The cell biology of fertilization: Gamete attachment and fusion

Abstract

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.

Figure 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.

Figure 2.

Structures of cellular factors involved in sperm–egg attachment and fusion. (A) Human IZUMO1 is shaped like a boomerang in an unbound state (Protein Data Bank [PDB] accession no. 5F4T). The 4HB, hinge, and IgSF domains are shown in orange, green, and cyan, respectively. (B) Human JUNO (PDB accession no. 5F4Q) belongs to the folate receptor family. (C) The structure of human IZUMO1–JUNO complex (PDB accession no. 5F4E) reveals that JUNO binds to IZUMO1 via the β-hairpin hinge, four residues from the 4HB domain and two from the IgSF domain. (D) Human CD9 (PDB accession no. 6K4J) adopts a conical shape formed by four transmembrane helices (TM1–TM4; blue) and two extracellular loops (SEL, pink; and LEL, red).

Figure 3.

Current model of sperm–egg attachment and fusion. (A) Acrosome reaction. After the acrosome reaction, IZUMO1 (blue), SPACA6 (purple), and TMEM95 (violet) colocalize to the equatorial regions of sperm. FIMP (pink) appears to function before the acrosome reaction. There are conflicting data on whether or not TMEM95 interacts with IZUMO1. SOF1 (turquoise) is a secreted sperm protein. DCST1 (green) and DCST2 (orange) are transmembrane proteins implicated in regulating the protein stability of SPACA6. (B) Initial attachment. After the sperm reaches the PVS, it attaches to the egg. IZUMO1 is localized on the equatorial segment of acrosome-reacted sperm and its counterpart receptor, JUNO (yellow), on the oocyte membrane. JUNO specifically recognizes and binds to IZUMO1 in a monomeric conformation. IZUMO1 binding to JUNO drives the accumulation of CD9 (pink) at the sperm–egg interface to form a physical anchor that holds the sperm and oocyte membranes in proximity. (C) IZUMO1 multimerization. After the initial IZUMO1–JUNO attachment, the complex undergoes a dimerization event. The trigger for IZUMO1 oligomerization is not fully understood; however, colocalization analysis revealed the presence of PDI (gray) on the sperm surface. JUNO is thought to be shed from the oolemma and into the PVS after fertilization. (D) Fusogen recruitment. The bona fide sperm–egg fusogen remains a mystery. However, data suggest that IZUMO1 forms a scaffold to recruit the gamete fusion complex. The roles of SPACA6, TMEM95, and SOF1 remain unclear, but these proteins likely play roles in fusion. (E) Fusion pore formation. The merger of the egg and sperm membranes requires modulation of the membrane architecture. The fusogen is thought to catalyze the formation of a hemifusion intermediate, which is a stalk-like structure where the outer leaflets of the sperm and egg membrane bilayers mix. Subsequently, the inner bilayer leaflets mix to form the fusion pore. The precise mechanism of this step will require the identification of the sperm–egg fusogen. Created with BioRender.