Perfect date—the review of current research into molecular bases of mammalian fertilization

The CatSper is a weakly voltage-gated and pH-sensitive ion channel with a high affinity to divalent cations [8, 9]. This multi-protein complex is composed of four pore-forming subunits CatSper1–4 and five accessory subunits encoded by at least nine genes in mammals [10, 11]. The structure and distribution of its subunits are integral for the channel function [11‐13]. The expression of CatSper proteins is testis-specific with localization confined to the surface of the sperm tail [14]. Genetic evidence shows that the function of CatSper channel proteins is indispensable for male fertility. The knockout mice were sterile despite having normal sperm count and morphology. Their spermatozoa exhibited impaired motility and failed to penetrate ZP [12]. In humans, genetic lesions and altered expression profiles of CatSper genes have been clinically linked to asthenoteratospermia and male infertility [15‐19].

To achieve fertilization, hyperactivated motility must be switched on at the right place and time. The ability of Ca²⁺ to evoke asymmetric flagellar beating is known to be primed by increased pH [20]. But how the capacitation-induced rise of intracellular pH regulates Ca²⁺ influx was unclear. Recently, SLC9C1, a sperm-specific Na/H⁺ exchanger mediating chemoattractant-induced intracellular alkalization, has been identified as a gatekeeper at the pore of the CatSper channel [21]. The elevation of intracellular Ca²⁺ in the sperm tail induces a conformational change of the blocker molecule. Subsequent dissociation of the SLC9C1 opens the CatSper channel for the Ca²⁺ entry [22] (Fig. 1). This finding provides molecular explanation for CatSper sensitivity to pH. In vivo, oviduct lumen pH increases after ovulation thus providing favorable conditions for sperm hyperactivation [23]. Yet, some researchers intuitively searched for a more specific physiological stimuli produced by the female reproductive tract.

Two simultaneously published studies revealed an unprecedented role of sex hormone progesterone as a hyperactivation-inducing factor [24, 25]. This female steroid hormone, secreted by cumulus cells that surround the oocyte after ovulation, is a well-known chemoattractant for mammalian spermatozoa [26]. The nanomolar concentration of progesterone has been shown to dramatically potentiate hyperactivated flagellar movement of human spermatozoa in an alkaline environment. Surprisingly, progesterone-induced hyperactivation does not involve a slow canonical pathway of interaction with nuclear receptor and alteration of gene expression. Instead, the steroid hormone binds to a membrane receptor on the extracellular side of the cell bringing about a CatSper-mediated influx of Ca²⁺. The non-genomic action of progesterone on CatSper is intermediated by α/β hydrolase domain-containing protein 2 (ABHD2) which serves as a progesterone receptor on the sperm membrane. Upon progesterone binding, lipid hydrolase ABHD2 degrades endocannabinoid 2-arachidonoylglycerol (2AG) present on the sperm membrane. The replenishment of AG2 leads to the CatSper opening and triggers sperm hyperactivation (Fig. 1). Since ejaculated human spermatozoa retain a substantial amount of 2AG, progesterone-stimulated removal of 2AG by ABHD2 is required for full activation of CatSper [27]. Similar to progesterone, prostaglandin E1 (PGE1) stimulates the activity of human CatSper, but apparently through a different binding site [24]. Structural similarity of PGE1 with a 2AG tail implies that there might be direct competition between PGE1 and 2AG [27]. Further research is needed to test this hypothesis and provide a robust insight into how PGE1 regulates the function of the CatSper channel. Although both mouse and human CatSper are pH-sensitive, mouse CatSper is not activated by progesterone or prostaglandins [24]. The interspecies differences in expression and function of this ion channel can be attributed to the low degree of sequence identity between orthologs of CatSper units [28]. For this reason, mice “knockout” models cannot be used to investigate and progesterone/PGE1-mediated hyperactivation pathway.

The ligand-sensitive nature of human CatSper activation predisposes human sperms to be modulated by various compounds which bind to the extracellularly accessible site of this channel complex. Physiological stimuli which have been recognized to induce CatSper-dependent Ca²⁺ entry include glycoproteins of ZP, serum albumin, and cyclic nucleotides [29, 30]. The predominant expression in sperm makes the CatSper channel an attractive target for modulation of male fertility [31‐33]. For instance, steroid-like plant terpenoids, lupeol and pristimentin, were identified as potent inhibitors of hyperactivation of capacitated spermatozoa. Both molecules compete with progesterone for its binding site on ABHD2 and can thus act as contraceptive compounds. Interestingly, both testosterone and hydrocortisone were found to antagonize the action of progesterone at physiological concentrations, which may explain why elevated levels of these steroids in the female organism adversely affect fertility [34]. Importantly, a range of structurally diverse molecules including environmental pollutants, endocrine disruptors, odorants, and common drugs have been shown to interact with the CatSper and interfere with hyperactivation signaling (Table 1) [30, 35‐41]. These exogenous chemicals desensitize spermatozoa to physiological CatSper agonists and thus may impair their functional competence. Future studies that would determine safety thresholds for acute and chronic exposure to these substances for menʼs reproductive health are urgently needed. Taken together, the CatSper channel serves as a polymodal sensor that integrates various chemical clues from the surrounding microenvironment and translates them into Ca²⁺ signaling patterns, which, in turn, modulates the amplitude of flagellar beating. The complex regulation of motility might be functionally important for navigation of sperm over long distances to reach the site of fertilization.

The vital role of CatSper-mediated influx of Ca²⁺ from the extracellular fluid is generally recognized. But there is also a piece of good evidence for the functional significance of Ca²⁺ stored in intracellular organelles. Unlike most cells, mature spermatozoa do not contain endoplasmic reticulum, which is a primary Ca²⁺ storage organelle. Potential areas for functional Ca²⁺ store in sperm include acrosome, mitochondria in the midpiece and redundant nuclear envelope in the connecting piece [42, 43]. The exact mechanism of Ca²⁺ mobilization from internal reservoirs remains to be elucidated. In the tentative model, CatSper-mediated elevation of flagellar [Ca²⁺]i spreads forward and stimulates the secondary release of stored Ca²⁺ thus leading to hyperactivation [43, 44]. Apart from the conventional store-mobilizing agonists, vitamin D or peptide hormones kisspeptin and ghrelin might be involved in the mobilization of Ca²⁺ in hyperactivated human sperm [43]. Although the evidence is preliminary, it is tempting to speculate that pharmacological activation of stored Ca²⁺ release might help to bypass the adverse effects on motility caused by impaired function of the CatSper.

CD9 was the first gene identified to have a sex-specific fertility effect. This ubiquitous membrane protein is localized to the oolemma of fertilization-competent eggs and required for normal microvilli morphology and distribution [58‐60]. The observation that the monoclonal anti-CD9 antibody potently inhibits sperm-egg binding in vitro [59] indicated that this surface protein is involved in gamete interaction. Three studies independently reported that female, not male, CD9-null mice exhibited a dramatic reduction of fecundity resulting from the specific failure of membrane fusion [60‐62]. The details of molecular mechanism by which the loss of the CD9 function impairs fertilization remain to be elucidated. Based on the experiments assessing the relationship between CD9 presence and adhesion phenotypes, Jegou et al. proposed a model in which CD9-driven formation of adhesion sites facilitate the tight sperm-egg contact necessary for fusion to take place [63]. Interestingly, CD9-containing vesicles secreted by wild-type oocytes have been reported to restore the fusing ability of the CD9-deficient mouse eggs and translocate CD9-positive membrane fragments onto the sperm head before its attachment to the egg membrane [64, 65]. These findings support the role of CD9 as an organizer of membrane architecture and suggest that not only the oolemma but also sperm membrane rearrangement is required for gamete fusion.

Izumo, named after the Japanese shrine dedicated to marriage, is the key fertilization element expressed on the sperm surface. The antigen was identified through the generation of a monoclonal antibody that effectively blocks fertilization. The importance of this molecule has been demonstrated by knockout experiments. Izumo−/− male mice are completely sterile despite normal mating behavior and sperm production. Mutant spermatozoa can penetrate the ZP but fail to fuse with the oocytes. However, when the fusion step was bypassed by intracytoplasmic sperm injection (ICSI), activation took place and the embryos developed to term [66]. Izumo is expressed on the inner acrosomal membrane and is thus undetectable on the surface of freshly ejaculated spermatozoa. At the time of acrosome reaction, Izumo becomes exposed and diffuses from the acrosomal cap to the equatorial segment of the sperm head where gamete fusion is initiated [66, 67]. Testis-specific serine kinase TSSK6 has been implicated in this translocation process. The inability to properly redistribute Izumo can explain the absence of fusion underlying sterility of Tssk6-null male mice [68, 69]. Izumo was found to be expressed also in human sperm [66, 70] but, to date, there is a paucity of clinical data supporting its involvement in etiology of idiopathic infertility [71, 72]. Although active immunization against Izumo causes contraceptive effect in female mice, the plausibility of targeting Izumo, or another sperm-specific protein, for development of vaccines for human contraception remains controversial and awaits further investigation [73‐75]. The discovery of two fusion-related antigens, a sperm-specific Izumo and oocyte-expressed CD9, stimulated further research into the molecular basis of sperm-egg recognition [58‐60, 64]. Although both Izumo and CD9 are enriched in the adhesion area, in vitro cell binding assays revealed that there is no physical interaction between the two molecules [76, 77]. Therefore, the existence of alternative Izumo-binding factor(s) on the eggʼs surface has been hypothesized [77].

In 2014, Bianchi and colleagues used the recombinant probe containing IZUMO domain to find its receptor on the oocyte plasma membrane. This experiment led to the discovery of the complementary receptor Juno which was named after the ancient Roman goddess of marriage and childbirth. The observation that Juno^−/− oocytes bind but do not incorporate the sperm head provided genetic evidence for the vital role of the Juno receptor in the fusion process [78]. In humans, the incidence of certain sequence variants of the Juno-encoding gene has been related to unexplained fertilization failure [79]. Analysis of the protein expression in unfertilized the metaphase II and in vitro matured human oocytes showed that Juno accumulates to the oolemma in the course of human oocyte maturation [80, 81]. Yet, fluorescent live imaging of good-quality oocytes is needed to characterize Juno dynamics prior to fertilization and explain the discrepancy between the localization patterns immunodetected in fixed and live human samples reported in this study. Of note, two recent studies reported that the exposure of the female mice to widespread environmental pollutants, melamine and benzo[a]pyrene, altered expression and localization of Juno and adversely effected mouse egg fertilizing capacity assessed in vitro [82, 83]. Future studies should address whether the similar risk of reproductive toxicity exists in humans.

The pairing of the sperm-specific protein Izumo and its oocyte-expressed counterpart Juno constitutes an initial step in gamete fusion. High-resolution imaging of ectopically expressed fluorescent proteins has been used to study receptor-ligand interaction in vitro. The results indicated that monomeric Izumo is recognized by Juno and then transferred to an unidentified egg receptor while generating robust intercellular adhesion [66, 77]. Yet, heterologous fusion assays demonstrated that the binding of Izumo and Juno facilitates membrane tethering but it is not sufficient to trigger the union of the two cells [76]. This indicates the requirement of additional factor(s) conferring adhesion complex fusogenic property. There are many open questions concerning the interplay of key molecular players. According to the current model, binding of Izumo and Juno induces accumulation of CD9 in the contact area and thereby promotes CD9-mediated clustering of the membrane proteins that participate in the assembly of putative fusion machinery [76, 78]. The current goal for researchers is to identify the crucial fusion-inducing component of this machinery.

The nature of Izumo-Juno interaction was found to be conserved in several mammalian species including humans [77]. Normally, the ZP establishes interspecies reproductive isolation. But at the absence of the protective layer, certain orthologues of Izumo and Juno proteins may directly interact allowing for cross-species gamete fusion in vitro. In particular, it has been demonstrated that mouse Izumo is capable of inducing adhesion with the human egg membrane [75]. Further, the recombinanat hamster Juno can bind human, mouse, and pig Izumo whereas human Izumo interacts with hamster but not mouse Juno [84]. This data provides a molecular explanation for a long-recognized ability of acrosome-reacted human sperms to penetrate zona-free hamster eggs. The phenomenon of heterologous fusion can be clinically exploited to assess the human sperm fertilizing capacity in vitro [85]. Due to the identification of surface proteins, vital for sperm-egg interaction, it may now be possible to develop better diagnostic assays, sperm selection techniques, and a new generation of contraceptives. It would be also interesting to investigate whether the altered expression of and/or misrecognition of key fertilization receptors contributes to human idiopathic infertility and unexplained fertilization failure.

The membrane block to polyspermy was intensively studied in marine animals. The experiments showed that the attachment of the first sperm induces a rapid shift in the egg membrane potential from negative to positive level [115]. This alteration of the electric charge hinders other sperm attachment to the oocyte surface. This observation led to the conclusion that post-fertilization membrane depolarization represents a fast block to polyspermy. Although commonly referred to as an evolutionarily conserved safeguard response, the contribution of an electrically mediated membrane block is virtually unfounded in mammals, including humans. It is likely that here, the fast membrane depolarization is less important than in invertebrates and amphibians because the female reproductive tract limits arrival of the sperm to the fertilization site and the sperm-to-egg ratio is thus much lower than during external fertilization [116,117]. Besides, the membrane potential overshot is only transient, lasting up to several minutes. To establish an effective barrier to secondary fertilization, the short-acting electrical depolarization must be complemented by a permanent block.

An alternative mechanism for the establishment of a fast membrane block against polyspermy has been proposed. When investigating molecular details of the gamete fusion process in mice, Bianchi and colleagues observed that after the initial Izumo-Juno contact was established, remaining oocyte receptors were shed off the egg membrane within extracellular vesicles [76]. Rapid loss of Juno from the oocyte surface renders the already fertilized egg refractory to additional sperm binding. The authors speculated that the presence of the Juno-containing vesicles in the perivitelline space might also serve as a “decoy” which neutralizes incoming spermatozoa and prevents secondary fertilization. Interestingly, when gamete recognition was circumvented by ICSI, Juno was not removed from the oocyte surface and the egg remained receptive to further sperm [78]. This finding is consistent with previous observations that ICSI does not induce an effective block to polyspermy in mammalian eggs [118, 119]. Studies are being undertaken to provide the evidence that the removal of Juno from eggʼs surface contributes to the establishment of a polyspermy block in humans.

The most studied strategy on how to reduce the incidence of polyspermy is the slow mechanical block at the level of the eggʼs external coat. Once the egg is fertilized, the surrounding ZP becomes impermeable to late-coming sperm. Although molecular details of this process remain unclear, the crucial role of cortical granules (CGs) has been recognized. These membrane-bound secretory organelles are derived from the Golgi apparatus early in development [120] and during oocyte maturation, they progressively translocate towards the periphery in an actin-dependent manner [121]. In the periovulatory egg, the electron-dense CGs are densely aligned underneath the oocyte cortex, hence the name “cortical granules” [120, 122]. The oocyte activation following the first sperm penetration evokes Ca²⁺ oscillations which in turn trigger CG exocytosis, known as a “cortical reaction.” The fusion of CGs with the plasma membrane leads to a burst of their content into the perivitelline space. The released proteins cause a structural modification of ZP that slowly transforms the receptive outer coat of the oocyte to the hardened protective layer of the developing embryo [123]. Post-fertilization aging of the oocyte creates the risk that cortical reaction is triggered spontaneously without fertilization. Premature or partial exocytosis of CGs compromises the effectiveness of the ZP block and contributes to a high incidence of abnormal fertilization in aged oocytes [124].

Until recently, the content of CGs has been largely unknown. The identification of mouse ovastacin has shed light on mechanistic bases of the zona hardening process. This protease is discharged from CGs and cleaves glycoprotein ZP2, a building component of the ZP. The destruction of the primary sperm binding ligand in the zona matrix establishes a definite post-fertilization block to polyspermy. The role of ovastacin was supported by the finding that in ovastacin-deficient female mice, ZP2 remains intact after fertilization and sperm bind even to 2-cell stage embryos [125, 126]. Recent research revealed that the slow block to polyspermy is further regulated on a higher level. It has been shown that, in mouse oocytes, CG exocytosis is prevented by the abundance of a liver-derived plasma protein fetuin [127]. In 2013, Dietzel and colleagues took advantage of targeted gene depletion and mouse transplantation experiments to demonstrate that a member of the fetuin family, fetuin B, controls ovastacin activity. This study concluded that fetuin B is required to specifically inactivate traces of ovastacin constantly leaking from unfertilized oocytes [128]. In other words, the direct inhibition by fetuin-B restrains basal ovastacin activity and keeps the ovulated egg fertilizable. However, once a sperm has penetrated, the oocyteʼs cortical reaction is unleashed, and ovastacin surge will overwhelm the fetuin-B buffer capacity, thereby initiating the zona hardening. The finding that fertilization can be controlled by a liver-derived plasma protein demonstrates extreme complexity of the regulatory circuits orchestrating mammalian fecundity. Based on the mice data, the authors proposed that the addition of exogenous fetuin-B into the oocyte culture media might increase in vitro fertilization (IVF) success in humans [129]. So far, only two clinical studies assessed the association between the level of fetuin-B and fertilization rate in human IVF patients. Both showed that serum fetuin B level can serve as a predictive marker which may help to make an informed decision whether the oocytes should be fertilized by conventional IVF or ICSI that overcomes the ZP barrier [130, 131]. Moreover, it has been reported that pharmacological down-regulation of fetuin-B caused reversible infertility in female mice [132] suggesting that this blood protein might represent a molecular target for the development of novel therapies for short-term modulation of female fertility. Although the current experimental data is very promising, more clinical studies performed by independent research groups are warranted to confirm the role of fetuin-B human reproductive biology.

During the last decade, zinc (Zn) has emerged as another essential element in ensuring monospermic fertilization. The work of Woodruff and colleagues has advanced our knowledge of the role of Zn in mammalian reproduction. In a series of experimental studies, the researchers used a combination of imaging and biochemical tools to study the Zn dynamics in mammalian oocytes before and after fertilization [133‐139]. Selective Zn indicators revealed that intracellular Zn concentration extensively increases during oocyte maturation and its localization tracks show similar pattern to CGs staining. Accumulation of Zn is a physiological imperative to avoid a premature exit from meiosis and sustain metaphase II arrest. On the contrary, the rapid loss of Zn at the time of fertilization is required for the egg-to-embryo transition [134, 137]. Explosive Zn efflux immediately following sperm entry has been visualized by a vital fluorogenic zinc probe [136, 137]. Due to the characteristic radial glare pattern of an activated egg, this phenomenon has gained the name a “zinc spark.” Apart from being an early hallmark of successful fertilization, the serial exocytosis of Zn-containing vesicles contributes to the ZP hardening. The released metal ions execute their role differently than proteases stored in the CGs. Instead of cleaving glycoproteins of the ZP, exposure to Zn alters the architecture of the eggʼs outer layer by stabilizing glycoprotein chains and bundling fibril components of the extracellular matrix. Conformational change turns ZP into a stiff mechanical barrier that obstructs further sperm access to the already fertilized egg [138]. The massive increase of extracellular Zn concentration establishes a “zinc shield” which could derail Zn signaling in late-coming spermatozoa thus adding a further component to anti-polyspermy defense [140].

The fertilization-induced zinc spark appears to be evolutionary conserved across mammalian species [133, 136, 137]. In mice, the amount of Zn released at the time of fertilization correlates with successful embryo development to the blastocyst stage [136]. This finding raises the intriguing option to develop the platform to quantify the zinc spark in humans. Non-invasive measurement of extracellular Zn in the culture medium would enable prospective selection of the best quality embryos for fertility treatment. Besides, the early detection of activation failure in oocytes conventionally inseminated in vitro would leave the opportunity to perform rescue ICSI during routine working hours. This approach would aid in the prevention of the overuse of ICSI in clinical practice. Furthermore, experimental treatment with Zn-specific chelator demonstrated that reduction of intracellular Zn is sufficient to induce human egg activation [137]. As shown in the porcine system, this kind of treatment may increase the efficacy of AOA, particularly in calcium ionophore-resistant cases [141, 142]. New findings regarding the Zn role in reproduction should be taken into consideration in search of optimal conditions for in vitro maturation (IVM) of human oocytes. It has been shown that supplementation of IVM media with adequate Zn concentration improved maturation and developmental potential of porcine oocytes. It would be interesting to examine if such treatment could also enhance cytoplasmic maturation and fertilization rate of oocytes in human IVM cycles [143].

There are still many unanswered questions regarding strategies mammalian eggs use to reduce the risk of multiple sperm penetration. Future studies might bring new revelations showing that the process is more complex than previously acknowledged. Yet, the importance of the polyspermy block in mammals is being disputed by some researchers who are raising concerns regarding the relevance of research data from in vitro inseminations. They point out that, in vivo, the female reproduction tract greatly limits the number of sperm reaching the fertilization site, thus reducing the risk of simultaneous fertilization. But, under laboratory conditions, high rates of polyspermy are artificially induced by increasing the sperm concentration [144]. Therefore, the experiments involving oocytes deprived of their vestments or exposed to an unnaturally high number of spermatozoa might be unrepresentative for the physiological situation and must be interpreted with caution [117].

Although most embryos inheriting an extra set of chromosomes die during the cleavage stage, some can give rise to a viable blastocyst and survive into gestation. Based on cytogenetic studies performed on spontaneous abortions, polyspermy is estimated to account for 2–3% of natural pregnancies [114, 145‐148]. However, the actual incidence of this phenomenon might be underreported since, in vivo, arrest in preimplantation development, implantation failure, and early pregnancy loss goes unnoticed. Interestingly, some cases of atypical twinning have been attributed to the dispermic fertilization [149‐151]. Detailed genotyping implicated the double sperm penetration in the case of live-born monochorionic but sex-discordant twins. The pair shared 78% of their parental genome while maternally derived genetic information was identical. This “sesquizygosity” is thought to arise from heterogoneic assortment of paternal genomes following fertilization of a single egg by two spermatozoa [151].