The earliest developmental events in several species, including mammals, sea urchin, fish some insects and plants, are largely controlled by the maternal genome (Flach et al., 1982; Laver et al., 2015). Maternal-effect genes are crucial for oogenesis and embryonic development, influencing processes such as oocyte growth and maturation, pronuclear formation, fusion, the establishment of the first cell division, embryonic genome activation and early embryogenesis (Li et al., 2013; Schultz, 1993). The absence of a single, versatile model system limits our ability to fully unravel the complexities of reproductive barriers, making it challenging to translate findings across species and to understand the cellular and molecular performances of these processes in humans.

In the post-genomic era, zebrafish have emerged as an outstanding model for studying maternal-effect genes due to their genetic similarity to humans and the relative ease of genetic manipulation. Numerous maternal-effect genes have been identified in zebrafish that play crucial roles in oocyte maturation, fertilization, and early embryogenesis (Fuentes et al., 2024; Kanagaraj et al., 2014; Li-Villarreal et al., 2015; Mei et al., 2009; Sun et al., 2018). Although zebrafish and humans have different reproductive strategies -zebrafish possesses external fertilization while humans experience internal fertilization-the underlying cellular and molecular mechanisms are remarkably similar. For instance, both zebrafish and human oocytes undergo CGE to modify the extracellular matrix of the cell and generate a barrier to additional sperm entry and protection (Becker and Hart, 1999; Sathananthan et al., 1985). Zebrafish, with 70% genetic homology to human genes, constitute a pivotal model organism for studying the molecular basis and genetics of numerous diseases [Reviewed in (Briggs, 2002; Fuentes et al., 2018)]. This substantial genetic similarity facilitates the extrapolation of findings from zebrafish to humans, offering profound insights into human biology and disease mechanisms. Consequently, zebrafish have become a prominent model for the investigation of several maternal-effect genes regulating CG biology (Table 1), as will be elaborated in the following sections.

As early oocyte growth progresses, CG are synthesized in the Golgi apparatus and accumulate beneath the oocyte’s PM (Trebichalska et al., 2021). Oogenesis in zebrafish is divided into five stages, distinguished by morphocellular features and oocyte size. CGs are synthesized during stage II and translocated to the oocyte cortex in stage III (Selman et al., 1993). These secretory vesicles are filled with enzymes, carbohydrates and proteins. In sea urchin, for example, CG contain a heterogeneous population of molecules, including hyalin, β-1,3-glucanase, glycosaminoglycans, Ca²⁺, and sulfated acid mucopolysacharides (Bal, 1970; Epel et al., 1969; Hylander and Summers, 1982; Schuel et al., 1974; Wessel, 1989; Wessel et al., 1987). In Xenopus laevis, CGs are associated with lectins and Ca²⁺ (Nishihara et al., 1986; Wolf, 1974). In mice, the metalloendoprotease Ovastacin, detected in CGs, is responsible for cleaving a structural glycoprotein ZP2 at its N-terminal portion, which hardens the ZP and prevents polyspermy (Burkart et al., 2012). Ovastacin function is necessary for post-sperm penetration; however, traces of this enzyme seep from unfertilized eggs before fertilization. During this time, its activity is inactivated by fetuin-B, a liver-derived plasma protein [Reviewed in (Stocker et al., 2014)].

CG are initially synthesized as immature secretory vesicles and subsequently mature through fusion with each other (Austin, 1956; Gulyas, 1980). The zebrafish maternal-effect mutant souffle (suf) was isolated from a forward genetic screen. Eggs from mutant females display defects in chorion elevation and PVS formation, indicative of impaired CG maturation (Kanagaraj et al., 2014). The suf gene encodes the zebrafish homolog of SPASTIZIN, which is also mutated in Hereditary Spastic Paraplegia, a neurodegenerative disorder characterized by progressive loss of lower limb motility due to axonopathy of corticospinal upper motor neurons (Blackstone, 2018; Finsterer et al., 2012; Kanagaraj et al., 2014). Post-activation, suf eggs retain CGs, leading to the chorion elevation defect. Further analysis revealed that suf oocytes possesses and increased number of smaller CGs, with Suf/Spastizin colocalized in a luminal microdomain of these vesicles, indicating its role in their formation (Kanagaraj et al., 2014). This accumulation of immature CGs and a delay in their exocytosis suggest that Suf/Spastizin is essential for proper CG maturation during oogenesis. In addition, suf mutant oocytes exhibit an accumulation of VAMP4 and clathrin-coated buds, highlighting Suf/Spastizin’s role in vesicle sorting and maturation. This function is likely mediated through the regulation of Dynamin-dependent fission of clathrin-coated buds, essential for CG maturation (Kanagaraj et al., 2014). Spastizin’s interaction with Beclin-1, a protein required for autophagy, further implicates it in vesicle maturation, with mutations leading to autophagosome accumulation (Sagona et al., 2011; Vantaggiato et al., 2013).

The zebrafish suf mutant highlights the importance of proper vesicle formation and maturation during oogenesis (Figure 1B). As CGs mature, they are poised for the next critical phase: translocation to the oocyte cortex. This event, which massively occur in mid oogenesis, is essential for positioning the CGs for eventual exocytosis following fertilization. Understanding the molecular details of CG maturation will provide a complete view into the orchestration of CG functions during early and late oogenesis.

CGs are synthesized in the oocyte center and then radially translocated to the cortex to effectively release their content into the PVS after fertilization or activation (Figure 1B). In mice, Rab27a has been identified as a key biomarker of CG translocation (Figure 2A). In fact, transgenic mice of the Ashen strain, which lacks functional Rab27a, fail to translocate the CGs to the cortex (Cheeseman et al., 2016). This finding underscores Rab27a’s essential function in CG transport. In mice oocytes, CG trafficking is an actin-dependent but microtubule-independent mechanism powered by myosin Va (Figure 2A) (Cheeseman et al., 2016). Oocytes from Fmn2 ^−/− mice, lacking the actin nucleation factor Fmn2, also display defects in CG translocation; thus confirming the involvement of the actin network in this process (Cheeseman et al., 2016). Rab11a vesicles, responsible for transferrin trafficking in oocytes, have been shown to cooperate in the translocation of CGs to the cortex, with Rab27a vesicles binding to Rab11a to facilitate their movement, dependent of the motor protein myosin Vb (Figure 2A) (Schuh, 2011; Vogt et al., 2019). Another maternal factor, Spire2, has been reported to interact with FMN2 and myosin Va, and it localizes in the oocyte cortex and in Rab11a-positive vesicles. This zinc-finger containing domain Spire2 protein, has a crucial role in oocyte asymmetric division, and actin mesh formation. It has been shown that oocytes treated with Zn²⁺ chelator as TPEN or SpireFull-Zinc^Mut injected, exhibited impairment in cortical and cytoplasmic actin network, highlighting the critical role of Zinc homeostasis in actin organization and, later as Zn²⁺ sparks, during egg activation (Bernhardt et al., 2011; Jo et al., 2019; Lee et al., 2020). In sea urchin oocytes, cytoskeleton depolymerization assays also confirmed that actin microfilaments are responsible for CG translocation to the cortex prior to fertilization (Wessel et al., 2001).

In zebrafish, CG translocation is associated with yolk globules (YGs), which fuse and generate an animal pole-directed cytoplasmic flow moving CGs to the cortex as oogenesis progresses (Figure 2B) (Shamipour et al., 2023). Although no direct association has been made between actin filaments and microtubules in this vesicle movement, CGs are associated with Rab11 at the oocyte surface, and this movement depends on peripheral microtubule asters (Figure 2B). In addition, overexpression of a dominant negative variant of Rab11 in zebrafish oocytes leads to defects in chorion elevation and PVS formation, highlighting Rab11’s essential role in CGE (Shamipour et al., 2023).

Recently, the zebrafish maternal-effect mutant krang was reported (Fuentes et al., 2024). Eggs from homozygous females fail to elevate the chorion and display delayed CGE after egg activation, retaining CGs in the cortex due to altered translocation dynamics (Fuentes et al., 2024). Also, krang transcript localizes with CG in early oogenesis, suggesting a function in their formation and translocation. Remarkably, several maternal-effect genes were also examined over easy (ovy), p33bjta, poached (poac), and black caviar (blac). These genes regulate key processes during oocyte maturation and egg activation in zebrafish, specifically are required for early steps in YG sizing, protein cleavage, independent of nuclear maturation (Fuentes et al., 2024). Analysis of CGs in activated mutant eggs revealed significant abnormalities in size, area, and number, which are correlated with defects in CGE and chorion elevation. Specifically, p33bjta and ovy mutants displayed reduced chorion elevation, with remaining CGs after egg activation, presumably due to defective CG translocation during oogenesis. This finding demonstrates that CG transport and exocytosis are regulated independently, highlighting a complex and parallel coordination of maternal genes functions during oocyte maturation and egg activation.

To gain exocytosis competence, CGs must anchor to the oocyte PM (Figure 1B). Such a competence is defined as the ability to undergo exocytosis in response to an increase in cytoplasmic Ca²⁺ levels (Ducibella et al., 1993). In mammalian oocytes, a subcortical maternal complex (SCMC) has been identified (Figure 2A), involving four maternal proteins: oocyte expressed protein (also known as FLOPED), transducin-like enhancer of Split 6 (TLE6), K-homology (KH) domain containing 3 (also known as FILIA) and the Nod-like receptor (NLR) family pyrin domain containing 5 (also known as MATER) (Li et al., 2008; Yu et al., 2014). Maternal MATER, in particular, is localized in the oocyte cortex beneath the PM, where it is involved in CG anchoring to the membrane by associating with myosin IIA. Loss of function of MATER in oocytes from null mice leads to defective CG accumulation at the PM, impaired exocytosis, and subsequent polyspermy (Vogt et al., 2019). Cortical actin reorganization is essential for CGs to fuse their membranes with the PM, a process linked to myosin IIA (Figure 2A). After exocytosis, actin also plays a role in compensatory endocytosis to retrieve the CG crypts from the egg surface (Becker and Hart, 1996; Becker and Hart, 1999; Sokac et al., 2003).

Despite our current understanding of the role of MATER and a few other proteins in CG anchoring within mammalian oocytes, our knowledge of additional maternal regulators involved in this process remains limited (Figure 1B). The genetic tractability of the zebrafish offers a unique opportunity to discover new factors involved in GC anchoring, in a different vertebrate system. By leveraging the advantages of zebrafish, we can deepen our understanding of the molecular mechanisms underlying CG anchoring and identify novel maternal factors that may be conserved across species.

CGs accumulate in the oocyte cortex until fertilization or egg activation, at which point they release their content into the PVS and harden the ZP to prevent polyspermy in mammals or chorion in fish, by fusing their membrane with the egg PM (Figure 1B). Several zebrafish maternal-effect mutants with altered CGE have been described. For example, the brom bones (brb) mutant eggs present a defect in egg activation, where mutant embryos fail to elevate the chorion due a blocked of CGE. The brb gene encodes HnRNP I, an RNA binding protein that regulates cytoplasmic Ca²⁺ levels after egg activation. The mutant phenotype can be rescued by providing Ca²⁺ or IP₃ to the eggs, indicating the important role of Ca²⁺ signaling in CGE (Mei et al., 2009).

The oocyte cortex is rich in cortical actin, a cytoskeletal network that must reorganize to allow CG release (Becker and Hart, 1996; Li-Villarreal et al., 2015; Mei et al., 2009). Two zebrafish maternal-effect mutants exhibit defects in CGE due to disrupted cytoskeleton dynamics. In the MZdchs1b mutant, embryos show defective chorion elevation due to a delay in CGE (Li-Villarreal et al., 2015). The dchs1b gene encodes Dchs1, an evolutionary conserved cadherin (Clark et al., 1995). This mutant displays abnormal actin and microtubules networks. Drug assays in wild-type embryos revealed that incubation with cytochalasin D, an actin depolymerizing drug, phenocopied the delay in CGE, confirming that this process is actin-dependent but microtubule-independent (Li-Villareal et al., 2015). Another maternal mutant, aura, shows complete embryo lethality and mid reduction in chorion expansion due to delayed CGE and retention of CGs in the egg cortex. aura encodes for Mid1ip11, and this mutant also present alterations cortical actin reorganization (Eno et al., 2016).

The disruptions in Ca²⁺ signaling and cytoskeleton dynamics highlight the importance of properly regulated protein synthesis for successful CG exocytosis. Transcription ceases during oogenesis and resumes only in the early stages of embryo development after zygotic genome activation [Reviewed in (Vastenhouw et al., 2019)]. Therefore, maternal mRNAs are the sole template for protein synthesis, underscoring the need for precise translational control to ensure the correct execution of molecular and cellular events in the time and space for oocyte maturation, fertilization and embryo development (Conti and Kunitomi, 2024; Lorenzo-Orts and Pauli, 2024). The zebrafish yxb1mutant also shows delayed CGE (Sun et al., 2018). The ybx1 gene encodes Ybx1, an RNA-binding protein involved in translational repression, RNA stabilization, and transcriptional regulation (Goodarzi et al., 2015). The absence of Ybx1 in mutant embryos leads to elevated translation, resulting in increased protein production during oogenesis, which may cause defects in CG transport and retarded CGE (Sun et al., 2018).

Rab3A, a GTPase known to control exocytosis in neurons (Schluter et al., 2004), pancreatic and chromaffin cells (Holz et al., 1994; Johannes et al., 1994; Lin et al., 1996; Olszewski et al., 1994; Yaekura et al., 2003), has been proposed as a CG biomarker. It colocalizes with CGs during oocyte maturation in mouse and sea urchin oocytes, and its absence affects CGE (Bello et al., 2016; Conner and Wessel, 1998). Rabphilin-3A, a Rab3A-binding protein with a C2 domain that interacts with Ca²⁺ and phospholipids (Yamaguchi et al., 1993), is also present in the cortical region of mouse oocytes. Due to its localization and interaction with Rab3A, it is proposed that together they regulate Ca²⁺-mediated CGE (Masumoto et al., 1996).

On the other hand, the fusion of secretory vesicles is mediated by SNARE proteins, which are categorized into v-SNAREs (vesicles-associated membrane proteins or VAMPs) and t-SNAREs (Syntaxin and SNAP-25) (Sollner et al., 1993; Weber et al., 1998). These proteins form a trimeric complex known as trans-SNARE, which becomes cis-SNARE after membrane fusion (Jahn and Scheller, 2006). To recycle these proteins for another round of vesicle fusion, the complex must be disassembled by alpha-SNAP and N-ethylmaleimide-sensitive factor (NSF) (de Paola, Bello and Michaut, 2015). Syntaxin2, SNAP23, VAMP1, and Vamp2 are expressed in porcine oocytes (Tsai et al., 2011), and SNAP23 is also expressed in mouse oocytes, where it regulates CGE. Syntaxin4 transcript is expressed in mouse oocytes as well, but its contribution to CGE has not been confirmed (Iwahashi et al., 2003). VAMP1 and VAMP3 are present in the cortical region of mouse oocytes, and both participate in CGE. The action of VAMPs is sensitive to tetanus toxin, which cleaves them and inhibits the fusion of CG membrane to the PM (de Paola et al., 2021).

In the context of CG biology, phenogenetics involves identifying and studying the genetic mutations that affect CG synthesis, maturation, translocation, anchoring, and exocytosis (Figure 1B; Table 1). By examining these genetic variations and their resulting phenotypic effects, we can elucidate the molecular mechanisms that regulate these critical processes in fertilization and early embryo development. This approach contributes in identifying key genes and pathways that ensure proper CG function and prevent polyspermy, which is essential for successful fertilization and embryo viability.

Building on the information obtained from phenogenetics, the study of CG biology not only advances our understanding of fertilization and early embryonic development but also has practical implications for assisted reproductive technology (ART) (Cappa et al., 2018). By characterizing the genes, phenotypes and pathways involved in CG regulation, we can address challenges such as the high incidence of polyspermy in IVF and the suboptimal competence of in vitro matured mammalian oocytes to undergo CG exocytosis. The aim of studying genes involved in CG regulation is to characterize maternal proteins involved in the vertebrate oocyte-to-embryo transition. On the other hand, integrating advanced techniques will deepen our understanding of CG biology and improve ARTs, ultimately enhancing fertility treatments (Figure 3).

For instance, CRISPR/Cas9 gene editing or TRIM-away can be used to create targeted knockouts and knock-ins in model organisms such as zebrafish and mice to study the role of specific genes in CG dynamics (Clift et al., 2017; Clift et al., 2018; Hang et al., 2016; Rezanujjaman et al., 2024). Recently, two zebrafish knockouts that fail to elevate the chorion have been generated by CRISPR/Cas9. The first is prss59.1, a gene upregulated during the induction of ovulation (Klangnurak et al., 2018; Klangnurak and Tokumoto, 2017). The mutation generated a truncated protein of a paralog of trypsin, Prss59.1. Both heterozygous and homozygous embryos display a defect in chorion elevation. Analysis of the chorion by electron microscopy showed that mutant embryos have smaller pores in their chorion compared with wild-type embryos (Rezanujjaman et al., 2024). The other knockout is val-opsin, affecting a gene encoding for VAL-opsin (Hang et al., 2016). The progeny of female knockouts also has a defect in chorion elevation upon activation, a high mortality rate, and a delay in hatching (Hang et al., 2016). Using TRIM-Away technology, it was demonstrated that SNAP23 is critical for CGE in mouse eggs (Mehlmann et al., 2019).

Zebrafish maternal null mutants for larp6a and larp6b were generated using CRISPR/Cas9-and TALEN-based genome editing, respectively (Hau et al., 2020). The La-related proteins (Larps) are a family of evolutionarily conserved RNA binding proteins (Maraia et al., 2017). Eggs from larp6a and larp6a;larp6b double mutant females display a defect in chorion elevation upon egg activation (Hau et al., 2020). Electron microscopy analysis of oocytes and eggs from double mutant females revealed no alterations in CGs; instead, defects were found in chorion formation and composition (Hau et al., 2020). While it is well-established that failure in PVS formation and chorion elevation post-fertilization or activation is a direct consequence of delayed or blocked exocytosis, CGs were not specifically evaluated in these mutants. Further studies are needed to elucidate if and how these genes are involved in CG biology and their broader role in fertility.

Looking ahead, integrating advanced technologies will significantly enhance our understanding of CG biology (Figure 3). Transcriptomics, including RNA-Seq, can reveal genes identities and differential expression patterns during oogenesis and after fertilization, helping identify key regulatory maternal genes of CG behavior (Bogoch et al., 2022; Fuentes et al., 2024; Smith et al., 2022). Single-cell RNA sequencing can uncover the heterogeneity in gene expression among individual oocytes and eggs, identifying subpopulations with distinct CG-related profiles (Hu et al., 2022; Liu et al., 2022). While CG function is largely regulated by maternal factors stored in the oocyte, the transcriptional and epigenetic landscape established during oogenesis plays a pivotal role in determining the availability and regulation of these genetic factors. ChIP-Seq and ATAC-Seq will provide complementary insights into CG biology by identifying the chromatin accessibility and transcription factor binding events that shape the maternal transcriptome involved in CG biogenesis, translocation, and exocytosis. Although transcription ceases upon initiation of oocyte maturation, the chromatin landscape established earlier determines the maternal mRNA and protein repertoire that supports CG biogenesis, translocation, and exocytosis. Thus, these approaches also have the potential to uncover novel regulatory pathways and maternal-effect genes critical for oogenesis progression and egg activation (Bogoch et al., 2022; Hanna et al., 2022; Liu et al., 2022; Yano et al., 2022; Zhang et al., 2020).

High-resolution live-cell microscopy and super-resolution imaging will provide detailed visualization of CG dynamics, including their movement, anchoring, and exocytosis (Fuentes et al., 2024; Morawiec et al., 2024). On the other hand, classical electronic microscopy of eggs from mutants allows the characterization of pattern, size and morphology of CGs giving insights of gene function. Microfluidic devices can recreate the fertilization environment in vitro, allowing for precise control and observation of CG behavior in response to different stimuli (Alias et al., 2021; Wu et al., 2024; Yanez and Camarillo, 2017). Finally, developing engineered living systems could be employed to design and test synthetic constructs that mimic or disrupt CG-related pathways, providing insights into their regulatory mechanisms (Cui et al., 2022; Hilburger et al., 2019). These innovative approaches will open new avenues for research, improving ARTs and broadening our knowledge of reproductive biology (Figure 2).