cultured cells have adhesion ability to the oocyte plasma membrane and IZUMO1 dimerizes at the adhesive surface, fusion of both membranes is not established10,11). Also, some fusion regulators, such as oocyte CD9, which are not affected by the IZUMO1-JUNO regulatory system, may provide additional regulatory mechanisms12). In fact, analyses using genome editing after 2020 have demonstrated that sperm acrosome associated 6 (SPACA6)13,14), transmembrane 95 (TMEM95)14,15), and fertilization influencing membrane protein (FIMP)16), and sperm-oocyte fusion required 1 (SOF1)14) are all essential for gamete fusion. Although spermatozoa lacking any of these factors commonly have defects in gamete fusion, the detailed molecular mechanism of how these factors are involved in gamete fusion is still unknown.
Meanwhile, we have identified DC-STAMP (dendritic cell-specific transmembrane protein) domains containing (DCST) 1 and 2, which are well conserved between invertebrates and vertebrates17). Spermatozoa from DCST1/2-deficient mice fail to fuse with oocytes, resulting in complete male infertility. Although DCST1/2-deficient spermatozoa have no defects in morphology or motility, when oocytes are retrieved from the oviduct 8 hours after mating, multiple spermatozoa that are unable to fertilize are observed in the perivitelline space between the zona pellucida and oocyte (Fig. 1C). In wild-type spermatozoa, the oocyte membrane proteins CD9 and JUNO assemble in the vicinity of the head of the adherent spermatozoa prior to gamete fusion12), and this phenomenon is also observed in DCST1/2-deficient spermatozoa17). This suggests that in DCST1/2-deficient spermatozoa, the physiological responses of spermatozoa (capacitation, acrosome reaction, zona pellucida binding and passage, sperm adhesion, etc.) until contact with the oocyte are considered to be normal.
In 2020, two studies reported that CRISPR/Cas9-mediated disruption of TMEM95 caused complete sterility in male mice due to gamete fusion failure14,15). However, we recently reported that deletion of the initial methionine codon of the Tmem95 gene is associated with male subfertility and not complete infertility18). This result is contrary to two papers published in 202014,15), and it is still unclear why there is a phenotypic difference. However, since sperm SPACA6, an essential factor for gamete fusion, is completely absent from the mature spermatozoa of fertile TMEM95 and FIMP-deficient male mice, SPACA6 is not required on the sperm surface and is not involved in so-called ligand-receptor interactions18). Rather, SPACA6 may be involved in the maturation of an unknown gamete fusion key factor.
For this to be elucidated, a persistent analysis of the properties of each factor is required. If all the essential components and molecular behavior of the fusion machinery are sufficiently identified, the elucidation of the mechanism of sperm-oocyte fusion will be greatly advanced.
CD9 (cluster of differentiation 9) is a four-transmembrane protein with four transmembrane regions, two extracellular regions of different sizes, and two intracellular regions. CD9-deficient mice have been studied in various fields and their abnormalities have been analyzed in detail. It first became apparent that the female mice exhibited severe infertility because they rarely fused with spermatozoa19-21). On the other hand, direct microinjection of wild-type spermatozoa into the cytoplasm of CD9-deficient oocytes resulted in litters, indicating that CD9-deficient oocytes are capable of normal development20).
Based on studies with various cell types, CD9 is known to interact with various proteins, such as integrins, and forms a dynamic complex termed the “tetraspanin web”22). In particular, oocyte CD9 is strongly associated with CD315 and IGSF823); however, IGSF8 is not functional in the fertilization process24). Using adhesion force measurements, Jegou et al. reported that the oocyte’s strong adhesion activity to the spermatozoon through the tetraspanin web to trigger sperm-egg fusion is impaired in Cd9-null oocytes25).
Oolemma is mainly divided into two areas: the microvilli-devoid region located on the surface of the cortical actin cap region, which anchors meiosis metaphase II chromosomes, and the microvilli-rich region, where the spermatozoon binds to the oocyte. Since microvilli are abnormally formed in CD9-deficient oocytes, CD9 is thought to be involved in the formation of microvilli23). The microvilli are thought to be the site where gamete fusion occurs, linking membrane proteins to the cytoskeleton22). We have newly found that CD9 function surface exclusion of glycosylphosphatidylinositol-anchored proteins (GPI-APs) such as JUNO and CD55 from the cortical actin cap region of oocytes12). Thus, surface compartmentalization by CD9 functions to confine GPI-APs to appropriate gamete fusion sites in the oocyte.
Recently, based on tertiary structural analysis of CD9, it was shown that CD9 has an inverted conical structure and may have the ability to change the curvature of the membrane26). CD9 is also thought to assemble by sensing membrane curvature27). Thus, since CD9 accumulates in the region of sperm-oocyte adhesion, inverted conical CD9 accumulation may increase the curvature of the oolemma and partially protrude its plasma membrane.
The essential processes of gamete fusion in fertilization can now be analyzed in detail at the molecular level. On the other hand, new experimental systems and analytical techniques must be developed to understand the essence of membrane fusion not only in fertilization but also in other membrane fusion. Furthermore, future research should go beyond the analysis of individual molecules to a comprehensive analysis of the membrane fusion system of fertilization.
Funding: This work was supported by JSPS KAKENHI Grant Number JP18H02453, JP22H02635 and JP23K23898.
I declare no competing interests.
