Introduction
The life cycle in mammals commences with the formation of a fertilized zygote resulting from the union of healthy spermatozoa and an oocyte. The oocyte is one of the largest cells in mammals. The oocyte itself is surrounded by a membrane referred to as the oolemma and is protected by a glycoprotein matrix, named the Zona Pellucida (ZP). The structure of spermatozoa is distinctive, featuring three areas: the head, the midpiece, and the tail. The head contains the nucleus with paternal DNA and an organelle named the acrosome, located above the nucleus. The midpiece houses mitochondria that produce chemical energy, which the tail converts to propel the spermatozoa through the female reproductive tract. Remarkably, this complex and precisely composed process of fertilization involves numerous critical stages. Each stage must occur correctly; otherwise, it can lead to implantation failure and miscarriage. Biologists have been intrigued by fertilization for hundreds of years, and the advancement of IVF methods in mammals has allowed for a comprehensive cellular analysis of spermatozoa and oocyte interactions. Based on these microscopic observations, fertilization can be conceptually divided into four consecutive stages: (i) capacitation: after ovulation, luteal cells release a chemical inducer, such as progesterone (Ramal-Sanchez et al., Reference Ramal-Sanchez, Bernabo, Tsikis, Blache, Labas, Druart and Saint-Dizier2020; Sharif et al., Reference Sharif, Kerns, Sutovsky, Bovin and Miller2021) to attract spermatozoa and activate the cation channel of spermatozoa (Lin et al., Reference Lin, Ke, Zhang, Yan and Wu2021; Prajapati et al., Reference Prajapati, Kane, McBrinn, Dean, Martins da Silva and Brown2022). This activation enables the spermatozoa to fertilize (Prajapati et al., Reference Prajapati, Kane, McBrinn, Dean, Martins da Silva and Brown2022); (ii) acrosome reaction: the acrosomal membrane combines with the spermatozoa plasma membrane, leading to the release of enzymes and cell surface ligands that were stored inside the spermatozoa head (Hirohashi & Yanagimachi, Reference Hirohashi and Yanagimachi2018); (iii) ZP penetration: the spermatozoa must penetrate the ZP to enter the perivitelline space, a significant gap located between the oocyte and the ZP; (iv) spermatozoa-oocyte interaction: spermatozoa must recognize and adhere to the oolemma, which is then followed by the fusion of the spermatozoa and oocyte membranes; spermatozoa entry into the oocyte triggers significant changes, facilitating the oocyte to transform into a developmentally competent zygote, with the second polar body extruded, a process known as oocyte activation (Krauchunas & Wolfner, Reference Krauchunas and Wolfner2013). Meanwhile, the fertilized zygote must ultimately prevent further spermatozoa fusions to avoid the creation of a nonviable polyploid embryo. Totally, anything that prevents the spermatozoa from changing properly during these processed mentioned above (i-iv) can lead to fertilization failure.
At present, infertility remains a significant challenge for human beings. What key factors are responsible for infertility? Gametogenesis, fertilization, and early embryonic development is the initiating events of life. Fertilization, in particular, is a key process in all sexually reproducing species. What crucial regulatory mechanisms are involved in fertilization? These interesting questions have been extensively studied by scientists across various disciplines. Fortunately, improper membrane fusion of gametes has been demonstrated to be one of the mechanisms of TFF (Enoiu et al., Reference Enoiu, Nygaard, Bungum, Ziebe, Petersen and Almstrup2022). Moreover, a growing number of studies have confirmed many sperm proteins (Roberts, Reference Roberts2005) are interacting in fertilization. However, there is no systematic summary of sperm protein in mammal fertilization up date. So this article mainly focus on sperm protein and it will provide a comprehensive overview of the role of sperm proteins in fertilization, which would provide a comprehensive understanding of the role of sperm proteins in fertilization in mammals and a reference for future studies in reproduction.
Sperm protein in spermatozoa-oocyte adhesion and fusion
Gamete adhesion and fusion is the process by which the spermatozoa penetrate the ZP of the oocyte and enters the perivitelline space after the acrosome reaction. During this reaction, the spermatozoa adhere to and fuse with the plasma membrane of the oocyte. Currently, it is believed that the acrosome reaction leads to the remodelling of the spermatozoa surface structure (Stein et al., Reference Stein, Primakoff and Myles2004). Some membrane proteins are relocated to other membrane regions, and this membrane change is necessary for gamete fusion (Primakoff & Myles, Reference Primakoff and Myles2007). Spermatozoa-oocyte adhesion and fusion is a sophisticated procedure involved in various factors, such as sperm proteins, maternal factors and others. Currently, several sperm proteins (see more detail in the following text) have been identified to act at this stage, and understanding the role of these proteins and the mechanisms of their action will contribute to a better comprehension of gamete interactions in fertilization and potential mechanisms of male infertility due to fertilization failure.
Spermatozoa-oocyte adhesion
IZUMO1 (Izumo sperm-oocyte fusion 1)
In 2005, Izumo sperm-oocyte fusion 1 (IZUMO1) is the first sperm protein identified to be essential for spermatozoa-oocyte recognition during fertilization (Inoue et al., Reference Inoue, Ikawa, Isotani and Okabe2005). The results proved that IZUMO1 is s a novel immunoglobulin superfamily protein and Izumo-/- mice were infertile. Although their spermatozoa appeared normal, binding to and penetrating the zona pellucida, but they couldn’t fuse with oocytes (Inoue et al., Reference Inoue, Ikawa, Isotani and Okabe2005). Moreover, the presence of anti-IZUMO1 antibody in the fertilization medium greatly impeded spermatozoa penetration into oocytes in vitro in pigs (Tanihara et al., Reference Tanihara, Nakai, Men, Kato, Kaneko, Noguchi and Kikuchi2014). Several other studies also indicate Izumo protein might serve as an exciting candidate for the development of antisperm contraceptive vaccines (An et al., Reference An, Huang, Wang, Xie, Ma and Chen2009; Naz, Reference Naz2009, Reference Naz2014; D. G. Wang et al., Reference Wang, Huang, Xie and An2008; Xue et al., Reference Xue, Wang, Liu, Tang, Xu and Xu2016). As its crucial role in reproductive medicine, a series of studies focused on IZUMO1 were performed to know its molecular architecture and the detail of its functional role in fertilization. An ellipsoidal helix dimer structure as the putative functional site (IZUMO1PFF) of IZUMO1 was first reported in mouse. The defective helical formation in the IZUMO1PFF mutant led to a loss of oocyte-binding and fusion inhibition, suggesting that the N-terminal domain is necessary for IZUMO’s functionality (Inoue et al., Reference Inoue, Hamada, Kamikubo, Hirata, Kataoka, Yamamoto and Hagihara2013). Next, the molecular architecture of IZUMO1 in human is also determined. The results indicated that IZUMO1 is a stable monomeric protein with a diverse combination of α and β secondary structures. IZUMO1 is mainly composed of two functional structural domains: a four-helix bundle at the N-terminal (4HB, α1, α2, α3 and α4) and an immunoglobulin-like structural domain at the C-terminal, also known as the IZUMO1 domain and the lg-like domain. The 4HB and Ig-like domains are connected by two antiparallel β-strands. These two antiparallel β-chains (β-hairpin) act as a hinge link (a device used to connect two objects and allow relative rotation between them) for the two domains (Saito et al., Reference Saito, Wada and Inoue2019). Moreover, this molecular structure is conversed in almost all IZUMO orthologues and other IZUMO family proteins (Halil Aydin et al., Reference Aydin, Sultana, Li, Thavalingam and Lee2016). Up to this point, the molecular structure of IZUMO1 is unambiguously solved, providing insights into the molecular basis of spermatozoa-oocyte recognition and cross-species fertilization.
The location of IZUMO1 is certainly a critical part during spermatozoa-oocyte adhesion. In fresh spermatozoa, IZUMO1 is not exposed on the surface, being localized in the anterior region of the spermatozoa in many species including human (H. Aydin et al., Reference Aydin, Sultana, Li, Thavalingam and Lee2016). After the acrosome reaction, IZUMO1 is located near the equator of spermatozoa and recognizes its receptor on the plasma membrane of oocytes. However, it was found that Tssk6 (testicular-specific serine kinase 6)-null spermatozoa fails to relocate IZUMO1 to the region near the equator of spermatozoa during the acrosome reaction, leading to fusion defects with oocytes in vitro and fertilization failure (Sosnik et al., Reference Sosnik, Miranda, Spiridonov, Yoon, Fissore, Johnson and Visconti2009).
The search and study of sperm proteins that interact with IZUMO1, as well as other receptors that play a role in adhesion and fusion, is more desirable as this represents the existence of a complex network that enables spermatozoa-oocyte adhesion and fusion. It is worth noting that CD9 is a protein expressed widely across the body and is anticipated to have functions in different areas. Moreover, CD9 was the first fusion-associated protein found to be indispensable in animals with manipulated genes. In 2000, three independent research teams simultaneously and unexpectedly discovered that the protein CD9 on oocyte is rather crucial for spermatozoa-oocyte fusion (Kaji et al., Reference Kaji, Oda, Shikano, Ohnuki, Uematsu, Sakagami and Kudo2000; Le Naour et al., Reference Le Naour, Rubinstein, Jasmin, Prenant and Boucheix2000; Miyado et al., Reference Miyado, Yamada, Yamada, Hasuwa, Nakamura, Ryu and Mekada2000). The results discovered CD9 -/- oocyte failed to fuse with spermatozoa, leading to fertilization failure. However, the defects caused by CD9 -/- oocytes can be rescued by intracytoplasmic spermatozoa injection (Miyado et al., Reference Miyado, Yamada, Yamada, Hasuwa, Nakamura, Ryu and Mekada2000). Yet, there is no direct interaction found between IZUMO1 and CD9, so the identification of the IZUMO1 receptor had been challenging. In 2014, the only IZUMO1 receptor, the JUNO protein, also known as a well-described protein Folate receptor 4 (FR4) (Liang et al., Reference Liang, Moskalenko, Van Roey and Jooss2013; Walker, Reference Walker2007; Yamaguchi et al., Reference Yamaguchi, Hirota, Nagahama, Ohkawa, Takahashi, Nomura and Sakaguchi2007), was discovered in oocytes through the oligomeric extracellular domain of IZUMO1. JUNO-IZUMO1 interactions are believed to be necessary for the adhesion event between spermatozoa and oocyte during fertilization (Bianchi et al., Reference Bianchi, Doe, Goulding and Wright2014). However, an interesting phenomenon emerged: JUNO is enriched at spermatozoa-oocyte junctions before the onset of membrane fusion but rapidly disappears after fusion begins, while a portion of IZUMO1 remains (Bianchi et al., Reference Bianchi, Doe, Goulding and Wright2014; Inoue et al., Reference Inoue, Hagihara, Wright, Suzuki and Wada2015). This suggests that IZUMO1 and JUNO are associated with spermatozoa-oocyte adhesion rather than fusion. Initially, IZUMO1 is recognized by JUNO (Inoue et al., Reference Inoue, Hagihara, Wright, Suzuki and Wada2015) and an additional ligand may serve as a secondary receptor after IZUMO1 binds to JUNO (Inoue et al., Reference Inoue, Hagihara, Wright, Suzuki and Wada2015; Merc et al., Reference Merc, Frolikova and Komrskova2021), facilitating subsequent membrane fusion. Meanwhile, Chalbi et al. proposed that the membrane fusion mechanism is built by inducing the adhesion of IZUMO1 to JUNO, accumulating and triggering CD9 at the fusion site. Both IZUMO1 and CD9 work through their own cis partners to set up the membrane fusion machinery in this process (Chalbi et al., Reference Chalbi, Barraud-Lange, Ravaux, Howan, Rodriguez, Soule and Gourier2014). However, another research reveals the distribution of JUNO and CD9 at the site where spermatozoa docks and attaches remains unaffected, yet a quick focal reduction in GM1, f-actin, annexin 2, and cholesterol occurs (Van Blerkom & Zimmermann, Reference Van Blerkom and Zimmermann2016), implying that JUNO and CD9 are not the factors involved in the earliest steps of fertilization.
In addition, generative cell-specific 1 (GCS1) is a well-established fusogen in flowering plants and protists and a demonstrated fusogen mammalian cells in culture. Notably, recent findings indicated that IZUMO1 was identified to be another fusogen besides GCS1 in mammal (BHK cells), which could play a role independently of JUNO bindings, mediating cell fusion via a viral-like mechanism (Brukman et al., Reference Brukman, Nakajima, Valansi, Flyak, Li, Higashiyama and Podbilewicz2023). Many studies have confirmed the importance of IZUMO1 as an essential sperm protein for spermatozoa-oocyte adhesion, however, more evidence is needed to demonstrate whether IZUMO1 mediates spermatozoa-oocyte fusion and whether it acts in conjunction with other fusion proteins (Brukman et al., Reference Brukman, Nakajima, Valansi, Flyak, Li, Higashiyama and Podbilewicz2023). The study of fugosen HAP2/GCS1 and IZUMO1 has given much information, leading to the belief that there may be a protein complex capable of mediating germ cell fusion in eukaryotic mammals, awaiting discovery.
The relations between IZUMO1 and other sperm proteins are also reported. For example, spermatozoa acrosome associated 6 (SPACA6) was absent in the spermatozoa of IZUMO1 KO mice, but it could be redetected after IZUMO1 supplementation (Inoue et al., Reference Inoue, Hagihara and Wada2021). This finding suggests a potential interaction between IZUMO1 and SPACA6. However, the sperm protein TMEM95, SPACA6 and SOF1 may not directly interact with IZUMO1 during spermatozoa-oocyte adhesion and fusion, because the knockout of TMEM95, SPACA6 and SOF1 did not affect the number and positioning of IZUMO1 in KO spermatozoa (Bianchi & Wright, Reference Bianchi and Wright2020). They likely regulate the dynamic continuity of IZUMO1 and downstream fusion events indirectly or through unknown factors (Lamas-Toranzo et al., Reference Lamas-Toranzo, Hamze, Bianchi, Fernandez-Fuertes, Perez-Cerezales, Laguna-Barraza and Bermejo-Alvarez2020).
In this part, we mainly summarize the molecular structure of IZUMO1, and its dynamic location, function and the interaction with other factors during fertilization, provide a missing link in our knowledge of mammalian gamete adhesion and fusion.
ADAM family
The ADAM protein family consists of more than 35 members that act as cell adhesion molecules and/or proteases (Black & White, Reference Black and White1998; Rocks et al., Reference Rocks, Paulissen, El Hour, Quesada, Crahay, Gueders and Cataldo2008; Seals & Courtneidge, Reference Seals and Courtneidge2003; White, Reference White2003). ADAMs are transmembrane glycoproteins that exist as functional heterodimers, consisting of α and β subunits, in various tissues and species (Bronson et al., Reference Bronson, Fusi, Calzi, Doldi and Ferrari1999; Edwards et al., Reference Edwards, Handsley and Pennington2008). Their multidomain structure is highly conserved (Edwards et al., Reference Edwards, Handsley and Pennington2008). ADAMs that have been associated with fertilization mainly include ADAM1 (fertilin α), ADAM2 (fertilin β), ADAM3, ADAM6, ADAM20, ADAM21 and ADAM24 (Han et al., Reference Han, Choi, Park, Lee, Jin, Kim and Cho2009; Hooft van Huijsduijnen, Reference Hooft van Huijsduijnen1998; E. Kim et al., Reference Kim, Nishimura and Baba2003; T. Kim et al., Reference Kim, Oh, Woo, Choi, Im, Yoo and Cho2006; Nishimura et al., Reference Nishimura, Kim, Nakanishi and Baba2004; Zhu et al., Reference Zhu, Myles and Primakoff2001). A collection of research from the 1990s led to the prevailing notion that fertilin on spermatozoa interact with integrins on the membrane of oocytes (Cho et al., Reference Cho, Bunch, Faure, Goulding, Eddy, Primakoff and Myles1998; D’Cruz, Reference D’Cruz1996; Evans, Reference Evans2012; Fenichel & Durand-Clement, Reference Fenichel and Durand-Clement1998; Snell & White, Reference Snell and White1996). Functional fertilin is a heterodimer of fertilin α and fertilin β. Located on the spermatozoa’s surface, the disintegrin domain of fertilin β facilitates the interaction between spermatozoa and integrins (most often is α6β1) on the oocyte’s membrane after spermatozoa penetrate through the zona pellucida (Almeida et al., Reference Almeida, Huovila, Sutherland, Stephens, Calarco, Shaw and White1995). In ADAM2 or ADAM3 knockout mice, it was found that the expression levels of ADAM4, 5, 6, and 7 in spermatozoa significantly decrease (Han et al., Reference Han, Choi, Park, Lee, Jin, Kim and Cho2009; T. Kim et al., Reference Kim, Oh, Woo, Choi, Im, Yoo and Cho2006). Alternatively, it is recently found that ADAM6-deficient mice can produce healthy spermatozoa, but spermatozoa adhesion is abnormal and cannot migrate from the uterus to the fallopian tubes (Voronina et al., Reference Voronina, Harris, Schmahl, Galligan, Oristian, Zamfirova and Macdonald2019). Moreover, the addition of ADAM7 in spermatozoa facilitates the acquisition of spermatozoa fertilization ability (Oh et al., Reference Oh, Han and Cho2009). Therefore, these ADAMs are interrelated during the process of spermatozoa-oocyte interactions in the form of complexes.
These updated discoveries reveal a sophisticated and fascinating puzzle of gamete adhesion and fusion, which involves multiple proteins evolved for this specific task. Further analysis of the sperm proteins that promote the expression of key players in spermatozoa-oocyte fusion may provide additional interesting perspectives on this unique event. This analysis is followed by an in-depth explanation of the sperm proteins identified in the current study, focusing on their function in spermatozoa-oocyte fusion.
Spermatozoa-oocyte fusion
SPACA6 and DCST1/DCST2 in spermatozoa-oocyte fusion
In 2014, Lorenzetti team demonstrated that the spermatozoa of the mutant mouse strain BART97b (SPACA6 was completely absent in the homozygous BART97b testis) exhibited a normal acrosome reaction capable of passing through the ZP. However, the mice showed infertility and defects in spermatozoa-oocyte fusion (Lorenzetti et al., Reference Lorenzetti, Poirier, Zhao, Overbeek, Harrison and Bishop2014). This gene is a testis-specific gene and encodes a conserved IgSF protein. SPACA6 gene belongs to the immunoglobulin superfamily and shares a similar extracellular domain composition with the spermatozoa-oocyte adhesion essential sperm protein (IZUMO1). SPACA6 features an N-terminal signal peptide, an α-helix domain, an IgSF domain, a transmembrane helix and a C-terminal short cytoplasmic domain (Binner et al., Reference Binner, Kogan, Panser, Schleiffer, Deneke and Pauli2021), so it is easy to assume that SPACA6 also participates in gamete fusion. SPACA6 KO mice exhibited normal copulatory behaviour leading to ejaculation and vaginal plug formation, allowing passage through the ZP, but the ability of spermatozoa-oocyte plasma membrane fusion was deficient (Barbaux et al., Reference Barbaux, Ialy-Radio, Chalbi, Dybal, Homps-Legrand, Do Cruzeiro and Ziyyat2020). Subsequently, transgenic mice expressing SPACA6 rescued the male infertility observed in SPACA6 KO (Noda et al., Reference Noda, Lu, Fujihara, Oura, Koyano, Kobayashi and Ikawa2020). SPACA6 was located on the cap of the acrosome before the acrosome reaction. Following the acrosome reaction, while SPACA6 was similarly transferred to the equatorial segment (Noda et al., Reference Noda, Lu, Fujihara, Oura, Koyano, Kobayashi and Ikawa2020), no evidence of its complex formation with IZUMO1 was observed, although SPACA6 was present in the post-acrosome reaction.
In 2021, the Inoue team discovered that dendrocyte-expressed seven transmembrane protein domain-containing 1 (DCST1) and dendrocyte-expressed seven transmembrane protein domain-containing 2 (DCST2) may play a key role in the fusion of spermatozoa and oocytes. DCST1/DCST2 are evolutionarily conserved in vertebrates and invertebrates (Inoue et al., Reference Inoue, Hagihara and Wada2021). DCST1/2 is a multipass transmembrane protein with six transmembrane helices, and six types of cysteines at the intracellular C-terminal form a non-canonical ring finger domain (T. Noda et al., Reference Noda, Blaha, Fujihara, Gert, Emori, Deneke and Ikawa2022). Male mice lacking Dcst1/2 -/-, Dcst1 -/- and Dcst2 -/-, similar to other spermatozoa fusion protein defects, accumulate in the perifollicular space of oocytes and show many unfertilized oocytes with normal acrosome-reacted spermatozoa displaying normal IZUMO1 localization. These spermatozoa are able to associate with the plasma membrane of the oocyte but are unable to fuse (Inoue et al., Reference Inoue, Hagihara and Wada2021). Interestingly, the expression of SPACA6 decreased significantly after spermatozoa knockout the IZUMO1, SPACA6, TMEM95, FIMP, DCST1/2, DCST1 and DCST2, respectively, while IZUMO1 was retained in all knockdown cases except in spermatozoa from IZUMO1 KO. After supplementing IZUMO1, the expression of SPACA6 was restored (Inoue et al., Reference Inoue, Hagihara and Wada2021; Inoue & Wada, Reference Inoue and Wada2022).
Interestingly, DCST1/2 transgenic mice exhibited preserved fertility, but no recovery of SPACA6 was observed. These results suggest that SPACA6 may not be necessary for the process of spermatozoa-oocyte recognition and fusion; instead, the essential fertility factors may function in mutual coordination, with the stability of SPACA6 potentially regulated by DCST1/2 (Inoue et al., Reference Inoue, Hagihara and Wada2021). This conclusion was further supported in 2022 by the Binner team in a vertebrate animal model (Binner et al., Reference Binner, Kogan, Panser, Schleiffer, Deneke and Pauli2021). Binner found SPACA6 -/- male sterility in zebrafish, which displayed defects in adhesion and fusion with oocytes. Concurrently, the level of DCST2 in SPACA6 -/- spermatozoa significantly decreased. Transgenic SPACA6 partially rescued the fertilization defect and restored DCST2 level to some extent (Binner et al., Reference Binner, Kogan, Panser, Schleiffer, Deneke and Pauli2021). The difference between mice and zebrafish led to a question: is SPACA6 regulated by DCST1/2 or DCST1/2 regulated by SPACA6? Does SPACA5 solely act as a stabilizing factor, or does it also play a role in spermatozoa-oocyte fusion? Conversely, the difference in the process of fertilization defects between mice and zebrafish after SPACA6 knockout may be related to the absence of JUNO on the surface of zebrafish oocytes (Grayson, Reference Grayson2015). Therefore, investigating whether SPACA6 and IZUMO1 combinedly regulate spermatozoa-oocyte adhesion and fusion during zebrafish fertilization warrants deep consideration. The study of the interaction and potential co-regulation between these key factors is crucial for understanding the critical event of the early life fertilization.
TMEM95, SOF1, and FIMP in spermatozoa-oocyte fusion
In 2014, through a genome-wide association study (GWAS), Pausch et al. found that the causal nonsense mutation of gene TMEM95 on chromosome 19 disrupted the ability of spermatozoa to bind to oocyte ZP and plasma membrane of bull, resulting in idiopathic low fertility (Pausch et al., Reference Pausch, Kolle, Wurmser, Schwarzenbacher, Emmerling, Jansen and Fries2014). Furthermore, Zhang et al. found that the conserved transcript TMEM95-SV1 of TMEM95 contains an arc or horseshoe-shaped repetitive C-terminal (LRRCT) leucine-rich domain and a Pfam: IZUMO, which encodes proteins closely related to protein-protein interaction and the acrosome reaction, respectively (S. Zhang et al., Reference Zhang, Cai, Yang, Shi, Pan, Lei and Lan2016). Studies in mice have found that TMEM95-deficient spermatozoa are normal until spermatozoa-oocyte fusion and that the fertilization failure in mice is the result of the failure of spermatozoa-oocyte fusion, resulting in complete male infertility (Lamas-Toranzo et al., Reference Lamas-Toranzo, Hamze, Bianchi, Fernandez-Fuertes, Perez-Cerezales, Laguna-Barraza and Bermejo-Alvarez2020). Studies on human spermatozoa revealed that TMEM95 fuses with the oocyte membrane through its positively charged surface in human spermatozoa (S. Tang et al., Reference Tang, Lu, Skinner, Sanyal, Lishko, Ikawa and Kim2022). However, the finding by the Inoue team in 2022 revealed that the absence of TMEM95 in mice did not lead to complete male infertility (Inoue & Wada, Reference Inoue and Wada2022), suggesting that even in the absence of TMEM95, other proteins may replace TMEM95 in this pathway. Interestingly, TMEM95 also relocates rapidly to the equator after the acrosome reaction, similar to IZUMO1 and SPACA6 (Y. Satouh et al., Reference Satouh, Inoue, Ikawa and Okabe2012). There may be an interaction between them, but studies have shown that TMEM95 does not interact with JUNO or IZUMO1 (Lamas-Toranzo et al., Reference Lamas-Toranzo, Hamze, Bianchi, Fernandez-Fuertes, Perez-Cerezales, Laguna-Barraza and Bermejo-Alvarez2020; Taichi Noda et al., Reference Noda, Blaha, Fujihara, Gert, Emori, Deneke and Ikawa2022).
SOF1 is considered well-conserved in almost all mammals, and its structure contains conserved ‘LLLL’ and ‘CFN (L or S) As’ motifs (Noda et al., Reference Noda, Lu, Fujihara, Oura, Koyano, Kobayashi and Ikawa2020), but the physiological function of this structure is still awaiting exploration. Noda et al. found that SOF1 KO mice could capacitate normally and cross the ZP to bind to the oocyte, but they failed to fuse with the oocyte membrane, resulting in male infertility (Noda et al., Reference Noda, Lu, Fujihara, Oura, Koyano, Kobayashi and Ikawa2020). Mice with transgenic positive SOF1 -/- mated with normal female mice successfully obtained 2PN (pronuclei) embryos, thereby saving the spermatozoa-oocyte fusion event (Noda et al., Reference Noda, Lu, Fujihara, Oura, Koyano, Kobayashi and Ikawa2020).
Fujihara’s team found that the testis-specific gene 4930451I11Rik on mouse chromosome 7 regulates mammalian spermatozoa-oocyte fusion, a process conserved in humans and other mammals. They subsequently renamed 4930451I11Rik to fertilization-affecting membrane protein (FIMP) (Fujihara et al., Reference Fujihara, Lu, Noda, Oji, Larasati, Kojima-Kita and Ikawa2020). Mouse 4930451I11Rik can be divided into two forms: TM (transmembrane) and secreted form, whereas the human homologous protein is exclusively expressed in the TM form (Fujihara et al., Reference Fujihara, Lu, Noda, Oji, Larasati, Kojima-Kita and Ikawa2020). The TM form of FIMP in mice is a transmembrane protein with a coding sequence for transmembrane function and an N-terminal cleavable signal peptide (Fujihara et al., Reference Fujihara, Lu, Noda, Oji, Larasati, Kojima-Kita and Ikawa2020). FIMP -/- male mice were obtained by using the CRISPR/Cas9 system. Although FIMP-TM KO male mice exhibited normal mating and ejaculation abilities and their spermatozoa could penetrate through the ZP, they failed to fuse with the oocytes, leading to a significant decrease in male mice fertility. This infertility was subsequently reversed by expressing a transgene for the TM form of FIMP (Fujihara et al., Reference Fujihara, Lu, Noda, Oji, Larasati, Kojima-Kita and Ikawa2020). Furthermore, co-expression experiments of FIMP with IZUMO1 revealed no evidence of FIMP directly adhering to the plasma membrane of oocytes or directly promoting the binding of IZUMO1 to JUNO on the plasma membrane of oocytes (Fujihara et al., Reference Fujihara, Lu, Noda, Oji, Larasati, Kojima-Kita and Ikawa2020).
Through gene knockout technology, SPACA6, DCST1/2, TMEM95, SOF1 and FIMP are currently considered as sperm proteins that play an important role in spermatozoa-oocyte fusion. While there is evidence linking these newly discovered sperm proteins to mediating spermatozoa-oocyte fusion through IZUMO1 (Inoue et al., Reference Inoue, Hagihara and Wada2021; Noda et al., Reference Noda, Lu, Fujihara, Oura, Koyano, Kobayashi and Ikawa2020), there remains a lack of evidence confirming the mechanism of action between them. This includes whether there is a complete protein interaction or the formation of a large protein complex pathway to facilitate the dynamic, continuous event of spermatozoa-oocyte adhesion and fusion.
Gamete fusion is mediated by a special protein called the fusogen (Brukman et al., Reference Brukman, Li and Podbilewicz2021). It is now generally believed that there may be an unknown gamete fusion protein that can induce cell membrane fusion, similar to the spermatozoa-oocyte fusion protein, HAP2/GCS1, in lower eukaryotes and protozoa (Pinello & Clark, Reference Pinello and Clark2021). Current evidence suggests to identify IZUMO1 and Syncytin-1 as a potential membrane fusogen during gamete interaction (Bjerregaard et al., Reference Bjerregaard, Lemmen, Petersen, Ostrup, Iversen, Almstrup and Ziebe2014; Brukman et al., Reference Brukman, Nakajima, Valansi, Flyak, Li, Higashiyama and Podbilewicz2023). However, these processes involve complex molecular mechanisms for which the other candidate fusogens remain to be explored.
Sperm proteins participate in oocyte activation
The oocyte undergoes meiosis cycle arrest at a specific stage before fertilization. For example, in most invertebrate species, oocyte stagnation occurs in the middle of the first meiosis (also known as MI stage), while in mammals and amphibians, oocyte stagnation primarily occurs at the second meiosis metaphase (MII stage) (Duncan et al., Reference Duncan, Schindler, Schultz, Blengini, Stein, Stricker and Williams2020; Swann & Lai, Reference Swann and Lai2016). Upon spermatozoa-oocyte fusion, the soluble sperm protein PLCζ diffuse into the oocyte, terminate the meiosis stagnation, and trigger oocyte activation (Swann & Lai, Reference Swann and Lai2016). This activation coordinates subsequent cortical granule exocytosis, prevents polyspermia, degrades maternal proteins, initiates translation of new proteins, facilitates meiosis recovery and completion, and leads to the entry into the first mitosis (Rojas et al., Reference Rojas, Hinostroza, Vergara, Pinto-Borguero, Aguilera, Fuentes and Carvacho2021). The continuous process of mammalian oocyte activation involves multiple complex signalling pathways (Heim et al., Reference Heim, Tischer and Mayer2018; Levasseur et al., Reference Levasseur, Thomas, Davies, Higgins and Madgwick2019; Liu et al., Reference Liu, Li, Labbe, Wang, Mao, Cao and Deng2019), making not only the initial step in embryo growth and development but also the pivotal event in the fertilization process. Failure in this process results in fertilization failure and the inability to form a normal embryo. The sperm proteins currently thought to cause oocyte activation are addressed next, as are the mechanisms of action in oocyte activation.
Oocyte activation pathway
Mature oocytes are stalled at mid-second meiosis (MII), and meiotic arrest depends on the maturation-promoting factor (MPF), which comprises cyclin B1 and cyclin-dependent kinase 1 (CDK1/Cdc2) (Daldello et al., Reference Daldello, Luong, Yang, Kuhn and Conti2019). Rapid inactivation of MPF is crucial for ending meiosis in oocytes. Activation of the anaphase-promoting complex/cyclosome (APC/C) and its co-activator Cdc20 disrupts cyclin B (Heim et al., Reference Heim, Tischer and Mayer2018). Concurrently, CaMKII inhibits CDK1 phosphorylation by activating Protein tyrosine kinase Wee1B (Oh et al., Reference Oh, Susor and Conti2011), resulting in the lifting of MII stalling. Calcium-activated CaMKII (Ca2+/CaM-protein kinase II) relieves meiotic arrest by degrading the APC/C repressor EMI2/XErp1 (Carvacho et al., Reference Carvacho, Piesche, Maier and Machaca2018; Heim et al., Reference Heim, Tischer and Mayer2018; Sako et al., Reference Sako, Suzuki, Isoda, Yoshikai, Senoo, Nakajo and Sagata2014). Injection of spermatozoa cytosolic extracts derived from mouse or bovine into mouse MII oocytes will raise calcium ions (T. S. Tang et al., Reference Tang, Dong, Huang and Sun2000), causing calcium alternation for several hours, a common trigger for mammalian oocyte activation that ceases as the pronuclei form which has been confirmed in mouse model (Marangos et al., Reference Marangos, FitzHarris and Carroll2003). Elevated calcium ions activate CaMKII (Kashir et al., Reference Kashir, Ganesh, Jones and Coward2022), and CaMKIIγ phosphorylates EMI2, leading to its destruction via the ubiquitin/proteasome system. In mature oocytes, APC/C inhibition further mediates cyclin B degradation and CDK1 inactivation, initiating meiosis resumption and completion (Backs et al., Reference Backs, Stein, Backs, Duncan, Grueter, McAnally and Olson2010; Bouftas et al., Reference Bouftas, Schneider, Halder, Demmig, Baack, Cladière and Mayer2022; Lara-Gonzalez et al., Reference Lara-Gonzalez, Moyle, Budrewicz, Mendoza-Lopez, Oegema and Desai2019).
Suzuki et al. proposed that when mouse oocytes do not release Ca2+, Zn2+ alone could induce the withdrawal of meiosis (Suzuki et al., Reference Suzuki, Yoshida, Suzuki, Okuda and Perry2010), indicating that, in the process of oocyte activation, in addition to calcium oscillations, ‘zinc sparks’ are also necessary and sufficient to trigger the cell cycle and exit mid-II stagnation (Garner et al., Reference Garner, Hester, Carothers and Diaz2021; Hu et al., Reference Hu, Duncan, Nowakowski, Antipova, Woodruff, O’Halloran and Wolfner2020). EMI2 contains a ZBR (zinc-binding region) at its C-terminal (Kong et al., Reference Kong, Bernhardt, Kim, O’Halloran and Woodruff2012), and current studies have shown that zinc sparks lead to a decrease in intracellular zinc, greatly reducing the level of binding and activation of EMI2. This relieves the inhibition of APC/C, degrades cyclin B, rapidly inactivates MPF, and finally resumes the process of meiosis (Garner et al., Reference Garner, Hester, Carothers and Diaz2021; A. M. Kim et al., Reference Kim, Bernhardt, Kong, Ahn, Vogt, Woodruff and O’Halloran2011; Kong et al., Reference Kong, Bernhardt, Kim, O’Halloran and Woodruff2012). An interesting study revealed that after bPLCζ cRNA enters the oocyte, each rapid fluctuation of calcium in the cytoplasm is followed by a transient change in zinc level (Que et al., Reference Que, Duncan, Lee, Hornick, Vogt, Fissore and Woodruff2019), which validated the importance of PLCζ in the embryo development (Detailed discussions about PLCζ is in the following). Additionally, a retrospective analysis of zinc spark profiles showed that parthenotes and zygotes which developed into blastocysts released more zinc compared with those that did not develop in mouse. Meanwhile, the prospective selection of embryos based on their zinc spark profile led to a significant improvement in developmental outcomes, more than doubling the percentage of embryos that achieved the blastocyst stage (N. Zhang et al., Reference Zhang, Duncan, Que, O’Halloran and Woodruff2016). This view aimed to select the highest quality embryos at a very early stage of development by non-invasive method (zinc spark detection), however, due to its sensitive microscope requirement，this technology has so far only been tested in animals and there are no clinical randomized trials to verify its safety and effectiveness. Nonetheless, we are now in the era of big data. By combining embryonic parameters obtained by non-invasive methods such as embryonic molecular parameters, genetics, morphology, embryo quality and live birth rate, a 4P (predictive, preventive, participative, and personalized) reproductive medicine system (Diaz-Gimeno & Meseguer, Reference Diaz-Gimeno and Meseguer2019) is expected to establish for embryo screening to greatly improve the success rate of ART. Perhaps the widespread clinical use of non-invasive pre-implantation embryo screening technology will be available in the near future.
PLCζ – Phospholipase C zeta as a spermatozoa-oocyte activation factor
The entry of mammalian soluble sperm proteins into the oocyte, leading to oocyte activation, is marked by the characteristic Ca2+ fluctuations induced in the oocyte (Limatola et al., Reference Limatola, Chun and Santella2022; Mehregan et al., Reference Mehregan, Ardestani, Akizawa, Carvacho and Fissore2021; Xu et al., Reference Xu, Yao, Niu, Fan, Ma, Shi and Sun2021). A new chapter in the study of oocyte activation has been opened since 2002 when Saunders’ team identified PLCζ (phospholipase C zeta) as the sperm protein that mediates Ca2+ alternations in mouse oocytes and oocyte activation (Saunders et al., Reference Saunders, Larman, Parrington, Cox, Royse, Blayney and Lai2002). Although PLCζ is the smallest of the mammalian PLCs, it has the typical X and Y catalytic structural domains, flanked by four EF-Hand structural domains at the N terminus and a C2 structural domain present at the C terminus. There is a linkage region between the X and Y catalytic structural domains (Saunders et al., Reference Saunders, Larman, Parrington, Cox, Royse, Blayney and Lai2002). The EF-Hand domain is associated with the sensitivity of PLCζ to calcium fluctuations (Nomikos et al., Reference Nomikos, Blayney, Larman, Campbell, Rossbach, Saunders and Lai2005), while the C2 domain plays a major role in the function of PLCζ; the mutation of C2 domain destroys the calcium alternation in oocytes (Theodoridou et al., Reference Theodoridou, Nomikos, Parthimos, Gonzalez-Garcia, Elgmati, Calver and Lai2013). It has been suggested that the C2 domain may interact with membrane phospholipids containing phosphatidylinositol 4-diphosphate (PIP2), which is responsible for targeting PLCζ to cytoplasmic vesicles (Nomikos et al., Reference Nomikos, Kashir, Swann and Lai2013; Rizo & Sudhof, Reference Rizo and Sudhof1998). X-Y linker region has a critical role in targeting PIP2. The absence of the X-Y linkage region would result in a significant decrease in the enzymatic activity of PLCζ, and the ability of PLCζ to induce cytoplasmic Ca2+ changes in oocytes would be lost (Nomikos, Elgmati, Theodoridou, Calver, et al., Reference Nomikos, Elgmati, Theodoridou, Calver, Nounesis, Swann and Lai2011; Nomikos, Elgmati, Theodoridou, Georgilis, et al., Reference Nomikos, Elgmati, Theodoridou, Georgilis, Gonzalez-Garcia, Nounesis and Lai2011).
After spermatozoa-oocyte plasma membrane fusion, PLCζ diffuses from the spermatozoa head to the cytoplasm of the oocyte, where it is recognized to bind phosphatidylinositol 4-diphosphate (PIP2) and hydrolysing it to produce inositol (1,4,5)-trisphosphate (InsP3). InsP3 can stimulate the opening of inositol (1,4,5)-trisphosphate receptors (InsP3R) at the endoplasmic reticulum, releasing the Ca2+ stored in the endoplasmic reticulum, leading to the Ca2+ changes in the cytoplasm and the activation of oocytes (Sanders et al., Reference Sanders, Ashley, Moon, Woolley and Swann2018; Swann & Lai, Reference Swann and Lai2016). With the rapid advancement of Assisted Reproductive Technology (ART), in vitro fertilization (IVF) and intracytoplasmic spermatozoa injection (ICSI), which enable the fertilization of spermatozoa and oocytes and develop normal and viable embryos in vitro, have helped many infertile couples to conceive. However, total fertilization failure (TFF) still occurs in ICSI and IVF; the main cause of TFF after ICSI is the oocyte activation defect (OAD) (Cheung et al., Reference Cheung, Xie, Parrella, Keating, Rosenwaks and Palermo2020; F. Wang et al., Reference Wang, Zhang, Kong, Li, Zhang, He and Zhu2020; Yan et al., Reference Yan, Fan, Wang, Yan, Li, Ouyang and Lyu2020). Previous studies have shown that Plcz1-/- low-fertility male mice constructed by CRISPR/Cas9 technology have the ability of normal spermatogenesis and spermatozoa-oocyte fusion but are unable to trigger the normal Ca2+ fluctuations of oocytes (Hachem et al., Reference Hachem, Godwin, Ruas, Lee, Ferrer Buitrago, Ardestani and Parrington2017). PLCζ, as an indispensable oocyte-activating protein, is closely linked to OAD resulting from male causes. Mutation in ACTL7A leads to acrosome defect, causing a decrease of PLCZ1 numbers and abnormal location (Xin et al., Reference Xin, Qu, Chen, Zhang, Chen, Tao and Sun2020), thereby contributing to male infertility. Similarly, ACTL9 mutation results in the absence of PLCZ1 in the mutant spermatozoa head (Dai et al., Reference Dai, Zhang, Guo, Zhou, Gu, Zhang and Lin2021), also leading to male infertility. Artificial oocyte activation (AOA) has proven successful in overcoming the embryonic arrest caused by reduced PLCZ1 expression and abnormal localization, enabling ACTL7A/ACTL9 mutant mice to produce offspring (Dai et al., Reference Dai, Zhang, Guo, Zhou, Gu, Zhang and Lin2021; Xin et al., Reference Xin, Qu, Chen, Zhang, Chen, Tao and Sun2020). By evaluating PLCZ1 expression and localization in human spermatozoa and integrating ICSI with artificial oocyte activation technology, personalized AOA management strategies can be developed to enhance fertilization rates, pregnancy outcomes, and live birth rates (Bonte et al., Reference Bonte, Ferrer-Buitrago, Dhaenens, Popovic, Thys, De Croo and Heindryckx2019).
Another oocyte activation factor in controversy – PAWP
Considering that various factors and pathways can induce oocyte activation, and given the low fertility rather than complete infertility of PLCζ KO male mice (Hachem et al., Reference Hachem, Godwin, Ruas, Lee, Ferrer Buitrago, Ardestani and Parrington2017; Y. J. R. Satouh, Reference Satouh2022), implying that other sperm factors may be present or that oocyte factors can participate in oocyte activation. It is currently believed that after the spermatozoa enters the oocyte, it releases sperm-borne oocyte-activating factor (SOAF), which is also one of the main mechanisms of oocyte activation. SOAF can cause changes in membrane potential, leading to the release and oscillation of calcium ions, which in turn leads to the resumption of meiosis and the production of pronuclei (Amdani et al., Reference Amdani, Yeste, Jones and Coward2015). One candidate SOAF at the centre of the debate is PAWP, also known as WBP2NL, which is expressed and assembled in elongated spermatozoa cells (A. Wu et al., Reference Wu, Sutovsky, Xu, van der Spoel, Platt and Oko2007). Its N-terminal is similar to WW domain-binding protein 2, while its C-terminal is proline-rich, containing PPXY consensus binding sites and many unique repetitive motifs like YGXPPXG (A. T. Wu et al., Reference Wu, Sutovsky, Manandhar, Xu, Katayama, Day and Oko2007). Wu identified the post-acrosomal sheath (PAS) WW domain-binding protein (PAWP) on the perinuclear membrane (PT) of mammalian spermatozoa in 2007 (A. T. Wu et al., Reference Wu, Sutovsky, Manandhar, Xu, Katayama, Day and Oko2007). Recombinant PAWP injection induced oocyte activation in pigs, cattle, rhesus monkeys and Xenopus. Aarabi’s team also reported on the role of PAWP in triggering calcium changes and protoplast formation in human and mouse oocytes, suggesting PAWP can be activated by binding to the WWI domain of proteins in oocyte cytoplasm (Aarabi et al., Reference Aarabi, Balakier, Bashar, Moskovtsev, Sutovsky, Librach and Oko2014). Two studies using co-injection of competitive PPGY peptides or anti-recombinant PAWP antibodies prevented oocyte calcium fluctuations, pronucleus formation and fertilization, supporting the crucial role of PAWP in mammalian oocyte activation. PAWP concentrations detected in spermatozoa showed a correlation with fertilization rates after ICSI treatment and normal embryo development (Vasicek et al., Reference Vasicek, Balazi, Svoradova, Vozaf, Dujickova, Makarevich and Chrenek2022), indicating a potential application of PAWP in diagnosing and treating spermatozoa quality and male infertility.
However, Nomikos’ team raised an objection: neither recombinant PAWP protein nor PAWP cRNA injected into mouse oocytes was able to trigger a Ca2+ response in oocytes (Nomikos et al., Reference Nomikos, Sanders, Theodoridou, Kashir, Matthews, Nounesis and Swann2014). Satouh’s team also found that PAWP-deficient mice were still fertile and did not affect oocyte Ca2 + changes in oocytes (Y. Satouh et al., Reference Satouh, Nozawa and Ikawa2015). The contradiction in these conclusions seems to have been addressed in the work of Hamilton (Hamilton et al., Reference Hamilton, Suzuki, Acteau, Shi, Xu, Meinsohn and Oko2018), who showed that WBP2, the ortholog of WBP2NL, shares the same domain and motif as WBP2NL, exhibits a similar testicular development pattern and location and can activate oocytes. When PAWP is competitively inhibited or knocked out, WBP2 may replace the intensifying effect of PAWP in oocyte activation, but further evidence is needed to confirm this.
Until now, many significant achievements have been made; however, numerous questions remain unanswered. Are there any oocyte-activating factors other than WBP2, WBP2NL, and PLCZ1 in spermatozoa? Are these three pathways complement each other? In the absence of WBP2 and WBP2NL, can oocytes rely on PLCZ1 to continue the activation process? Is this mechanism conservative across different species? We require substantial evidence to unveil the secret of oocyte activation.
Conclusion
According to statistics, about 15% of couples worldwide are affected by infertility, with approximately 50% of cases related to male factors (Bardhi & Drakopoulos, Reference Bardhi and Drakopoulos2021; Baskaran et al., Reference Baskaran, Agarwal, Leisegang, Pushparaj, Panner Selvam and Henkel2021). 5%–15% of the couples that are selected for in vitro fertilization (IVF) experience a total fertilization failure (TFF), where no zygotes develop despite oocytes and semen parameters appear to be normal. There may be some variation in this data in different regions of the world, but the overall trend highlights a growing concern regarding male infertility (Baskaran et al., Reference Baskaran, Agarwal, Leisegang, Pushparaj, Panner Selvam and Henkel2021). Despite the advancements in ART, which have aided numerous cases of infertility stemming from spermatozoa abnormalities, there remains a significant incidence of fertilization failure. Consequently, it is of great theoretical and clinical practical value to reveal the mechanisms in fertilization in human. However, human gametes, especially the mature oocytes, are exceedingly valuable precious materials that are scarce and involve ethical issues, so it would be rather hard to conduct these functional experiments in Human. Generally, most of the current knowledge regarding early embryo development mainly comes from mammals. Here, our review offers a comprehensive analysis of the sperm factors crucial in the sequential process of spermatozoa-oocyte adhesion, fusion and oocyte activation in mammals. A hypothetical model of adhesion/fusion gametes and oocyte activation is presented in Figure 1. Nonetheless, several gaps persist in understanding the complete pathway involving various molecules and signalling pathways in the mammalian fertilization process, such as whether a synapse between the spermatozoa and oocyte involved in fertilization (Krauchunas et al., Reference Krauchunas, Marcello and Singson2016; Maniates & Singson, Reference Maniates and Singson2023), which is essential for the successful fertilization and formation and development of the embryo.
Hypothetical model of adhesion/fusion gametes and oocyte activation in the mammal. A. IZUMO1 recognizes and binds with JUNO on the oocyte membrane, causing spermatozoa and oocytes to adhere to each other. So that the sperm protein can interact with the corresponding protein on the oocyte to cause spermatozoa-oocyte fusion. Also, ADAMs on sperm and α6β1 on oocytes are players in spermatozoa–oocyte adhesion. B. Following spermatozoa-oocyte fusion, the oocyte activation factor in spermatozoa diffuses into the ooplasm through the fusion pores produced by spermatozoa-oocyte fusion, PLCZ binds and recognizes phosphatidylinositol(4,5)bisphosphate (PIP2), and hydrolyses it to produce inositol (1,4,5)-trisphosphate (InsP3) and diacylglycerol (DAG). InsP3 binds to InsP3R in the endoplasmic reticulum and stimulates InsP3R opening, releasing Ca 2+ from the endoplasmic reticulum stores and resulting in increased Ca 2+ and calcium oscillation in the cytoplasm. In turn, WBP2NL binds to a protein containing the WWI structural domain in the oocyte cytoplasm, activating oocyte PLC γ (PLCG) to produce InsP3, which causes calcium oscillations. Calcium oscillation triggered both zinc sparks and CaMKII activation, and CaMKII γ and zinc sparks caused a significant decrease in cytoplasmic zinc levels, which greatly reduced the level of EMI2, which activated APC/C. APC/C was inhibited in the metaphase of meiosis of mature oocytes, activated APC/C further mediates the degradation of cyclin B and the inactivation of CDK1, and finally resumes the process of meiosis. ‘?’ is representative of unknown currently.
The mechanisms of interaction or upstream and downstream regulation between spermatozoa-oocyte fusion and oocyte activation remain a mystery. Fertilization is an extremely important event in mammals; spermatozoa and oocyte fusion usually depend on the synergistic effect of several proteins. It is speculated that there may exist several similar signal pathways that play analogous functions during spermatozoa and oocyte fusion. Most current studies have focused on the necessity of a single sperm protein for this process, and comprehensive analyses of multiprotein complexes at the cell surface are currently lacking. Future studies should focus more on the mechanisms involved in sperm protein complexes during spermatozoa-oocyte fusion and whether the localization and expression of other sperm proteins are normal or disturbed during spermatozoa-oocyte fusion when IZUMO1 is knocked out. Is the same knockout of other known spermatozoa-oocyte fusion proteins impactful for other spermatozoa-oocyte fusion protein localization as well as the amount of expression? If there is an effect, validating the existence of a regulatory network between them, does giving a stoichiometric compensation at this time lead to the recovery of the expression of other sperm proteins? Perhaps between the two proteins, it is also possible that several proteins form large protein complexes to mediate one of the important events of fertilization. Are there specific correlations between these sperm proteins that are known to be essential in this process? If one sperm protein is knocked down, is there an effect on the amount of expression of other factors, including whether there is a role between upstream and downstream factors? These research questions, which require a large amount of scientific research and clinical data to verify, are of great importance in improving the accuracy of clinical diagnosis of male factor-induced infertility and in enhancing the safety and efficacy of assisted reproductive technologies.
The definitive method for evaluating a gene’s importance in fertilization involves testing the fertility of an animal that has been genetically modified to lack the gene. Following the rapid advancement in gene knockout and genome editing technologies in recent years (Gemberling et al., Reference Gemberling, Siklenka, Rodriguez, Tonn-Eisinger, Barrera, Liu and Gersbach2021; Miyata et al., Reference Miyata, Castaneda, Fujihara, Yu, Archambeault, Isotani and Matzuk2016), several genes (IZUMO1, JUNO and CD9; see more detail in the above) encoding surface protein have been known to be required for fertilization in mammals. Despite being able to produce spermatozoa and oocyte that seem normal in terms of number, appearance, and behaviour, animals without those surface proteins display a unique phenotype, as fertilization fails during the final adhesion and fusion phase. Identifying the molecules required for fertilization and understanding how they cooperate to ensure spermatozoa-oocyte interaction and oocyte activation could be of great significance. In our review, we characterize the role and molecular mechanisms of sperm proteins in the three key processes of gamete adhesion and fusion and oocyte activation, which would provide directions for the improvement of assisted reproductive technology and save the endangered species whose survival may depend on such approaches, identify molecular abnormalities that could lead to developmental defects in embryos, thereby providing strategies for prevention, contribute to the advancement of superior infertility diagnostic tests and promote the development of new non-hormonal contraceptives potentially.
Acknowledgments
Conceptualization, Xiangrui Meng and Kaiyue Hu; writing – original draft preparation, Kaiyue Hu and Xiangrui Meng; writing – review and editing, Kaiyue Hu, Bo Dong, Yugang Wang, and Kaichen Hu; All authors have read and agreed to the published version of the manuscript.
Funding
This study was supported by the National Natural Science Foundation of China (31870817), the National Key R&D Program of China (2019YFA0110900 and 2019YFA0802200), the Science and Natural Science Foundation of Henan Province (22100026/20), the Science Research Project of Luoyang Maternal and Child Health Hospital (FYJH202408) and Luoyang City Science and Technology Development Plan Project (2401139B).
Competing interests
The authors declare that they have no competing interests.
