Gametes surfaces, like other cell surfaces, are covered by a thick dense coat known as the glycocalyx, formed by a complex network of glycoconjugates including glycoproteins and glycolipids representing up to 30 μm for echinoderm oocytes (Cohen and Varki, 2010; Schjoldager et al., 2020). The terminal position of oligosaccharide chains (i.e. glycans) is occupied by a variety of negatively charged acidic monosaccharides, the Sias, all derived from Neu5Ac (N-Acetylneuraminic acid) and Kdn (2-keto-3-deoxynononic acid). Interestingly, these sialylated glycoconjugates are characterized by a high structural and functional diversity, which can be analyzed at different levels (Cohen and Varki, 2010).

A first level of diversification of Sia molecules is due to various substitutions with acetyl, glycolyl, methyl, lactyl, phosphate, or sulfate groups found on Sias hydroxyl or amino groups (Figure 1A) leading to over 50 structurally different Sia derivatives in nature (Schauer, 1982). The most abundant members of the Sia family are Neu5Ac and Neu5Gc (5-N-glycolylneuraminic acid), followed by Kdn and Neu (neuraminic acid) (Figure 1B). However, there are marked differences in the distribution of these Sias in nature and some of these Sias completely disappear from a lineage as for Neu5Gc in human and platypus (Schauer et al., 2009; Chen and Varki, 2010). Although the biological significance of these modified Sia residues is still mostly unknown, it is interesting to note that the most Sia diversity is encountered in invertebrate deuterostomes such as echinoderms (sea urchin and starfish) and the simplest profiles are found in human (Gagneux et al., 2017). The natural occurrence of O-acetylated Sias is more frequently documented than other modified Sias and O-acetylation at C-4, C-7, or C-9 of Sias has been observed in bacteria and eukaryotes including humans (Angata and Varki, 2002; Nguyen et al., 2020). A neuraminic acid molecule may contain one to three O-acetyl residues, but N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2) is the predominant form (Schauer and Kamerling, 2011). As for O-sulfated Sia (Sias) structures, 8-O-sulfation of Neu5Ac and Neu5Gc have been described in sea urchin gametes where they are involved in sperm–egg interaction and in the regulation of sperm motility, but so far almost nothing is known about the occurrence of Sias in vertebrates (Ertunc et al., 2020).

Additional levels of diversity arise from 1) the type of glycosidic α-linkage through which sialic acid moieties are attached to other glycan units, 2) the underlying sugar chains and 3) the type of glycan (Cohen and Varki, 2010). Sias can be linked in α2,3- and in α2,6-linkage to galactose (Gal)-containing glycans or in α2,6-linkage to N-acetylgalactosamine (GalNAc) and N-acetylglucosamine (GlcNAc) residues (Figure 1C). These Sias occur as monosialyl residues at the nonreducing terminal ends of glycan chains of either N- or O-glycoproteins and glycolipids. A very unique feature of Sias is that they can be linked to each other through α2,8 or α2,9-linkages forming oligo- and polySia chains found on a few glycoproteins that are called disialic acid (diSia), trisialic acid (triSia), oligosialic acid (oligoSia), and polysialic acid (polySia) according to their degree of polymerization (DP) (Troy, 1992; Sato, 2013; Sato and Kitajima, 2013). These combinations of Sia structures, modifications, DP, linkages, underlying glycans and glycan types lead to a huge diversity of sialoglycans and sialoglyconjugates constituting the so-called sialome (Varki, 2017; Sato and Kitajima 2021).

A final layer of complexity stems from the various glycosylation sites within a given protein (macroheterogeneity and microheterogeneity), and the three-dimensional organization of sialoglycoconjugates on cell surfaces (Figure 1D). Sialoglycoproteins and gangliosides can assemble into micro-domains on cell surfaces leading to the formation of glyco-cluster (Figure 1E) that could mediate differential biological functional events.

Metabolism of Sias and sialoglycoproteins schematized in Figure 2A divides into several steps diversely localized in the animal cell and a recent phylogenetic analysis reported the double origin of Sias in eukaryotic cells (Petit et al., 2018). An extrinsic Sias source in Metazoa is likely ensured by an intake of Sias from the extracellular space via the lysosomal SLC17A5 transporter also known as sialin associated with neuraminidase 1 (NEU1). The canonical biosynthetic pathway of Sias takes place within the cytosolic compartment of vertebrate cells (Figure 2A). Sias formation is catalyzed by a bifunctional enzyme, glucosamine UDP-GlcNAc-2-epimerase/N-acetylmannosamine kinase (encoded by the human GNE gene), which converts UDP-GlcNAc (uridine diphosphate N-acetylglucosamine) to ManNAc-6-P (N-acetyl- mannosamine 6-phosphate) and UDP (uridine diphosphate). Then, ManNAc-6-P is condensed with phosphoenolpyruvate (PEP) by the Neu5Ac-9-P synthase (NANS) forming N-acetylneuraminate 9-phosphate (Neu5Ac-9-P). Neu5Ac-9-P is then dephosphorylated by the cytosolic N-acetylneuraminate-9-phosphatase (NANP), resulting in the release of Neu5Ac to the cytoplasm. These two enzymes are widely distributed in most eukaryotic lineages (Petit et al., 2018). In prokaryotes, the condensation of ManNAc (N-acetylmannosamine) and PEP is catalyzed by an aldolase. The same pathway can use Man-6-P instead of ManNAc-6-P leading to Kdn.

Subsequent use of Sias depends on their activation to the sugar-nucleotide cytidine monophosphate sialic acid (CMP-sialic acid or CMP-Sia) which is catalyzed by the cytidine monophosphate N-acetylneuraminic acid synthetase or CMP-sialic acid synthetase (CMAS or CSS) in the nucleus of most vertebrate cells (Figure 2A). In Metazoa, orthologous sequences of human CMAS gene were retrieved in the Cnidaria, deuterostomes, and Arthropods (Di et al., 2017; Petit et al., 2018). Interestingly, in the zebrafish Danio rerio, there are two CSS with different substrate specificity and spatial expression one localizing in the nucleus and showing higher specificity towards Neu5Ac and the other localizing to the cytosol and showing higher specificity towards Kdn (Schaper et al., 2012).

Neu5Ac can be transformed into Neu5Gc by hydroxylation of the methyl group in the N-acetyl moiety of the activated CMP-Neu5Ac donor through a reaction catalyzed by cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) (Shaw and Schauer, 1988). Orthologues of the CMAH gene were found in Porifera (Kapli et al., 2021), and chordates including tunicates, lancelets, reptiles, amphibians, most fish, and a number of mammals (Peri et al., 2018). Only a few deuterostome lineages do not have a functional CMAH gene as reported in humans, New World monkeys, ferrets, several bats, the platypus, the perciform fish lates calcarifer (Simakov et al., 2015; Peri et al., 2018), Sauropsids (birds and reptiles), Pinnipedia, and members of Musteloidia (Altman and Gagneux, 2019) that lack Neu5Gc.

The various activated sugar-nucleotide CMP-Sias are then transported from the cytosol to the sialylation site within the Golgi lumen (Lepers et al., 1989; Lepers et al., 1990) by the SLC35A1 antiporter (Figure 2A). SLC35A1-like sequences are found in deuterostomes from cephalochordates to human and in protists further suggesting that it is ancestral in eukaryotes (Petit et al., 2018).

Then sialylation steps and glycosidic linkage formation are carried out by specific α2,3-, α2,6- or α2,8-sialyltransferases confined to the Golgi apparatus that belong to glycosyltransferase family 29 (GT29) according to the Carbohydrate Active EnZymes database (CAZy, http://www.cazy.org) (Lombard et al., 2014) (Figure 2B). Four large families of sialyltransferases were described in Metazoan i.e. the ST6GAL, ST6GALNAC, ST3GAL, and ST8SIA (Harduin-Lepers et al., 2001; Harduin-Lepers et al., 2005; Harduin-Lepers, 2010; Teppa et al., 2016). A burst of novelties was acquired in each family after the two rounds of whole genome duplication (WGD) at the root of vertebrates leading to several subfamilies that were maintained or lost during vertebrate evolution (Figure 3) (Harduin-Lepers et al., 2008; Petit et al., 2010; Petit et al., 2015). An interesting case study is the one of teleost ST8SIAs that shows an extended repertoire in fish mostly shaped by the WGD events and a particular distribution in fish species (Chang et al., 2019; Venuto M. T. et al., 2020). Moreover, sequence-based analysis suggested that molecular evolution of polysialyltransferase-related sequences could account for the extraordinary diversity in polySias encountered in fish (Venuto M. T. et al., 2020). Additional modifications of Sias like O-acetylation are thought to take place in the Golgi apparatus and involve intraluminal O-acetyltransferase and acetyl-CoA transporter proteins (Figures 2B,C).

Sialoglycoconjugates exported to the cell surface function as ligands for sialic acid-binding proteins including selectins, a class of calcium-dependent C-type lectins, and Siglecs. Siglecs are Sia binding immunoglobulin (Ig)-like lectins that belong to the immunoglobulin superfamily and act as transmembrane cell surface immune regulatory receptors predominantly found on hematopoietic cells including B and T cells, natural killer cells, dendritic cells, granulocytes as well as mast cells and macrophages (Varki and Angata, 2006; Crocker et al., 2007). At least 15 human Siglecs are known to be expressed in human tissues which include CD33-related Siglecs and Siglec-1 (Sialoadhesin), Siglec-2 (CD22), Siglec-4 (myelin-associated glycoprotein, MAG), and Siglec-15. Siglec-1, Siglec-2, Siglec-4, and Siglec-15 constitute a group of highly conserved and ancient molecules which could be found in the common ancestor of vertebrates (Bornhöfft et al., 2018). The remaining Siglecs including the CD33-related Siglecs show marked inter-species differences in repertoire (a loss of Siglecs genes was reported in rodent), sequence, and binding preference (Angata, 2006).

Finally, sialidases, also known as neuraminidases (NEU genes) release α-linked Sias from glycoconjugates and polysaccharides. These enzymes are common in Metazoa and also in microorganisms. The human enzymes NEU1, NEU2, NEU3 and NEU4 are classified in the glycoside hydrolase family GH33 of the CAZy classification (Henrissat and Bairoch, 1996). They are specific of sialylated glycoconjugates and show characteristic tissue and cell expression pattern (Monti et al., 2010). Interestingly, NEU1 is found in the lysosome of Metazoa where it is involved in the hydrolysis of exogenous sialyloglycoconjugates (Figure 2D) and recent phylogenetic analyses described NEU1 as the most ancient neuraminidase whereas the clade NEU2/NEU3/NEU4 would have another origin in deuterostomes (Giaccopuzzi et al., 2012; Petit et al., 2018).

Oocytes from non-aquatic vertebrates do not bear PolySias (Simon et al., 2013) but display unique Sias structures compared to aquatic species localized in the vitelline envelope and the zona pellucida. In addition, humans and several mammalian species lack Neu5Gc (Altman and Gagneux, 2019) due to a single exon deletion/mutation in the gene encoding CMAH (Varki, 2017) (Figure 4). The loss of vertebrate-specific Neu5Gc is puzzling and evolutionary scenarios include the proposal for a strong selection by various parasites, but also a selection via females towards paternal Neu5Gc. Humans also have circulating antibodies specifically targeting Neu5Gc which is immunogenic and hypothesized to lead to chronic inflammation and other pathologies (Padler-Karavani et al., 2008). Neu5Gc antibodies do not seem to have a biological function in gametes and reproduction (Altman and Gagneux, 2019), but it cannot be excluded that they could play a role in the female genital tract to avoid interspecies fertilization.

The transparent extracellular ZP surrounding oocytes of non-aquatic vertebrate species is a viscous fibrogranular structure at the border between the oolemma plasma membrane and the inner layer of follicle cells of the cumulus matrix (corona radiata) (Sinowatz et al., 2001). ZP matrix proteins have been described in granulosa cells and are supposedly involved in folliculogenesis (ZP3 in rabbit, mice, and human, and both ZP3 and ZP1 in bovine and porcine (Prasad et al., 2000; Hasegawa et al., 2007; Gook et al., 2008). ZP is also involved in the protection against physical damages as it is a thick elastic matrix composed of ZP proteins assembled into polymers, which hardens upon fertilization after a series of changes that also provide a polyspermy block (Papi et al., 2010; Monné and Jovine, 2011; Fahrenkamp et al., 2020; Wassarman and Litscher, 2021). In some mammals, ZP proteins have been demonstrated to be involved in species-specific recognition and binding (mouse and human ZP2) to the spermatozoa (Avella et al., 2014; Bhakta et al., 2019), and probably play a role in the ZP matrix modification associated to the polyspermy block (mouse and human ZP1 crosslinker of ZP filaments (Greve and Wassarman, 1985; Nishimura et al., 2019). The composition of ZP proteins varies among species. In mammals, ZP is composed of three to eight proteins. In most mammals including humans, ZP is composed of four proteins: ZP1, ZP2/ZPA/gp69/64, ZP3/ZPC/gp41, and ZP4/ZPB, while in mice it is composed of ZP1, ZP2, and ZP3, and in cows, dogs, dolphins, and pigs, ZP2, ZP3, and ZP4. Marsupials from South America have ZP1, ZP2, ZP3-1b, and ZP3-1c or from Australia ZP1, ZP2, ZP3-1a, ZP3-1b, ZP3-1c, ZP4, ZPAX, while monotremes contain ZPY, ZP1 ZP2, ZP3-1a, ZP3-1b, ZP3-2, ZP4, ZPAX (Wassarman and Mortillo, 1991; Lefièvre et al., 2004; Moros-Nicolás et al., 2021). ZP is also composed of acidic glycoproteins with N-linked and O-linked carbohydrate chains and various sialylated oligosaccharides (Noguchi et al., 1992; Nakano and Yonezawa, 2001; Dell et al., 2003; Pang et al., 2007; Velasquez et al., 2007), (Figure 4). In mammals, sialylated ZP has also been proposed to act in immune recognition as oocytes lack major histocompatibility (MHC) class I molecules that mediate self-recognition, which makes them vulnerable to natural killer cells (Clark, 2010).

In Birds, ZP1, ZP3, and ZPD are the major egg-coat ZP proteins (Nishio et al., 2018). In chicken, ZP2 is concentrated around the germinal disk region (Nishio et al., 2014; Nishio et al., 2018. Mutation of the T168 O-glycosylated site reduces the sperm-binding activity of recombinant chicken ZP3 (Han et al., 2010). The highest concentration of Sias is present in the vitelline membrane, but Sias are also found in chalaza, yolk and white (Nakano et al., 1994).

Chimeric engineered mice that express human ZP3 and acquire mouse-type O-linked glycans deprived of SLe^x structures are capable to bind murine but not human sperm (Dell et al., 2003; Gahlay et al., 2010). In addition, murine oocytes ZP3 deficient in core 1-derived O-glycans (mutated on T-synthase) undergo fertilization (Williams et al., 2007). Mutated mouse oocytes lacking N-glycans (mutated for Mgat1, encoding N-acetylglucosaminyltransferase I) (Shi et al., 2004), or deficient in core 2-derived O-glycans support mouse sperm binding but not human sperm binding compared to wild-type mouse oocytes suggesting a glycan-independent process, and other determinants necessary for taxon-specificity (Hoodbhoy et al., 2005). The respective role of the different oligosaccharide ligands remains to be elucidated as the majority of the O-glycans linked to murine ZP3 are sialylated including core two O-glycans, LacNAc (Galβ1-4GlcNAc), LacdiNAc (GalNAcβ1-4GlcNAc), Galα1-3Gal, and Neu5Acα2-3(GalNAcβ1-4)Galβ1,4GlcNAc (Sd^a antigen), and SLe^x (Dell et al., 2003; Dall'Olio et al., 2014).

In porcine, the major O-linked and N-linked oligosaccharides found in the ZP matrix are bi-antennary complex type glycans with antennae containing sialylated polyLacNAc sequences (Dell et al., 1999; Nakano and Yonezawa, 2001). Glycoprotein ZP2 display six, ZP3 three, and ZP4 five potential N-glycosylation sites, while ZP4 contains three and ZP3 six potential O-glycosylation sites (Yurewicz et al., 1991; Yonezawa et al., 1997). N-glycosylation of porcine ZP glycoproteins seems crucial in sperm penetration and binding to the ZP (Lay et al., 2013). While the role of sialylated glycoproteins was not established, N-glycosylation of ZP3 might increase sperm binding (Lay et al., 2011). Pig Sias are shown to be synthesized by α2,6-sialyltransferases (Kimura et al., 1999).

In bovine, 77% of the carbohydrate chains of ZP glycoproteins are acidic chains containing Sias (Katsumata, 1996), with mainly Neu5Ac (84.5%) and Neu5Gc (15.5%) linked to Galβ1-4GlcNAc and GalNAc with α2,3- and α2,6-linkages, on N- and O-glycans respectively (Velasquez et al., 2007). A ZP3/ZP4 association mediates interaction with the spermatozoa and depends on N-glycans (Suzuki et al., 2015), with ZP4 having the strongest sperm-binding activity (Yonezawa et al., 2001). The N-glycosylation of ZP3 (Suzuki et al., 2015), and the Sias sequence of Neu5Ac (α2-3)Gal (β1-4)GlcNAc (Velasquez et al., 2007) are also implicated in sperm-ZP binding.

In human, mutations in ZP genes encoding ZP1, ZP2, ZP3, and ZP4 are involved in cases of female infertility (Wassarman and Litscher, 2021; Wassarman and Litscher, 2022). Recombinant studies implying glycosylation of human ZP3 revealed this glycoprotein is essential to induce acrosomal exocytosis but not for sperm binding (Chakravarty et al., 2008). Human oocytes ZP N- and O-glycans display unique SLe^x structures that were initially proposed to act in the sperm interaction with the oocyte but later experiments showed they were dispensable (Miranda et al., 1997; Oehninger et al., 1998; Pang et al., 2011; Wassarman, 2011; Clark, 2013; Gupta, 2018; Bhakta et al., 2019; Gupta, 2021; Tumova et al., 2021). ZP mutated mouse oocytes rescued by human ZP2, and deprived of SLe^x in their zona pellucida matrix were shown to bind to human sperm, and ZP2 sperm-binding domain was determined to be required for human sperm-oocyte recognition and zona pellucida penetration (Baibakov et al., 2012; Avella et al., 2014). Transgenic mouse oocytes ZP composed of mouse ZP1, ZP3, and human ZP4 do not bind mouse sperm, and transgenic mouse ZP containing human ZP1, ZP3, and ZP4 do not bind human sperm and are infertile (Avella et al., 2014; Bhakta et al., 2019), supporting ZP2 as necessary and sufficient for human and mouse sperm binding. Genetic removal of ZP2 N-terminus glycan does not impede sperm binding or female fertility (Tokuhiro and Dean, 2018), showing that ZP2 N-terminus is dispensable for gamete recognition (Avella et al., 2014; Tokuhiro and Dean, 2018). Chimeric mouse ZP2 proteins, with a human N-terminal domain, support human sperm binding evidencing that the N-terminus of ZP2 being involved in human species-specificity binding of sperm. (Avella et al., 2014). Morevover, the human sialyltransferase ST3GAL4 gene is present in ovaries but its precise role is not understood (Tanagushi et al., 2003). ZP Sias could mediate other functions related to immune recognition (Clark, 2010). Indeed, SLe^x structures are ligands for Siglec nine tyrosine-based immunoreceptors that can transmit signals to a variety of different types of leukocytes and lymphocytes to allow oocyte survival and block the female immune response (Angata and Varki 2002; Avril et al., 2004).

A clue to understanding this diversity could come from the ZP glycoproteins synthesis study that differs between several vertebrate species. Mammalian ZP glycoproteins are transcribed in human, monkey, rabbit, dog, and cow both by the oocyte and the follicular cells, while in mice the oocyte exclusively contributes to their synthesis (Sinowatz et al., 2001). In birds, ZP1 is from the liver and both ZP3 and ZPD are from follicular granulosa cells (Nishio et al., 2018).

In spawning freshwater and marine organisms, the dilution that reduces the probability of gamete encounters is counteracted by the release of diffusible chemoattractant by the oocyte jelly (Bader, et al., 1998; Olson et al., 2001; Tholl et al., 2011; Guerrero et al., 2020). Once into close contact, the spermatozoa interact with the oocyte.

Sea urchin sperm (Miyata et al., 2004) interacts specifically with the oocyte jelly coat containing polySias and sulfated oligoSias chains inducing the acrosomal reaction (Yeşilyurt et al., 2015). These oligo/polySias act not only in sperm activation but are also involved in a species-dependent recognition by the release of a sperm major species-specific recognition effector called bindin after the contact with the oocyte sperm receptor and the subsequent acrosomal reaction (Lopo et al., 1982; Kitazume et al., 1996; Sato and Kitajima, 2013). The oocyte bindin receptors are aggregated into complexes in lipid rafts and the sperm polySias receptors mediate specific interactions (Hakomori, 2004; Cohen and Varki, 2010). However, the spermatozoa receptor for polyNeu5Gc is not identified and questions remain regarding carbohydrate-based sperm-oocyte recognition. In addition, polySias modify the spermatozoa intracellular pH and have been proposed to be involved in the extension of the acrosomal protrusion formed by actin filaments (Summers and Hylander 1975; Hirohashi et al., 2008).

In amphibians, except for some Urodeles where the sperm is released in the cloaca and stored in a spermatheca (Watanabe and Onitake 2002), fertilization occurs in the external medium. In newt Urodele, spermatozoa are directly inseminated on the oocyte jelly (rich in sialic acid and cations) before motility is triggered. In anuran, sperm motility is instead triggered by the external environmental composition changes and followed by a sperm-oocyte binding involving ZP, Sias and other glycoproteins.

Fish also show diverse reproductive strategies with most oviparous species having external fertilization, and few other zygoparous displaying internal fertilization. In several fishes having external fertilization, glycans can guide the spermatozoa to the oocyte into the micropylar canal playing a role similar to cell surface receptors. In the rainbow trout, S. gairdneri, oocytes express a 500 kDa Kdn-glycoprotein located in their vitelline envelope around the micropyle through which the spermatozoa can penetrate (Kanamori et al., 1990; Tezuka et al., 1994). This Kdn glycoprotein contains complex N-glycans as minor components and is heavily O-glycosylated with α2,8-linked Sias. In the cherry salmon vitelline envelope, the major component is a Neu5Gc mucin-type glycoprotein (Yanagimachi, 2011; Yanagimachi et al., 2017). Other teleost fishes present different sperm entry mechanisms through their micropyle being simply physical by an indented chorion around the micropyle in zebrafish and loach or requiring grooves in goldfish. In the case of flounder, herring, and Alaska pollock, the micropyle is a simple funnel devoid of sialoglycoprotein (Yanagimachi et al., 2017). The spermatozoa attached to the oocyte through the micropyle triggers the cortical alveoli discharge into the perivitelline space leading to PSGP cleavage, and an increase in the osmotic pressure which causes an influx of external water that swells the perivitelline space. This further ensures the chorion hardening and the closure of the micropyle to avoid polyspermy (Mengerink and Vacquier 2001; Gallo and Constantini, 2012). In the teleost Sebastidae blackbelly rosefish Helicolenus dactylopterus, the male possesses a urogenital copulating organ and fertilization occurs in the female genital tract where mucous cells from the papilla secrete sialoglycoproteins, O-linked mucin-type glycans terminated with α2,3-linked Sias, and mannose type N-linked glycans terminated with sialic acid α2,6-linked Sias to galactose/N-acetylgalactosamine, which role remains to be clarified (Accogli et al., 2018).

In mammals, fertilization takes place in the ampulla of the fallopian tubes, and sperm binding to the oocyte occurs in the ZP made of N- and O-glycoproteins (Tecle and Gagneux, 2015). The molecular model proposed to explain sperm-oocyte interaction in mammals has changed through the years, going from ZP2 and ZP3 acting as sperm receptors (Bleil and Wassarman, 1980; Bleil and Wassarman, 1988; Bleil and Wassarman, 1990), to the possibility that sperm interaction with the ZP depends on the supramolecular structure of the oocyte coat rather than a single ZP subunit (Rankin et al., 2003), to a more recent view with one domain of ZP2 playing a central role in gamete recognition by physically interacting with counterpart molecule(s) on sperm (Avella et al., 2014). Currently available data do not fully exclude the role of ZP glycosylation and Sias in fertilization in one of its multistep processes such as the formation of the membrane block to polyspermy, or ZP hardening, but this involvement differs from what was initially suggested and varies between species.

In pig, gamete recognition depends more on N- than O-glycans (Noguchi et al., 1992; Yonezawa et al., 1997), and particularly the main ZP-neutral di-antennary N-glycans (Nakano et al., 1996; Topfer-Petersen, 1999). The N-glycosylation of ZP glycoproteins was shown necessary for sperm-ZP interaction, including sperm binding to ZP as discussed in chapter 3.2 (Lay et al., 2013).

In bovine, Sias of the ZP are physiologically implicated in the binding of the sperm to the ZP (Velasquez et al., 2007).

Mice genetically modified, lacking N- and O-glycans (and without Sias), are fertile as discussed in chapter 3.2, showing sperm binding is independent of N- and O-glycans from the ZP (Thall et al., 1995; Shi et al., 2004; Williams et al., 2007; Raj et al., 2017; Tokuhiro and Dean, 2018). N-glycosylation was shown important for the normal secretion of ZP2 (Roller and Wassarman, 1983).

For human, terminal SLe^x sequences, present on human ZP, are not essential for sperm binding as discussed in chapter 3.2 (Miranda et al., 1997; Oehninger et al., 1998; Pang et al., 2011; Bhakta et al., 2019; Tumova et al., 2021), and requires other molecular effectors (Siu et al., 2021), even if lectin-like and protein–protein interactions were initially shown to play a role in gamete interaction (Clark, 2013). Human ZP2 without SLe^x antigen, expressed in transgenic mice, is sufficient for human sperm binding and penetration in the ZP (Baibakov et al., 2012; Avella et al., 2014). Chimeric ZP2 proteins composed of the human N-terminus instead of the mouse terminal region allow human sperm binding showing a role for this region in species-specificity (Avella et al., 2014; Tokuhiro and Dean, 2018).

Interestingly, in Drosophila, oocytes are surrounded by a thick protective chorionic eggshell that presents a cone-shaped specialized projection, or micropyle, through which the sperm enters at fertilization as in teleosts (Horne-Badovinac, 2020). While Drosophila polySias homopolymers are present during embryonic development, no Sias nor polySias glycoproteins were detected before fertilization in oocytes and spermatozoa (Koles et al., 2004). It is hypothesized that Sias could be unnecessary at fertilization or act in a pre-mating step with a preferential selection of the spermatozoa through transport and storage in the specialized female spermatheca. This selection would build an efficient reproductive barrier by preventing heterospecific fertilization by a sperm selection before gamete encounter (Intra et al., 2009).

In sperm, the outer acrosomal membrane at the apical tip of the head fuse with the plasma membrane allowing exocytosis of the acrosomal contents originating from the spermatid’s Golgi complex and containing a variety of lytic enzymes and ZP-binding proteins including acrosin, hyaluronidase, and sialidases (Srivastava et al., 1988; Yanagimachi, 2011; Khosravi et al., 2016). Acrosome-reacted sperm digest the protective oocyte vestment and bind to the oocyte plasma membrane (Okabe, 2015). Upon contact with the spermatozoa, several vertebrates and invertebrates oocytes exhibit polyspermy-preventing reactions. The membrane block to polyspermy is a rapid reaction that converts the oocyte jelly coat to a form that does not support sperm interaction. Urodeles amphibians, cartilaginous fish, and birds accept polyspermy. The total content of Sias between the vitelline membrane compared to the fertilization membrane does not vary (Wolf et al., 1976). It is not known if this absence of level changes is restricted to some species and if it reflects the sum of sialylation/desialylation processes or is simply a low level of Sias dynamical change.

In sea urchin, as seen in chapter 3.1.2, the acrosomal reaction is potentiated by fucose-sulfated polymers and also requires the oocyte Sia-rich polysaccharides found on the 350 kDa sperm receptor that belongs to the heat shock protein 110 family (Kitazume et al., 1996; Hirohashi et al., 2008; Sato and Kitajima, 2013; Yeşilyurt et al., 2015). The oocyte 350 kDa sperm-binding protein is largely localized in the vitelline layer and lipid rafts and acts for a large part in the acrosome reaction (Maehashi et al., 2003). In sea urchin and non-mammalian species, the membrane block to polyspermy is mediated by the exocytosis of oocyte cortical granules to convert and elevates the oocyte jelly coat. It is accompanied by multiple biochemical changes and by a transient depolarization of the oocyte plasma membrane potential (Jaffe and Cross 1986; Longo et al., 1986; McCulloh and Chambers, 1992).

In mammals, sperm capacitation destabilizes the acrosomal sperm head membrane for penetration in the outer layer of the oocyte in addition to the chemical changes in the tail necessary for greater mobility. Sialylated acrosin released from the sperm acrosome digests the ZP allowing part of the sperm plasma membrane to fuse with the egg plasma membrane, in the boar (Schleuning et al., 1975), or hamsters (Hirose et al., 2020). Acrosin homologs have been identified in quail bird (Sasanami et al., 2011), and Xenopus (Kubo et al., 2010) to bind to the oocyte. However, in mice, acrosin null mutation did not impair fertilization indicating it is not a necessary molecular effector in this process (Wassarman, 2011). The polyspermy-preventing reaction in mammals is composed of complex slow calcium-dependent reactions in the oocyte jelly and plasma membrane, requiring cortical granule exocytosis and ZP conversion (Evans, 2020). The cortical granules exocytosis allows ZP mechanical properties to change from elastic to plastic, also called hardening (Papi et al., 2010; Gupta and Bhandari, 2011; Fahrenkamp et al., 2020). However, to prevent polyspermy mammals’ oocytes have been observed to preferentially use a ZP coat block or a plasma membrane block. In sheep or dog, no sperm is found in the perivitelline space, between the ZP and the plasma membrane, suggesting an effective ZP block while in some other species such as rabbit, or mole sperm is found in the perivitelline space suggesting a plasma membrane block rather than a ZP block (Evans, 2020).

In pig, the N-glycosylation of ZP glycoproteins was shown necessary for the induction of acrosomal exocytosis in the ZP-bound sperm (Lay et al., 2013). The number of oocyte Sias decreases after fertilization (Rath et al., 2005), and after the exocytosis of cortical granules (Sun, 2003). Desialylation of the oocyte decreases sperm acrosome reaction and polyspermy block (Lay et al., 2011).

In bovine, it was hypothesized that neuraminidase released from the cortical granules would participate in the polyspermy block by removing Sias from the ZP (Velasquez et al., 2007). Sias need to be removed from bovine oocyte coat to allow sperm interaction but are required to avoid abnormal fertilization (Fernandez-Fuertes et al., 2018), pointing to a fine-tuned regulation of Sias.

In mice, two sperm sialidases (neuraminidases 1 and 3), shed during capacitation, interfere with ZP binding but are not associated with the polyspermy block (Ma et al., 2012). By using a transgenic mouse line expressing GFP in the acrosome, it was determined that most sperm undergo an acrosome reaction before contacting the ZP in vitro (Hirohashi et al., 2011; Jin et al., 2011). The timing of the acrosome reaction before the ZP binding is flexible, and exocytosed enzymes are dispensable for sperm penetration in the ZP (Okabe, 2015). The majority of sperm has undergone acrosome exocytosis in the upper segments of the oviductal isthmus before the ampulla (La Spina et al., 2016) and the cumulus (Hino et al., 2016; Muro et al., 2016), suggesting that sperm acrosome exocytosis could take place in the female oviduct during sperm migration, and questioning about the role played by Sias in the female reproductive tract.

In human, terminal SLe^x sequences have been involved in the sperm acrosomal reaction (Miranda et al., 1997; Oehninger et al., 1998; Pang et al., 2011; Tumova et al., 2021). Recently a protein bearing terminal SLe^x, C1orf56, localized in the human acrosomal region of the spermatozoa was proposed to act in the ZP-induced acrosome reaction (Wang et al., 2021). In addition, three of the four reported glycodelin (Gd) sialylated glycoproteins (acquired by the sperm in the female genital tract) GdA, GdF, and GdC, but not GdS coating the spermatozoa, are involved in the regulation of the acrosomal reaction. GdF blocks premature acrosomal reaction during Fallopian tube migration. GdF is subsequently converted into GdC by the oocyte cumulus cells when a sperm-oocyte contact is established, removing GdF inhibition (Sawyer, 2021).