In the same work describing for the first time the existence of MPF [9], Masui and Markert also reported that injection of cytoplasm taken from *Xenopus* oocytes arrested in M-II in a blastomere at two-cell stage, blocked division of the injected blastomere; therefore, they supposed the existence of a cytoplasmic factor, present in mature oocytes, able to inhibit cell division [9]. Functional tests of CSF activity have been described from decades [9, 57] and they have been confirmed in model systems of vertebrates (*Xenopus, Rana*) [58-60]. Unlike MPF, the biochemical characterization of CSF has been intensively pursued, but remained elusive. According to recent reviews, the three main features necessary to define CSF activity from a functional point of view are: (i) CSF must appear during oocyte maturation; (ii) CSF must be present during M-II arrest (at least in vertebrates); (iii) CSF must be degraded at the time of fertilization [7, 61-62]. From a molecular point of view, CSF includes Mos kinase, a 39 kDa protein encoded by c-*mos* gene, a cellular proto-oncogene [63]. Mos is a Ser/Thr kinase originally identified as cell homologue of the *v-*mos viral gene product, which is responsible for Moloney's murine sarcoma [64] and is expressed specifically in the ovary and in the testicle [65]. In 1989, it was discovered that Mos was required for the maintenance of CSF activity, as the degradation of the c-*mos* mRNA coincided with the loss of CSF activity [63]. Subsequently, the essential function of Mos has been confirmed in all vertebrate oocytes investigated, where it is responsible for the M-II arrest, operating as an essential component of CSF [5, 63, 66-69]. In the same years, it was demonstrated that Mos was responsible for activation of p42 MAPK during oocyte maturation [70-72]. This suggested that MAPK might be a different modulator of Mos activity, as confirmed later in mammals. In fact, in mos-/- mice, MAPK was not activated in oocytes [73]. Previously, MEK1 dual kinase from the same pathway was identified upstream of MAPK and able to phosphorylate and activate both tyrosine (Tyr-185) and threonine (Thr-183) residues in the regulative loop of MAPK [74-76; 77-78]. In fact, MEK1 injection in immature *Xenopus* oocytes caused the activation of MAPK, while inhibition of MEK1 prevented oocyte maturation induced by progesterone [71]. The phosphorylation cascade is initiated by Mos which activates MEK1 by phosphorylating Ser-218 and Ser-222 residues [71, 79]. Later it was noticed that MAPK phosphorylated ribosomal S6 kinase, p90Rsk [80]. In mammals, three different isoforms p90Rsk have been identified, named Rsk1, Rsk2, Rsk3, while in *Xenopus*, only two, Rsk1 and Rsk2, are present, both active during oocytes maturation [81]. The molecular feature of CSF seems to be defined by the pathway Mos/MEK1/MAPK/p90Rsk (Fig. **3**). During oocytes maturation in *Xenopus*, each component of this pathway is synthesized or activated after progesterone-induced maturation. Furthermore, an active form of each factor is sufficient to induce oocyte maturation [61]. At fertilization, owing to the increase in the cytoplasmic concentration of calcium ions (Ca2+), Mos is degraded and its mRNA is deadenilated, while other components of the CSF pathway are inactivated by dephosphorylation, suggesting that in *Xenopus*, Mos is the only MAPKKK (MAPK kinase kinase) active during oocyte maturation [61]. The regulative pathway activated by Mos, and therefore responsible for the CSF-mediated M-II arrest, operates by inhibiting the activity of Cdc20/p55CDC/Fizzy, an activator of the anaphase-promoting complex or cyclosome APC/C, which degrades cyclin B and inactivates MPF at fertilization [61-62]. Most studies concerning the regulation of Mos, have been focused on the translation of its mRNA and the stability of the protein.  The translational recruitment of c-*mos* and several other mRNAs is regulated by cytoplasmic polyadenylation, a process that requires two 3' untranslated regions, the cytoplasmic polyadenylation element (CPE) and the polyadenylation hexanucleotide AAUAAA. Although the signalling events which trigger c-*mos* mRNA polyadenylation and translation are unclear, they probably involve the activation of CPEB, the CPE binding factor. In response to progesterone, the Eg2/Aurora kinase A phosphorylates CPEB, which regulates c-*mos* mRNA translation [82-83]. This allows an increase in the synthesis of Mos [84]. Some authors believe that the stability of Mos protein is due to phosphorylation of the residue Ser-3 and among the kinases responsible for this phosphorylation event, potential candidates are Cdk1 [85], p42 MAPK [86] and Mos itself [87]. Chen and Cooper [88] have shown that phosphorylation of Ser-3 facilitates interaction of Mos with MEK1, but also promotes its activation [89]. This suggests that Mos is regulated at the level of expression and by post-translational mechanisms, although the Ser/Ala mutation in position 3 does not prevent oocyte maturation or maintenance of CSF [90]. Chen *et al.* also showed that CK2β, the regulatory subunit of CK2 protein kinase, can act as a negative modulator of the Mos activity [91-92]. In 2004, Lieberman and Ruderman demonstrated the interaction between Mos and CK2β identifying the CK2β docking site on Mos sequence [93]. It remains to be defined if the inhibition of Mos by CK2β is constitutive or regulated and how this interaction regulated Mos function during oocyte maturation.

**Figure 3:** Regulation of CSF during maturation and at fertilization (see text for description)

More recently, the activity of CSF has been linked to the control of the spindle assembly, since p90Rsk can phosphorylate Bub1, a Ser/Thr kinase which inhibits APC/C activating components of the spindle checkpoint, such as Mad1, Mad2, BubR1 [61-62, 94]. However, this hypothesis remains largely circumstantial and partially elusive. In fact, the demonstration of a CSF arrest independent of the Mos/MEK1/MAPK/p90Rsk [95] indicated the existence of other activities, independent of the Mos/MAPK pathway. An alternative candidate, has been identified in the Cdk2/Cyclin E [96-98]. More recently, a great importance has been attributed to the APC/C inhibition by Emi/Erp factors. Initially it was thought that the protein Emi1 (*Xenopus* early mitotic inhibitor) was a component of CSF because it is necessary and sufficient to induce CSF activity [99]. However, the belonging of Emi1 to CSF has been questioned, since it has not been detected in CSF-arrested oocytes, nor its timing of degradation is consisted with its potential role in CSF activity [62, 100]. In the same context, more interesting appears the role of Erp1/Emi2, an inhibitor of the APC/C, in the maintenance of CSF [101-102]. Both in *Xenopus* and in mouse [102, 103] Erp1/Emi2 is destroyed at fertilization similarly to Mos [104]. Erp1/Emi2 is a zinc finger protein with an F-box domain in the C-terminal region. F-box proteins are part of the complex SCF (Skp1/cullin/F-box) ubiquitin ligase that, similarly to APC/C, mediates the degradation of cell cycle regulators [62]. In mammals, phylogenetic analysis of protein containing the F-box domain revealed a close relationship between Emi1 and Erp1/Emi2 [105]. Schmidt and colleagues demonstrated that Emi2 is essential for the maintenance of CSF and it is rapidly destroyed at fertilization [102], where the calcium-dependent degradation of Erp1/Emi2 occurs. However, the activity of Erp1/Emi2 required phosphorylation by Plx1 (Polo-like kinase) following Ca2+ increase at fertilization and CaMKII (Ca2+/calmodulindependent protein kinase II) activation [62] which facilitates the phosphorylation-dependent Emi2 recognition by the proteasome machinary [106]. The final evidence linking Mos to Erp1/Emi2 emerged from two parallel papers showing that, in *Xenopus*, p90Rsk directly phosphorylates Erp1/Emi2 on Ser-335/Thr-336 both *in vivo* and *in vitro*  upregulating Erp1/Emi2 stability and activity [107-108].

### **OOCYTE MATURATION IN LOWER VERTEBRATES**

In *Xenopus*, progesterone induces completion of the first meiotic division and maturation in P-I arrested oocytes. Briefly, after GVBD oocytes complete meiosis I emitting the first polar body, enter M-II without resting in interphase and remain blocked in M-II until fertilization [10]. MPF activity oscillates during meiotic maturation: it is high in M-I, decreases during M-I/M-II transition, and increases again in M-II, where remains stable and enzymatically active due to the activity of CSF, which inhibits APC/C-mediated cyclin B degradation and other events leading to M-II exit [22]. In immature oocytes blocked in P-I, MPF is present as pre-MPF, where Cdk1 is associated with cyclin B, but enzymatically inactive since phosphorylated on Tyr-15 and Thr-14 by Myt1/Wee1 kinases [40]. As described above, Cdc25 phosphatase removes these inhibitory phosphorylation and fully activates Cdk1/cyclin B, which in turn phosphorylates Cdc25, causing a rapid amplification of MPF [2, 109]. The activation of Cdc25 is catalyzed by a complex signalling (see above for details details), which includes xPlk1 (*Xenopus* pololike kinase 1) and its upstream activator xPlkk1 (polo-like kinase kinase 1) or homologous kinases in mammals [110-113]. It has been shown that MPF activation induced by progesterone depends upon two mechanisms related to PKA: i. inhibition of PKA activity [114]; and ii. synthesis of new proteins which induce a decrease in the levels of cAMP resulting in decreased activity of PKA. This latter kinase is a potent inhibitor of meiotic maturation and its inhibition is sufficient to induce maturation of *Xenopus* oocytes [115]. A similar response was also described in mice, rats and fish. Inversely, oocytes from pigs, sheep and rabbits exhibit a transient increase, instead of a decrease in cAMP levels [116]; finally, even in the jellyfish, the increase in cAMP can induce oocyte maturation [116]. The link between decrease in cAMP levels and MPF activation loop seems to be the dual PKA-dependent phosphorylation of Wee1 kinase and Cdc25 phosphatase. These events lead to an increase in Cdk1/cyclin B kinase activity and, consequently, oocytes undergo GVBD [41].

Several studies reported the existence of a parallel pathway in MPF activation that involves the synthesis of Mos kinase which indirectly activates p90Rsk (according to a mechanism described above), which in turns phosphorylates and inhibits Myt1 and simultaneously promotes the activation of Cdc25 [2, 114, 117]. Briefly, in addition to the pathway xPlkkl/Pxl/Cdc25C, progesterone may activate a second, independent pathway, the Mos/MEK/MAPK/p90Rsk phosphorylation cascade. p90Rsk phosphorylation of Myt1 may facilitate Cdc25-mediated activation of MPF and entry into meiosis I [40]. Other evidence suggest that Mos can directly phosphorylate Myt1 [118]. The exit from M-I is associated with cyclin B degradation mediated by APC/C complex. Synthesis of new cyclin B will occur before entry into M-II and its stability is maintained by the CSF [95]. At fertilization, the calcium increase induces entry into anaphase and promotes the degradation of Emi2 which causes activation of APC/C and the cyclin B degradation, as described above.

In fishes (*Carassius auratus*) it has been initially reported that the MPF is not present as pre-MPF, but Cdk1 appears as a monomer dissociated by cyclin B which is synthesized only after hormonal stimulation. The complex formed is only activated by phosphorylation of residue Thr-161, as inhibitory phosphorylations on Thr-14 and Tyr-15 residues are lacking by a mechanism which does not require the intervention of the phosphatase Cdc25 [119]. These results have been questioned by a study showing that in teleost fish *Anabas testudineus* a pre-MPF exists and that the transition G2/M induced by Cdk1/cyclin B complex dependents upon Cdc25 activation [120]. In fish oocyte, maturation occurs in three phases which are regulated by specific factors: gonadotropin (LH), maturationinducing hormone (MIH) and MPF [119]. LH acting on the follicle induces the production of MIH (17, 20 dihydroxy-4-pregnen-3-one, 17, 20-DP); in the majority of teleosts MIH leads to new synthesis of cyclin B by translational activation of masked mRNA after a signalling which involves cAMP/PKA pathway, while Cdk1/cyclin B is activated by phosphorylation on Thr-161 [119]. After egg activation, MPF is inactivated by degradation of cyclin B by the 26S proteasome through the first cut in its NH2 terminus at Lys-57 [119].

### **OOCYTE MATURATION IN INVERTEBRATES**

In invertebrates the transition from P-I to GVBD is induced by different stimuli depending on the species. In sea urchin, oocytes complete meiosis before fertilization and arrest at the pronucleus stage; in starfish, maturation is induced by 1-methyladenine and fertilization occurs at the P-I stage [121]. In molluscs, two different situations have been described: i. oocytes, such as those of bivalves *Spisula* and *Barnea*, which are fertilized at the P-I stage, the maturation is induced by unknown molecules and proceeds with meiosis completion followed by the fusion of male and female haploid pronuclei [12]; ii. oocytes, such as those of the gastropod *Patella* and bivalves *Ruditapes* and *Mytilus*, which are also arrested in P-I, undergo meiosis after hormonal or other stimuli and secondarily arrest in M-I until fertilization occurs with the release of M-I arrest and meiosis completion [12]. Serotonin induces oocytes maturation in molluscs *Crassostrea* and *Ruditapes* (Moreau *et al*., 1996), whereas in *Spisula* fertilization causes meiotic completion [12]. In the nematode *C. elegans,* a cytoskeletal protein involved in sperm motility (major sperm protein) causes oocyte maturation and ovulation [122]. Starfish oocytes are arrested at the G2/M-phase border of meiosis I. Exposure to their natural mitogen, 1-methyladenine, leads to the activation of MPF and MAP kinase, resumption of the meiotic cell cycle, and fertilization competency [123]. In addition, in starfish, unlike the wellstudied case in *Xenopus* oocytes, where MAP kinase is an essential component of the MPF activation pathway, MAP kinase is not required for either MPF activation or subsequent oocyte maturation. Instead, its major role appears to be suppression of DNA synthesis in unfertilized eggs [123]. Sea urchins are members of a limited group of animals in which meiotic maturation of oocytes is completed prior to fertilization. Sea urchin oocytes accumulate significant amounts of cyclin B mRNA and protein during oogenesis. Cyclin B synthesis is not necessary for the entry of G2-arrested oocytes into meiosis; however, it is required for the proper progression through meiotic divisions. Mature sea urchin eggs contain significant cyclin B protein following meiosis that serves as a maternal store for early cleavage divisions [124]. In ascidians, the signals that trigger oocytes maturation are unknown. It has been suggested that follicle cells surrounding oocytes secrete a meiosis-inducing substance in several species [125].

In general, mechanisms regulating maturation and meiotic completion in invertebrates appear heterogeneous and are also difficult to reconstitute from an evolutionistic point of view. Probably, the unique conserved feature common to all species is the presence of MPF and its oscillation during maturation. As an example, in *Patella vulgata* [126] and *Mitilus edulis* [127], the metaphase block is guaranteed by the stability of M-I cyclin B which is ensured by the continuous synthesis of short lived proteins, not yet characterized. This hypothesis has been formulated based on the observation that exit from M-I was induced by treatment of oocytes with protein synthesis inhibitors [12]. In starfish, the pathway that leads to meiotic maturation does not require protein synthesis and the MPF, present as pre-MPF, is immediately activated by autoamplification mechanisms [1]. MPF promotes oocytes maturation in ascidians [4, 15]; however, this process is different in other invertebrates because the maintenance of M-I block seems to be independent from the presence of newly synthesized proteins [4, 128-129]. Similarly to *Xenopus* oocytes [130], but differently than other invertebrates where inhibition of protein synthesis causes the release of M-I block and chromosomes decondensation [1].

The role of calcium in meiosis completion has been extensively investigated in vertebrates and has been recently analyzed in excellent reviews in this book [131] and elsewhere [132-134]. In ascidians, we firstly correlated calcium oscillation at fertilization with changes in MPF [135] and sperm factor [136]: Cdk1/Cyclin B activity is maximal at M-I and M-II, and decreases at exit from meiosis I and II. A series of calcium oscillations occur simultaneously with the decrease in Cdk1 activity at M-I exit, while a second group of intracellular Ca2+ transients precedes the Cdk1 increase at M-II [135]. This calcium signaling system however does not appear to be the central cell cycle control mechanism during meiosis I, since inactivation of Cdk1/Cyclin B was Ca2+-independent at this stage. However, oocytes did not extrude a polar body after fertilisation in the presence of calcium chelators, suggesting that calcium is involved in the completion of meiosis I. Later, others confirmed our finding [137- 138], although a major controversy regarded the role of Ca2+ oscillation before the M-II peak of MPF activity. We reported that inhibiting the second phase of sperm-triggered Ca2+ oscillations in *C. intestinalis* oocytes with calcium chelator, the reactivation of MPF that accompanies formation of the MII spindle was inhibited [135]. These data imply that the second phase of Ca2+ oscillations was required to reactivate MPF prior to extrusion of the second polar body. On the opposite, others showed that MPF activity initially decreases then increases even in the absence of further Ca2+ signals. In fact, a monotonic Ca2+ signal leads first to the inactivation then the reactivation of MPF activity [138-139]. These data may be revised and reconciled considering the concentration of Ca2+ chelator applied. In addition, differences among independent studies might be attributed to the employment of different ascidian species. More recently, our original observations on the functional link between the second wave of calcium oscillation at fertilization and MPF activity has been partially reevaluated. In fact, McDougall's group demonstrated that Cdk1/Cyclin B activity is both necessary and sufficient for the generation of second phase Ca2+ oscillations. The new and important observation regards the correlation between in Cdk1/Cyclin B activity sperm factor triggered calcium release. In fact, their data suggest that Cdk1/Cyclin B activity promotes IP3 production in the presence of the sperm factor, rather than sensitizing the Ca2+ releasing machinery to IP3 [140]. The hypothesis formulated is that, following fertilization, the ascidian sperm factor is delivered into the egg and triggers Ca2+ release, resulting in the first phase of Ca2+ oscillations. The sperm factor is then inactivated by an unknown eggderived mechanism pausing Ca2+ oscillations; it is the rise in Cdk1/Cyclin B activity following the metaphaseanaphase transition of meiosis I that restores the Ca2+-releasing activity of the sperm factor, resulting in the second phase Ca2+ oscillations. The decrease in Cdk1/Cyclin B activity at meiotic exit results in the inactivation of the sperm factor and in the block of Ca2+ oscillations. The Authors hypothesized that Cdk1 modulates sperm factor activity directly by phosphorylation [140].

Regarding the role of CSF in meiotic maturation in invertebrates, to date Mos kinase has been identified in starfish, where it seems to be essential for the maintenance of meiotic block [69] and a homolog of Mos has also been cloned in *Drosophila* [141], where the deletion of the gene does not prevent meiotic completion, suggesting the existence of a redundant pathway in meiotic regulation by CSF in *Drosophila* [141]. More recently, a phylogenetic survey has revealed that c*-mos* genes are conserved in cnidarians and ctenophores, but not found outside the metazoa or in sponges [142]. Mos orthologs from *Pleurobrachia* (ctenophores) and *Clytia* (cnidarians) activated MAPK and blocked by classical RNA injection assay into *Xenopus* blastomeres. Unusually, cnidarians were found to possess multiple Mos paralogs. In *Clytia*, one of two maternally expressed paralogs accounted for the majority MAPK activation during maturation, whereas the other may be subject to differential translational regulation and have additional roles [142]. Very recently, the genome of *C. intestinalis* has been used as an essential tool to reconstitute, from a phylogenetic point of view, CSF pathway in ascidians. In this study, the existence of an active CSF in ascidians has been experimentally proved by microinjection of cytoplasmic extracts from *C. intestinalis* oocytes blocked in M-I into embryos at the two-cell stage [129]. The Authors also suggest that the cascade reaction leading to CSF arrest is common to vertebrate species for the presence in *C. intestinalis* genome of the main protein kinases involved in the pathway, from Mos to p90Rsk. The rapid decrease of Cdc2/Cdk1 and MAPK enzymatic activities following fertilization can be interpreted as the expected inactivation of MPF and CSF observed in meiotic arrested oocytes. In *C. intestinalis*, continuous protein synthesis appears not required to maintain an active CSF and MPF as in lower vertebrates (*Xenopus*, fishes) and differently than other invertebrates. On the opposite, protein synthesis is essential to permit M-II entry, probably for the strong requirement of newly synthesized cyclin B [129]. This study suggests that, in ascidians, CSF is essential to maintain high MPF and M-I block, but it is not essential to remove the metaphase arrest and complete meiosis, since this event occurs in the presence of high MAPK activity. This apparent contradiction is explained evoking the presence of alternative pathways. In fact, redundant pathways exist in *Xenopus* independent from Mos to ensure CSF arrest and APC/C inhibition, *e.g.* Cdk2/Cyclin E [61]. In addition, in invertebrates such as *Drosophila*, the presence of Mos did not affect meiosis completion [141]. Furthermore, the presence in *C. intestinalis* Mos of an N-terminal domain not present in vertebrate homologs, suggests extra functions for this kinase to be explored. Perhaps, in *C. intestinalis* the Mos/MEK1/MAPK/p90Rsk is necessary to establish CSF arrest, but not to maintain it. Future studies are necessary to address this aspect. The other main difference emerging in CSF regulation between ascidian and *Xenopus* derive from the *in silico* analysis of Mos pathway. Russo and colleagues [129] reported that the cascade of phosphorylation Mos/MEK/MAPK/p90Rsk/Bub1/Cdc20 could be active in *C. intestinalis*, differently than the parallel pathway Mos/MEK/MAPK/p90Rsk /Emi2/Cdc20 established in *Xenopus*. In fact, neither Emi1, nor Erp1/Emi2 has been found in *C. intestinalis* genome; therefore, it is possible that the Mos/Emi-Erp pathway controlling APC/C inhibition and M-II arrest in vertebrates [62] is not active in Urochordata. On the opposite, the presence of Bub1 downstream to Mos might represent the only way to ensure CSF stability in invertebrates, while in vertebrate, although still present [94], this alternative pathway could have become redundant during evolution [129]. A further convergence between ascidian and *Xenopus*, not yet investigated in other invertebrates, regards the role of CK2 in regulating CSF. As depicted in Fig. **3** and reported above, CK2 seems to negatively regulate CSF function acting directly on Mos. Our group recently characterized the independent regulation of the catalytic, CK2, and regulatory, CK2 subunits of CK2 during meiosis completion in *C. intestinalis* at fertilization [16]. An interesting observation emerged from the alignment of the carboxyl-terminal region of CK2 in invertebrates versus all vertebrates. Briefly, we observed that Ser-209 of CK2 is absent in all invertebrates having a *bona fide* meiotic block in metaphase of the first meiotic division, such as insects (*Drosophila*, *Anopheles* and *Spadoptera*) and worms (Platyhelminthes *Schistosoma*). Ser-209 is embedded in a perfect consensus site for Cdk1/cyclin B phosphorylation and it is conserved among vertebrates, all having mature oocytes arrested in M-II. As an example, *H. sapiens* and *C. intestinalis* CK2are highly homologous, except for few residues, including Ser-209. In all invertebrates sequences we analyzed, including *C. intestinalis*, Ser-209 is substituted by other residues although the remaining part of the consensus site for Cdk1/cyclin B phosphorylation is conserved. Since many invertebrates present a meiotic block in P-I/M-I, these data support the hypothesis that Ser-209 might be an important requirement to ensure a M-II arrest in vertebrates, or, more in general, be involved in regulating CSF in oocytes [16]. This hypothesis remains to be supported by experimental data.

#### **CONCLUSION**

One aspect of biology that fascinated and still fascinates biologists is undoubtedly fertilization and meiosis. Thanks to the investigation of excellent experimental models, including *Xenopus*, it has been possible to outline most of the mechanisms regulating these fundamental biological processes. On one hand, it has been possible to understand, from a physiological and molecular point of view, how regulatory mechanisms of fertilization and meiotic completion have been conserved during evolution; on the other hand, many questions remain unsolved.

#### **88** *Oocyte Maturation and Fertilization Russo et al.*

The present review underlined the heterogeneity and redundancy existing in the mechanisms regulating maturation and meiosis completion in invertebrates. Although the "universal" role of MPF is confirmed, more complex appears the function of CSF in these organisms. Neglected until few years ago, the presence of Mos and its function as CSF component in invertebrates has been demonstrated by recent studies. However, "alternative" functions of Mos in oocytes are emerging, as well as its different cross-talks with other CSF components. In this view, it must be kept in mind that even if MPF and CSF have been identified together and have been often considered "twin" complexes from a physiological point of view, they strongly differ from a biochemical point of view, being MPF an enzyme constitutes by two structurally different subunits, while CSF represents a complex pathway of signal transduction which includes several proteins with multiple functions. This difference accounts for the enormous difficulty in characterizing CSF in oocytes from different species. Although recent data suggests that Mos kinase appeared early during animal evolution in regulating meiosis in oocytes, the function of CSF seems to change among invertebrate species. As an example, CSF machinery in *C. intestinalis* oocytes is closer to *Xenopus* and other vertebrates, than to invertebrates, possibly reflecting the position of ascidians in the evolution of Chordata.

The introduction of new experimental models supported by the availability of tools and sequences from complete genomes will certainly help in the next future to trace phylogenetic patterns and clarify unknown aspects of meiosis and fertilization.

# **REFERENCES**

#### **90** *Oocyte Maturation and Fertilization Russo et al.*

© 2011 The Author(s). Published by Bentham Science Publisher. This is an open access chapter published under CC BY 4.0 https://creativecommons.org/licenses/by/4.0/legalcode

# **Gamete Binding and Fusion**

**Young-Joo Yi1,3 and Peter Sutovsky1 , 2,\***

*1 Division of Animal Sciences, and <sup>2</sup> Department of Obstetrics, Gynecology & Women's Health, University of Missouri-Columbia, Columbia, MO 65211, USA; <sup>3</sup> Research Center for Transgenic Cloned Pigs, Chungnam National University, Daejeon 305-764, South Korea*

**Abstract:** Fertilization is a stepwise process that starts before the actual gamete binding and fusion. Spermatozoa travel through the female reproductive system and respond to chemotaxis and thermotaxis to reach the oocyte. In most mammals, zona pellucida (ZP) glycoprotein ZPC is the primary sperm receptor that mediates sperm-ZP binding and acrosomal exocytosis (AE). During AE, the outer acrosomal membrane, already primed for AE during capacitation, fuses with the plasma membrane and undergoes vesiculation. The acrosomal matrix (AM) is exposed and dispersed in a step-wise manner. Sperm-ZP penetration is supported by sperm motility and by enzymatic activity of the hypothetical egg coat "lysin", an acrosomal protease that digests the fertilization slit. Acrosin has been considered as a zona lysin candidate. However, male mice lacking the *Acr* gene are fertile. Recently, researchers have been focusing on the 26S proteasome as a mammalian and non-mammalian egg coat lysin. Following zona penetration, spermatozoa reach the perivitelline space and adhere to and fuse with the oolemma. Tetraspanin superfamily members CD9 and CD81 appear to act as sperm receptors on the oolemma, possibly supported by integrins and other elements within the cortical tetraspanin web. IZUMO, a member of immunoglobulin superfamily is a sperm ligand candidate for oolemma tetraspanins. Both CD9 and IZUMO are essential for gamete adhesion and fertility in the mouse. However, there is no evidence yet supporting the involvement of IZUMO and CD9 in sperm-egg plasma membrane fusion. After sperm-oolemma fusion, the fertilizing spermatozoon is incorporated into the ooplasm, a process aided by oocyte cortex microfilaments.

#### **EVENTS IMMEDIATELY PRECEDING GAMETE BINDING**

#### **Sperm Migration/Transport in the Female Reproductive System**

Depending on the species, mammalian spermatozoa are deposited into the female vagina, cervix or uterus. In vaginal depositors such as primates and ungulates, spermatozoa first contact cervical mucus which is thought to be able to filter out spermatozoa with poor morphology and motility. Only good (morphologically normal and motile) spermatozoa pass through the cervix and uterus. Finally, a few thousand spermatozoa reach the oviducts where they are stored transiently for several hours or even days in a segment referred to as the sperm reservoir. Bound by their heads to the apical surfaces of the oviductal epithelial cells, these spermatozoa are maintained in a fertile state until ovulation. In response to ovulation, spermatozoa become capacitated and hyperactivated, which releases them from the sperm reservoir and allows them to approach the fertilization site. At this stage, spermatozoa move toward the oocyte by a combination of chemotaxis and thermotaxis [1].

#### **Sperm Chemotaxis**

Chemotaxis, defined as a reaction to chemoattractant or chemorepellent, is the migration of a cell or organism to/from a chemical source [2]. Sperm chemotaxis has mainly been studied in marine species because females release eggs into seawater, and the released spermatozoa need guidance to find them [3]. In mammals, high numbers of spermatozoa released during ejaculation enter the female reproductive tract and move spontaneously while aided by peristaltic action. However, a low number of spermatozoa reach the fertilization site, e.g., in humans, only about 251 (range 79–1386) spermatozoa are found in both fallopian tubes [4]. Although their number decreases dramatically during sperm migration, multiple spermatozoa still must contact the egg to complete fertilization. Chemotaxis of spermatozoa in mammals is directed toward follicular fluid in mice and oviductal fluid in humans, as well as toward medium conditioned by eggs and cumulus cells [5]. In humans and rabbits, minute concentrations of progesterone seem to attract spermatozoa. The low concentration gradient of progesterone (1-100 pmol/L) stimulated human

**Elisabetta Tosti and Raffaele Boni (Eds) © 2011 The Author(s). Published by Bentham Science Publishers**

**<sup>\*</sup>Address correspondence to Dr. Peter Sutovsky:** Department of Obstetrics, Gynecology & Women's Health, University of Missouri-Columbia, Columbia, MO 65211, USA; E-mail<EMAIL_ADDRESS>
spermatozoa chemotactically [6]. Follicular fluid contains progesterone; however, only a small amount of follicular fluid was released at ovulation [7]. After ovulation, cumulus cells continue synthesis and secretion of the sperm chemoattractant progesterone [8-11] and its carrier protein [12], which maintains progesterone in soluble form. In the rabbit, cumulus oophorus also seems to secrete progesterone as the chemotactic response was inhibited by antiprogesterone antibody *in vitro* [13]. Progesterone secreted by cumulus cells also appears to be a sperm chemoattractant in humans [14]. At present, progesterone is a strong candidate for mammalian/human sperm chemoattractant, while other mechanisms may work in synergy with it.

### **Sperm Thermotaxis**

Capacitated spermatozoa respond to a temperature gradient, a reaction called thermotaxis that is also found in other cell types [15]. Thermotaxis in mammalian spermatozoa plays an important role for guiding spermatozoa from the cooler sperm reservoir site toward the warmer fertilization site, a difference of approximately 2°C. Thermotaxis and chemotaxis are thought to act in synergy in guiding spermatozoa toward the fertilization site near the oviductal isthmus. Thermotaxis is a long-range guidance mechanism; by contrast, short-range guidance is by chemotaxis [13- 15]. The difference in temperature between the sperm reservoir and the fertilization site is observed at ovulation, mediated by a temperature rise from cooler sperm reservoir toward warmer fertilization site. In rabbits, approx. 2°C difference has been observed between the isthmus and the isthmic ampullary junction [16], and in pigs, 0.7°C difference has been measured between the isthmus and the ampulla [17]. Both rabbit and human spermatozoa respond to even small temperature differences (0.5°C) [15]. Several mechanisms have been postulated to produce a temperature drop at the isthmus [18]. First, Luck *et al.* [19] suggested endothermic hydration, a process by which the acidic mucus glycoprotein in the isthmus is hydrated by circulating steroid hormones [20] and causes a temperature drop during ovulation. Secondly, cold blood from the ovary may decrease the blood temperature of the vasculature surrounding the sperm reservoir [17].

#### **Sperm-Cumulus Oophorus Interactions**

During oocyte growth, cumulus oophorus cells play an important role in maintaining the oocyte in meiotic arrest. Following the preovulatory LH-surge, cumulus cells convey the signal for meiotic resumption and support nuclear and cytoplasmic maturation of the oocyte [21]. The cumulus oophorus cells are embedded in an extracellular matrix (ECM) rich in water-binding glycan, hyaluronan. Mammalian spermatozoa carry a hyaluronan-receptor and bind to hyaluronan-rich ECM [22]. Spermatozoa circle a cumulus enclosed oocyte before fertilization [23]. Stock *et al.* [24] suggested that a high rate of acrosomal exocytosis (AE) can be induced by sperm co-incubation with cumulus cells, and the human cumulus cell-conditioned medium also induced acrosomal activity [25]. Cumulus cells secrete progesterone, a sperm attractant discussed above [26], and hyaluronic acid present in cumulus ECM can increase the intracellular calcium concentration of spermatozoa [27]. It is reasonable to conclude that cumulus oophorus is involved in fertilization [28].

### **SPERM-ZONA PELLUCIDA INTERACTIONS**

### **Sperm-ZP binding**

Following penetration through cumulus ECM, spermatozoa bind to another type of highly organized ECM, the mammalian egg coat, zona pellucida (ZP). The ZP is composed of three (most mammals) or four (murids, primates) ZP-glycoproteins sharing the conserved, immunoglobulin-like ZP domain [29]. Based on sequence similarity, mouse (m) and human (h) ZP proteins are designated mmZP1 (hZPB), mZP2 (hZPA), mZP3 (hZPC) and ZP4 (hZPB2) [30-32]. The general mouse model of zona assembly suggests that ZP2-ZP3 (ZPA-ZPC) heterodimer fibers are cross-linked with ZP1 (ZPB) molecules, and ZPC serves as the primary sperm receptor on the egg coat [33]. In pigs, a heterodimeric complex of ZPB/ZP3alpha (homologue of hZPB2/ZP4) and ZPC seems to act together as a sperm receptor [34]. Such a synergy may also exist between ZP3 and ZP4 in humans [35]. The sperm receptor has a dual function of sperm-ZP binding and induction of AE. The latter may start even before sperm-ZP binding, but it seems to be completed during that process. This dual function is somewhat, but not universally, species-specific [36]. Evidence suggests that the branched triantennary and tetraantennary N-glycans and O-glycans decorating the sperm receptor, rather than its protein backbone, mediate sperm-ZP binding [37]. Consequently, transgenic mouse ova carrying human sperm receptor ("humanized" mouse zona) still bind only mouse spermatozoa [38-39], most likely because the human ZPC protein secreted by these murine ova carries mouse-like ZPC-associated glycans. Furthermore, recombinant human ZPC protein will only bind to capacitated human spermatozoa and induce AE if it carries glycans consistent with those naturally occurring on the human ZP surface [40]. It is important to note that the soluble zona proteins are capable of inducing AE, a well known fact that is sometimes ignored when alternative models of sperm-ZP interactions are proposed.

Both spermatozoa with an intact acrosome and those progressing through AE can bind to ZP [41], at least in some species. A number of proteins on the acrosomal surface were proposed as ZP-binding ZPC-receptors, but the evidence for any candidate remains inconclusive. These include SP56/ZP3-receptor [42], β1,4-galactosyl transferase (GALT) [43], zonadhesin (ZAN) [44], several spermadhesins [45], arylsulfatase A (ASA) [46-47], P43H [47], proacrosin [48], SP38/IAM38/ZPBP1&2 [49-50] and PH20/SPAM1 [51].

**Figure 1:** A low magnification (A) and a detailed view (B) of sperm-zona pellucida binding in the pig, visualized by scanning electron microscopy (SEM). Magnification is indicated by scale bars.

### **Acrosome Reaction Versus Acrosomal Exocytosis**

The process by which spermatozoa respond to sperm receptors on the ZP has historically been referred to as acrosome reaction (AR), while it is now being reinterpreted based on new research and perhaps more appropriately called acrosomal exocytosis (AE) [52]. Exocytosis in general is a process by which cargo-carrying vesicles dock to the inner face of the plasma membrane, fuse with it and release their cargo in the extracellular space. This is typically a continuous process that can be turned on/off as needed. The AE/AR is unique in this context as it is an irreversible, one time event by which the sperm acrosome is altered and part of it is lost. While vesicle fusion during AE may employ pathways similar to those observed in somatic cells [e.g. SNARE hypothesis proteins of synaptic vesicle fusion [53-54], the purpose of AE is to expose the acrosomal matrix rather than to deliver a cargo concealed inside the vesicles to the extracellular space. The vesicle fusion during AE is heterologous, thought to arise from the fusion of inner acrosomal membrane with plasma membrane, forming what is called "hybrid vesicles" of the acrosomal shroud [41,55].

The acrosome is thought to originate primarily from the Golgi during spermiogenesis, while the perinuclear theca components that eventually form a capsule around the sperm nucleus are also thought to contribute, and lysosomelike enzymes are found in the acrosomal matrix [56-58]. During AE, the outer acrosomal membrane (OAM) fuses with the acrosome-covering plasma membrane, giving rise to said vesiculation event. The acrosomal matrix (AM) is exposed and partially dispersed, while the inner acrosomal membrane (IAM) with some adhering acrosomal matrix components remains intact throughout the process of sperm-ZP binding and sperm-ZP penetration [41].

Gerton [52] summed up the difference between AE and AR definitions as follows: AR is a binary (1/0 or ON/OFF) event (acrosome is either intact/ON, or reacted/OFF) that has no intermediate steps and starts with sperm-ZP binding. The AE is an "analog" event, a progression of distinct steps that starts with the priming of the outer acrosomal membrane and plasma membrane already occurring during sperm capacitation. Then the AE progresses through sperm-ZP binding, fusion of the PM with OAM, formation of the acrosomal shroud and a stepwise exposure/dispersion of AM layer-after-layer. Contrary to most exocytotic events observed in somatic cells that are continual and can be turned off and resumed, AE is an irreversible, one-time event. Thus, caution is in order when the mechanism of AE is extrapolated from exocytosis studied in other cell types.

Signaling during the AE is mediated by second-messenger molecules such as IP3, cAMP and Ca2+ ions. Two distinct pathways have been proposed by Breitbart to be activated during AE [59]. In both cases, a central event is the increase of Ca2+ ion content inside the acrosome. In one scenario, the influx of external Ca2+ ions in the acrosomal interior is controlled by gating of Ca2+-channels via activation of the cAMP-dependent kinases (PKA), which is a consequence of ZPC binding to a Gi-coupled receptor on the sperm plasma membrane. An alternative pathway that could cause cytosolic calcium increase inside the acrosome is through activation of a phospholipases enzyme (PLCgamma) by a tyrosine kinase associated with the sperm receptor for ZPC. PLCgamma hydrolyses phosphatidyl inositol di-phosphate (PIP2) into inositol 3-phosphate (IP3) that can trigger intra-acrosomal Ca2+ release, and diacyl-glycerol that activates protein kinase C (PKC), causing the opening of Ca2+-channels. In both scenarios, the influx of Ca2+-ions triggers microfilament depolymerization in the acrosome to promote fusion and vesiculation of OAM.

The pH of the acrosome is acidic [60-61] and increased during capacitation [61]. The contents of sperm acrosome are alkalinized by calcium influx, and sperm begin acrosomal exocytosis [62]. Kim and Gerton [63] classified the progression of mouse sperm AE based on epifluorescence microscopy of GFP-acrosin expressing transgenic spermatozoa and anti-sp56 coated beads binding to the acrosome. At stage 1, the acrosome is acidic and maintains intact OAM and plasma membrane (PM). Next, the pH around the acrosome begins to increase and OAM and PM start to vesiculate. In the third stage, the acrosomal components disperse due to vesiculation of OAM and PM. Finally, at stage 4, the OAM and PM vesiculated completely, and acrosomal components are released from acrosomal matrix [63]. During acrosomal exocytosis, some acrosomal enzymes/components undergo proteolysis and post-transitional modifications, exemplified by proteolytic cleavage of pro-acrosin into enzymatically active acrosin [64-65].

#### **Sperm-Zona Penetration**

To what extent does the sperm-zona penetration rely on flagellar motility and proteolysis, respectively? Two opposing but not necessarily irreconcilable hypotheses address this question: the mechanical and the enzymatic hypotheses of sperm-zona penetration. Only sperm motility is required for sperm-ZP interaction in the "mechanical" scenario, while in the enzymatic paradigm, the acrosomal enzymes are of primary importance and sperm motility is secondary. In general, spermatozoa will only penetrate the zona once they have undergone the acrosome reaction. This morphological and biochemical change activates and exposes the proteolytic enzymes present in the acrosomal matrix. Possibly the most studied acrosomal protease, acrosin, has properties similar to trypsin, and both trypsin and crude acrosin extracts remove the zona from oocytes [66]. Because of this, acrosin has for many years been regarded as the principal contender for the role of zona "lysin", a fertilization slit digesting acrosome borne enzyme, the existence of which was proposed as early as 1958 [67]. If the fertilizing spermatozoa make the penetration slit by proteolysis rather than by mechanical disruption or cutting movement, as proposed by Bedford [68], the egg coat "lysin" would be expected to remain on the IAM after AE.

Acrosin has been studied extensively as a candidate for mammalian zona lysin [70-71], but acrosin gene ablation studies demonstrated that it is not essential for mouse fertility. Male mice homozygous for the targeted mutation in the *Acr* gene were mated with homozygous F2 female mice and produced normal litters. In addition, mouse oocytes were fertilized with acrosin-lacking spermatozoa *in vitro* and showed normal sperm penetration and fertilization rates (98% sperm penetration and 79% fertilization), similar to wild type mice (99% sperm penetration and 76% fertilization). The only difference observed was a slight delay (approx. 30 min) in sperm penetration and fertilization, observed in homozygous mutant mice [72]. A similar result was later reported by another group: Homozygous *Acr- /-* mice produced offspring, but showed a somewhat delayed fertilization [73]. Therefore, acrosin, while perhaps facilitating acrosomal remodeling during AE, is not an essential factor in sperm ZP-penetration. This conclusion led to re-examination of the mechanical hypothesis of fertilization, while results of *Acr* gene deletion led to renewed interest in other acrosomal enzymes.

The enzymatic hypothesis has been revived recently by Honda *et al.* [74]. Besides acrosin, several acrosome-borne trypsin-like enzymes have been implicated in sperm-ZP penetration. It was shown earlier that trypsin inhibitors inhibit sperm-ZP binding and penetration [75-77]. The list of acrosomal proteases identified in mammalian spermatozoa includes a collagenase-like peptidase [78], a cathepsin D-like protease [79], trypsin-like proteases different from acrosin [80-81], a serine protease [82], dipeptidyl peptidase II [83], the TESP proteases [84] and the 26S proteasome [85]. In particular, the 26S proteasome, an ubiquitin-dependent protease with trypsin-like, chymotrypsin-like and caspase-like (peptidyl-glutamyl-peptidase-like) protease activities, has been a focus of intense research in mammals, ascidians and echinoderms. Data show that enzymatically active proteasomes are present in the sperm acrosome and participate directly, by degrading a ubiquitin-tagged sperm receptor on the egg coat [85-87], or indirectly, by facilitating acrosomal remodeling during capacitation and AE [88-89], in the process of sperm-egg coat penetration.

# **SPERM-OOLEMMA INTERACTIONS**

#### **Sperm-Oolemma Binding & Fusion**

After spermatozoa penetrate the zona pellucida, they reach the perivitelline space and adhere to and fuse with the oocyte plasma membrane, the oolemma. The ADAM (a disintegrin and metalloprotease) family of integrin-binding proteins present on sperm head surface and integrins on the egg plasma membrane, the oolemma, were the early candidates for mediators of sperm-oolemma binding and fusion [90-93]. Specifically, fertilin (α and β) and cyritestin, ADAM family proteins found on the plasma membrane of spermatozoa, were thought to interact with oolemma integrins α6β1 or αvβ1 during fertilization. Fertilin-β (ADAM-2) was proposed to participate in sperm binding to eggs through its disintegrin domain, and fertilin-α (ADAM-1) was hypothesized to induce sperm-oolemma fusion through a virus-like fusion domain. However, the importance of fertilin-integrin interaction has not been confirmed by gene deletion/mutation studies, as these mutants are either fertile or have a fertility defect due to failed sperm transport in the female reproductive system, rather than to failed sperm-egg fusion [94]. Consequently, new candidates emerged, including tetraspanin superfamily members CD9 and CD81, initially considered as potential sperm receptors on the oolemma because of CD9's proposed association with integrin α6β1.

Female *Cd9*-/- mice showed reduced fertility and sperm-egg fusion [95-97]. The *Cd9*-/- mice eggs injected with *CD9* mRNA recovered their sperm-fusion ability. Based on these experiments, the large extracellular loop of CD9 was involved in gamete fusion [98]. In addition, the soluble ligand PSG17 which is a member of the immunoglobulin superfamily (IgSF) has been identified as a CD9 receptor [99-100]. Knockout of a related tetraspanin-encoding gene, *Cd81,* also reduces female fertility and sperm-oolemma fusion, although the effect is only partial. Altogether, it appears that tetraspanins CD9 and CD81 act synergistically during sperm-oolemma adhesion/fusion. Tetraspanins cooperate with integrins within a cortical network composed of cytoskeletal elements, cell surface receptors and signaling proteins, called the tetraspanin web. Such a close association may explain why anti-integrin antibodies and disintegrin domain-mimicking peptides interfering with extracellular domain of integrins hindered sperm-oolemma adhesion in earlier studies.

At the current level of knowledge, the likely binding partners of oolemma tetraspanins are the sperm-expressed proteins from the immunoglobulin superfamily (IgSF). In particular, the IgSF member IZUMO shows testis and spermatid-specific expression, and the monoclonal anti-IZUMO antibodies inhibit sperm-egg fusion *in vitro* [101]. *Izumo*-null males are sterile, as their mating with wild-type females produces no pups; *Izumo-/-* spermatozoa show normal motility and ability to undergo ZP-induced acrosomal exocytosis; the null females are fertile. Consequently, the *Izumo-/-* mutant spermatozoa penetrate the ZP, but accumulate in perivitelline space without fusing with the oolemma [102].

The IZUMO protein is composed of 397 amino acid residues with a single Ig domain in roughly the middle of the extracellular domain [102]. While IZUMO is apparently involved in sperm adhesion to oolemma, it is not clear if or how it participates in membrane fusion. Other IgSF proteins participate in cell-cell adhesion, and CD9, the likely oolemma receptor for IZUMO, is one of its *cis-*partners in tetraspanin web, an intricate network of tetraspanins, integrins and associate cytoskeletal and signaling proteins underlying the plasma membrane [103].

Most of IgSF members act as cell adhesion molecules in *Drosophila* and *C. elegans* [104-105]. Tetraspaninassociated IgSF members, IgSF8 and prostaglandin F2 receptor negative regulator (PTGFRN, or EWI-F and CD9-P-1), have been identified in mouse eggs [106-107]. IgSF *cis-*partners EWI-2 and EWI-F may be linking CD9 to microvilli of the oocyte, and microvilli could support, directly or indirectly, sperm-oolemma adhesion/fusion [107]. The mechanism governing sperm oolemma fusion is not known, although sperm surface ligands, such as viral envelope–like proteins and members of the SNARE hypothesis, discussed with regard to membrane fusion during acrosomal exocytosis, could be involved. We do know, however, that the fertilizing spermatozoon contributes membrane components to the oolemma and that the fusion of the respective sperm and oocyte plasma membranes has an effect on subsequent fertilization steps. In the absence of sperm-oolemma fusion, mouse ova fertilized by intracytoplasmic sperm injection (ICSI) fail to develop a competent anti-polyspermy defense observed during natural fertilization. There is some evidence suggesting that the interaction between sperm plasma membrane receptors and oolemma integrins elicits this response during fertilization [108].

#### **Sperm Incorporation**

After sperm-oolemma adhesion and fusion, the sperm tail motility ceases and the sperm head, and subsequently sperm tail, are incorporated in the cortical ooplasm. The oocyte cortex is supported by actin microfilaments forming microvilli that cover most of the oocyte surface except the smooth surface over the metaphase chromosomes, located cortically in the mature oocytes [109]. In zebrafish eggs, sperm equatorial surface binds to the egg microvilli [110]. In mammals, the equatorial segment and subsequently the post-acrosomal sheath contact with microvilli during sperm binding [107]. With the help of cortical microfilaments, the mouse oocye cortex raises around the sperm head, forming a distinct fertilization cone, a structure that is not seen in mammals. This swelling of the oocyte cortex disappears only after the entire sperm head and flagellum enter the ooplasm [110]. The actin microfilaments around the sperm entry site in zebrafish eggs consist of a tight mesh formed before fertilization. This filament network forms the fertilization cone within 15-20 seconds of fertilization, and actin is rearranged into a thick network around the sperm nucleus during incorporation [111]. The sperm incorporation site of non-rodent mammalian oocytes, in contrast, is small and flat [112-114].

In zebrafish eggs pre-incubated with microfilament disruptor cytochalasin B (CB), spermatozoa fail to enter eggs [115]. Similarly, CB-treatment during fertilization prevents sperm incorporation without affecting sperm-oolemma fusion and oocyte activation during bovine and porcine fertilization [116-117]. Microfilament modulator jasplakinolide (JAS) also inhibits sperm incorporation in the mouse [118]. Reduced sperm incorporation has been shown in mouse zona-free oocytes in the presence of CB, JAS and latrunculin B [119]. However, the rate of polyspermy increased during fertilization in the presence of a related microfilament disruptor, cytochalasin D (CD). Somehow, CD-treatment interrupted cytoskeletal rearrangements and led to polyspermy [119]. Rho protein(s) regulating actin-based cytoskeletal reorganization have been implicated in sperm incorporation [120]. It can be concluded that the activity of microfilaments in the oocyte cortex, including the formation of a protruding fertilization cone in rodents, plays an important role in sperm incorporation. It is during the sperm incorporation step of fertilization that the spermatozoon releases the sperm borne, oocyte-activating factor(s) that convey full oocyte activation. These factors are embedded in the dense matrix of the sperm perinuclear theca that solubilizes completely upon sperm head entry in the oocyte cytoplasm [56].

#### **CONCLUSIONS AND CHALLENGES**

To achieve fertilization, gametes adapted, in a unique fashion, many mechanisms also studied in somatic cells. While such mechanisms as membrane fusion, motility and exocytosis are reversible in somatic cells, they are one time, point-of-no-return events during fertilization. Consequently, gametes are irreversibly altered during the fertilization process. Already spermatozoa commit to their fate when they undergo sperm capacitation and hyperactivation during passage through the female reproductive system, which leads to sperm death unless the capacitated spermatozoon meets the oocyte. Sperm-egg coat binding is mediated by glycans decorating the sperm receptor protein(s) on the zona pellucida such as ZPC and ZPB. While multiple candidates have been identified, the essential sperm partner of this receptor is not known with certainty. Sperm-ZP binding induces acrosomal exocytosis via one or more pathways leading to calcium influx into the acrosomal interior. These signaling pathways are only partially understood. Acrosomal exocytosis involves irreversible fusion of sperm plasma membrane with the neighboring outer acrosomal membrane, possibly mediated by proteins similar to those involved in synaptic vesicle fusion. Sperm penetration through the zona is most likely achieved by a combination of enzymatic zona digestion by acrosome-associated proteases and mechanic propulsion provided by the motile force of sperm flagellum. Further work is necessary to characterize acrosomal enzymes involved in this process. Upon completion of sperm-ZP penetration, the adhesion of sperm plasma membrane to oolemma is mediated by IZUMO on the sperm equatorial segment and tetraspanins CD9 and CD81 on the oolemma. It is not clear at present if any of these proteins also has a role for sperm-oolemma membrane fusion. The functioning of tetraspanins during fertilization is probably supported by the tetraspanin web that is organized in the oocyte cortex with support from plasma membrane integrins connected to the microfilament cytoskeleton and its underlying signaling pathways. The oocyte cortex also plays a central role in sperm incorporation and facilitates the release of sperm-contributed factors necessary for oocyte activation. The complexity of gamete interactions during fertilization will undoubtedly reveal many surprises through focused study of regulatory pathways and protein-protein interactions.

**Figure 2:** Mouse fertilization visualized by triple immunofluorescence labeling (A, C, E) of ubiquitin-C-terminal hydrolases UCHL1 (green) and UCHL3 (red), and sperm and oocyte DNA (blue; DAPI stain). Corresponding differential interference contrast (DIC) images are shown in panels B, D, F, with arrows pointing to sperm tails. (A, B) Sperm-zona penetration stage; the oocyte is still in metaphase of second meiotic division, showing a typical metaphase-II spindle *(MII)*. (C, D) Normal monospermic fertilization with one male *(mpn)* and one female pronucleus, two polar bodies *(pb1, pb2)* connected with the oocyte through a microtubule-based midbody *(mb),* and one sperm tail inside the oocyte cytoplasm. (E, F) Aberrant, polyspermic fertilization showing multiple male pronuclei *(mpn)* and multiple tails inside the ooplasm, some only partially incorporated.

#### **ACKNOWLEDGMENTS**

We thank Shawn W. Zimmerman (Division of Animal Sciences, University of Missouri) and Lou Ross (Electron Microscopy Core Facility, University of Missouri) for the preparation of the picture for electron microscopy

#### **REFERENCES**

#### **100** *Oocyte Maturation and Fertilization Yi and Sutovsky*

#### **102** *Oocyte Maturation and Fertilization Yi and Sutovsky*

© 2011 The Author(s). Published by Bentham Science Publisher. This is an open access chapter published under CC BY 4.0 https://creativecommons.org/licenses/by/4.0/legalcode

# **CHAPTER 7**

# **Ionic Events at Fertilization**

#### **Brian Dale\* and Martin Wilding**

# *CFA-Italia, Via Manzoni 15, Naples, Italy*

**Abstract:** The difference in ionic balance between the cytoplasm and the extracellular environment in all cells is maintained by ion channels and transporters located in the plasma membrane. In this chapter we review the voltage-gated channels and transporters common to both gametes and somatic cells and describe gamete specific ligand-gated channels in spermatozoa and oocytes. In lower deuterostome oocytes the fertilization potential is biphasic. The first event may be the result of gamete fusion, while the second larger depolarization is the result of the activation of several hundred non-specific channels in the oocyte plasma membrane. The fertilization channel is one of the largest channels known in biological membranes with a single channel conductance of upto 400pS and a reversal potential of +20mV. A soluble sperm factor appears to gate this channel via the ADPr/NO pathway. In higher deuterostomes the fertilization channel is a calcium-gated potassium channel. The type, number and topographical distribution of channels and transporters change continually during gameteogenesis, through fertilization to early embryonic cleavage stages indicating both the importance of ionic homeostasis and the role of second messengers in early development.

#### **ION CHANNELS**

The difference in ionic constitution of the cytoplasm and intracellular organelles with the extracellular medium is maintained by the hydrophobic lipid bilayer and the trans-membrane proteins - ion channels and transporters. All cell types, including gametes, survive in this manner, in fact one of the first manifestations of cell death is loss of this ionic homeostasis. It has been estimated that 15-30% of all membrane proteins are involved in transport. Transporters have moving parts to transport specific molecules across membranes and may be coupled to an energy source, while channels form a narrow hydrophilic pore, allowing passive movement of small inorganic ions. By generating ionic concentration differences across the lipid bilayer, cell membranes can store potential energy in the form of electrochemical gradients; however, the cell is electrically neutral, e.g., it must contain equal quantities of positive and negative charges. Up to 100 x 106 ions can pass through one open channel each second, 105 times greater than the fastest rate of transport mediated by transporters.

Ion transport through channels is passive, i.e., not linked to energy sources, and is often specific for a type of ion. Although channels are essentially hydrophilic pores they are not continuously open, but gated, i.e., they open and close briefly in response to a change in voltage, mechanical stress or the binding of a ligand. Protein phosphorylation and dephosphorylation also regulates the activity of many ion channels. When there is no net flow of ions across the plasma membrane the resulting transmembrane voltage is called the resting membrane potential which, using the Nernst equation, may be calculated knowing the ratio of internal and external ion concentrations. The resting potential in most cells depends on the gradient of K+ across the membrane together with the characteristics of K<sup>+</sup> ion channels. Since there is little Na+ inside the cell, this has to be balanced by an increase in cations, mainly K <sup>+</sup> , which is actively pumped into the cell by the Na+ /K+ pump and can also move freely in or out through the K+ leak channels in the plasma membrane. Very few ions adjacent to the plasma membrane (<1nm) actually contribute to the resting potential and a small flow of ions carries sufficient charge to cause a large change in the membrane potential. Generally, the more permeable the membrane is to a specific ion, the closer the potential will be to the equilibrium potential for that ion. Since, 1 microCoulomb of charge (6x 1012 monovalent ions) per square centimeter of membrane, transferred from one side of the membrane to the other, changes the membrane potential by roughly 1 V, then in a spherical cell of 10 microns diameter about 1 /100,000 of the total number of K+ ions in the cytosol have to flow out to alter the membrane potential by 100 mV.

The plasma membrane of many cells also contain voltage-gated cation channels, which are responsible for depolarizing the plasma membrane i.e. to a less negative value inside. Voltage-gated Na+ channels, allow a small

**<sup>\*</sup>Address correspondence to Dr. Brian Dale:** CFA-Italia, Clinica Villa Del Sole, Via Manzoni 15, 80123, Naples, Italy; Tel: 0039-081641689; E-mail<EMAIL_ADDRESS>
amount of Na+ to enter the cell down its electrochemical gradient and this then depolarizes the membrane further, thereby opening more Na+ channels, and so on in an auto amplification mode. In an action potential this may shift the membrane from -70mV to +50mV in a fraction of a second. The Na+ channels then inactivate and voltage-gated K+ channels open to return the plasma membrane potential back to its original resting potential. The efflux of K+ is much more powerful that the influx of Na+ and quickly drives the membrane back toward the K+ equilibrium potential of -70mV. Voltage-gated sodium channels are primarily responsible for action potential propagation in neurons [1]. The channel family consists of 9 members. These channels have two subunits, α and β and several membrane-spanning regions [2]. The primary effect of voltage-gated sodium channels in the plasma membrane is a depolarisation of the cell through the influx of sodium ions. In contrast to that of sodium channels, the primary effect of the opening of voltage-gated potassium channels is a hyperpolarisation of the plasma membrane. This occurs because potassium ions leave the cell cytoplasm, causing a net increase in negative charge in the cell cytoplasm. The role of these channels is to regulate the depolarisation caused by voltage-gated sodium channels – causing the cell to repolarise after an action potential. Potassium channels have a tetrameric structure consisting of four subunits [3]. Voltage-gated calcium channels allow the entry of calcium ions into the cell after depolarisation. The channel is a complex structure consisting of α 1 , α 2 δ, β 1-4, and γ subunits. Voltage-gated calcium channels are commonly involved in muscle contraction, gene expression and neurotransmitter release. There are four common types of voltage-gated calcium channels; L-type, N-type, P/Q type, R-type and T-type [4]. Voltage-gated chloride channels also exist, and play a role in resetting the action potential caused by the opening of other voltage-gated channels.

Cell membranes contain thousands of ion channels and recording with an intracellular microelectrode allows a qualitative measurement of the membrane potential. To quantify and measure the actual currents underlying these voltage changes it is necessary to voltage clamp the membrane with a second intracellular micro-electrode. In 1976, Neher and Sakmann [5] refined a much superior technique for voltage clamping with a single electrode called patch clamping. This new technique, using a fire polished micropipette of about 1micron in diameter, demonstrated current flow through a single channel and took electrophysiology to the molecular level. Breaking the G ohm seal by suction gives access to the cell interior and enables the researcher to whole cell voltage clamp the cell with a single micropipette. Since that date many channel types have been classified in a variety of cells. Patch-clamp recording showed that individual voltage-gated Na+ channels open in an all-or-nothing fashion. A channel opens and closes at random, but when open, the channel always has the same conductance, allowing 1000 ions to pass per millisecond. Therefore, the total current across the membrane reflects the total number of channels that are open at any one time [6].

Voltage-gated Na+ , K<sup>+</sup> , and Ca2+ channels have positively charged amino acids in one of their trans membrane segments that responds to depolarization by opening the channels. Despite their diversity, all these voltage channels belong a large super family of related proteins. Another gene family embraces Cl channels and a class of ligand gated channels activated by ATP [7]. Ligand-gated ion channels are relatively insensitive to the membrane potential and therefore cannot by themselves produce a self-amplifying depolarization. The best example, of a ligand-gated ion channel is the acetylcholine receptor, which was the first channel to be sequenced. The acetylcholine receptor of skeletal muscle is composed of five trans membrane polypeptides encoded by four separate genes, and is nonspecific for ion selectivity. Na+ , K<sup>+</sup> , and Ca2+ may pass through the acetylcholine-gated channel [8].

#### **Transporters or Pumps**

Transporters are long polypeptide chains that cross the lipid bilayer several times and transfer bound solutes across the membrane either passively or actively. Transporters are often called pumps since they are able to "pump" certain solutes across the membrane against their electrochemical gradients [9]. This active transport is tightly coupled to a source of metabolic energy, such as ATP hydrolysis or an ion gradient [9]. The solute binding sites are alternately exposed on one side of the membrane and then on the other. Transporters may be categorized into uniporters that move the solute from one side of the membrane to the other, symporters, that simultaneously transport two solutes in the same direction or, antiporters that transfer two solutes but in opposite directions [9].

# **Antiporters, pH and Ca2+ Regulation**

Marine and mammalian cells need to maintain their pH around a value of 7.2 for the correct functioning of enzymes. To do this they employ one or more Na<sup>+</sup> driven antiporters in their plasma membranes, which use the energy stored in the Na+ gradient to pump out excess H+ . One example is the Na+ /H<sup>+</sup> exchanger*,* which couples an influx of Na+ to an efflux of H+ , while another is the Na+ driven Cl- / HCO3 exchanger that couples an influx of Na+ and HCO3 to an efflux of Cl and H+ [10]. Free cytosolic calcium needs to be maintained at very low levels in all cells. Ca2+ transporters that actively pump this ion out of the cell are the Ca2+ ATPase and the Na+ / Ca2+ exchanger [11].

#### **The Na+ / K<sup>+</sup> Pump**

K+ is typically 20 times higher inside cells than outside, whereas the reverse is true for Na+ (Table **1**) The Na+ / K<sup>+</sup> pump maintains these concentration differences, by actively pumping Na+ out of the cell against its steep electrochemical gradient and pumping K+ in [12]. This pump is vital for survival and it has been estimated that 30% of cellular energy is devoted to its activity. Since the pump is an enzyme it can work in reverse to produce ATP [12]. The electrochemical gradients for Na+ and K<sup>+</sup> and the relative concentrations of ATP, ADP and phosphate determine whether ATP is synthesized or Na<sup>+</sup> is pumped out of the cell. This pump is also involved in regulating osmolarity [12].

**Table 1:** Distribution of the major ions across the plasma membrane of marine and mammalian cells.

**Notes:** In/Out refers to the concentration of the ion species (in mM) inside and outside of the cell cytoplasm.

Ca2+ 10-4

# **ION REGULATION IN GAMETES**

Not surprisingly, gametes possess a wide variety of ion channels and transporters that are essential for their function. Modifying or eliminating, partially, or totally, any of these plasma membrane elements will of course be detrimental to development. Echinoderms have been used extensively to study the electrophysiological properties of the oocyte plasma membrane. In germinal vesicle stage starfish oocytes three types of voltage-dependent currents have been identified. An inward Ca2+, a fast transient K+ and an inwardly rectifying K<sup>+</sup> current [13]. During hormone-induced *in vitro* maturation, the Ca2+ current increases, whereas both K+ currents become smaller [13-14]. These changes are also associated with a decrease in membrane conductance and a depolarization of the resting potential [15-17]. A similar situation was reported for sea urchin GV oocytes [18]. Ca2+ channels were first reported in sea urchin oocytes by Okamoto and collaborators [19].

/2 10-4

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Block and Moody [20] described a transient inward Na+ current, a transient inward Ca2+ current, and an inwardly rectifying K<sup>+</sup> current in ascidian oocytes. Later, this calcium current was shown to be composed mainly of L-Type calcium channels, suggesting a role in regulating cytosolic calcium during early developmental processes [21]. Common voltage gated channels in the oocyte membrane are sodium, potassium and chloride channels. L-type calcium channels are also widely present [20-25].

In amphibians, K<sup>+</sup> and Cl voltage-gated currents present in immature oocytes decrease in mature oocytes, and are replaced by a Na+ current [26-31]. A voltage-dependent hydrogen current has been described in axolotl oocytes [32]. Ion currents have also been measured in molluscs [33-35], and marine polychaetes [36-37]. Ion channels are known to be activated both during germinal vesicle breakdown and during meiosis. For example, in ascidians, a Na+ current is present in prophase I oocytes and this current diminishes as oocytes progress towards germinal vesicle breakdown (GVBD). This is accompanied by a Ca2+ current that reaches its' peak at the time of GVBD [38-40]. The Ca2+ current is driven by L-type calcium channels [38-40]. Prevention of calcium entry reduced the efficiency of both GVBD and the release of calcium after activation [38-40], suggesting that calcium currents during prophase I potentiate GVBD, and fill the internal Ca2+ stores of the oocyte in preparation for fertilisation [38-40]. These observations have been made in many species to date [33, 41-48]. Although the calcium current appears common to many species, the sodium current may be specific to ascidians since it has not been yet demonstrated in other species [20,22,24]. In Xenopus and *Caenorhabditis elegans*, a chloride current that increases the efficiency of GVBD has been found [49-50].

Mammalian oocytes are in connection with their cumulus cells via gap junctions [51-52], however the two cell types have different membrane potentials [53]. Gap junctions are in fact low resistance channels [54] and are involved in meiotic maturation of the oocyte [55-60]. During meiosis progression there are changes in ionic permeability of the mammalian plasma membrane [58-59,61-62] which appear to be due to L-type Ca2+ channels. In bovine oocytes, the activity of the L-type Ca2+ channels decreases throughout meiosis progression [58]. Since Ca2+ is necessary for meiotic progression [63-65], there may be a role for external sources of Ca2+ in the mobilization of intracellular stores during oocyte activation and fertilization.

In human oocytes, the first studies on membrane potential were performed by intracellular recordings in immature oocytes collected by ovariectomy [68-69], while the patch clamp technique showed that the most frequently observed channel in mature oocytes was a 60 pS non-inactivating, K+ -selective pore, which was activated by depolarization [70]. The membrane potential in mitochondria in situ in human oocytes can be measured indirectly by using fluorescent probes, where it has been shown that this parameter may be used to indicate oocyte competence [71-72].

Despite their size, techniques such as voltage and ion-sensitive fluorescent indicators, immunocytochemistry, pharmacology and DNA recombinant technology, have demonstrated the role for ion channels in sperm function [73-75]. Single channel recording first identified K<sup>+</sup> and Cl- channel activity in the sea urchin sperm plasma membrane [76] and this was extended to several animals revealing the presence of K<sup>+</sup> , Ca2+ and Cl channels [77]. Immature spermatogenic cells offer a technical advantage, for the patch-clamp technique. Hagiwara and Kawa [78] first demonstrated the presence of K+ and Ca2+ currents in rat spermatogenetic cells, and later it was shown that the negative resting potential of rat spermatids was determined by Cl and K+ conductance with a minor contribution of Na+ conductance [79]. A role for Cl- conductance in spermatogenesis was shown in *Caenorhabditis elegans* [80], while in mouse spermatogenetic cells [81], a pH dependent Ca2+ permeability factor and a series of K+ -selective currents were correlated with the function of mature sperm [82-83]. Recently, evidence for the regulation of T-type Ca2+ channel expression during mouse spermatogenesis [84] was presented. Spermatozoa are less well studied than oocytes due to the small size and difficulty of applying the patch clamp technique. However, in these cells, both voltage and ligand gated channels are now known to be present [73, 85-92]. Voltage gated channels include sodium, potassium, calcium and anion channels. Voltage gated sodium channels appear to be required for motility [93] whereas voltage-gated potassium and anion channels are associated with the poorly understood capacitation process. Calcium channels are required for the motility, the calcium influx that primes hyperactivation and the acrosome reaction necessary for fertilisation [94]. Spermatozoa appear to have two specific calcium channel types. The first – a sperm-specific channel group termed CatSper located on the flagellum of human spermatozoa [95], appears more involved in hyperactivated motility, whereas L- type voltage gated calcium channels located on the acrosome, [85,88] control the rapid influx of calcium required for the acrosome reaction. Ligand gated channels in spermatozoa are almost completely undefined. However, an ATP –gated sodium channel has been defined, although its' function is not known [96]. Given the fact that spermatozoa must complete several events prior to fusing with the oocyte such as chemotaxis and species specificity, it is likely that other ligand-gated channels will be discovered as techniques improve.

Rothschild [97] demonstrated that K<sup>+</sup> concentration, pH and oxygen tension maintain sea urchin spermatozoa quiescent in the testis. After spawning, a change in these physical parameters induced spermatozoa to swim by acting on the axonema in the tail, while peptides from the outer oocyte layers also change sperm motility [98]. These peptides bind to a sperm plasma membrane receptor which then is transduced to change the modulation of the K<sup>+</sup> channel [76, 99]. K<sup>+</sup> efflux causes a hyperpolarization of the membrane potential, a Na+ /H+ exchange, a rise in pH, increase in cyclic nucleotides, Na+ influx, depolarization of the membrane potential and Ca2+ efflux [74]. In ascidians it has been suggested that K+ permeability increases when the spermatozoon interacts with the oocyte investment, and this induces a -50 mV hyperpolarization of the plasma membrane which in turn elevates cAMP. A cascade of cAMP dependent kinases may then activate sperm motility [100]. T- type Ca2+ channels were also found to be related to the elevation of cAMP [101], suggesting a role for Ca2+ in ascidian sperm chemotaxis. Osmolarity and K<sup>+</sup> concentration are the main factors regulating sperm motility in teleosts. K+ efflux and a rise in intracellular Ca2+ both appear to be responsible for initiation of motility in marine and freshwater teleosts, salmonid and rainbow trout [102-105]. Finally, in mammals, where the role of follicular factors as chemo attractants for sperm is less clear [106], there is some evidence to indicate that intracellular Ca2+ release from stores in the mid-piece is involved that mediates flagellar beating [107-109].

In order to interact and fuse with the oocyte plasma membrane the spermatozoon must undergo the acrosome reaction. In echinoderms, several compounds with acrosome inducing activity have been isolated. These include, fucose sulphate polymers [110], sulphated fucose, galactose, xylose and the acrosome reaction inducing substance (ARIS) [111-113]. The acrosome reaction occurs following binding of these substances to a specific receptor on the sperm plasma membrane and is an ion channel-regulated event. Ca2+ influx is an absolute requirement for the acrosome reaction in the sperm of all species [73], since it seems to be involved in the dehiscence of the acrosomal vesicle and in membrane fusion [74]. Potassium ions also play a role in the acrosome reaction [114-117]. In sea urchin spermatozoa after contact with the jelly layer there is an immediate influx of Na+ and Ca2+ and an efflux of H+ and K+ leading to a change in membrane potential and an increase in intracellular pH [73]. Evidence for the presence of two different Ca2+ channels and their synergistic action in inducing the acrosome reaction has been reported by Guerrero and Darszon [118]. The Na<sup>+</sup> /H<sup>+</sup> exchange and rise in intracellular pH is induced by mobilization of K<sup>+</sup> channels causing a fast and transient hyperpolarization followed by a Ca2+-mediated depolarization [76,119-120]. Cl selective anion channels also identified in the sea urchin sperm plasma membrane may also have a role in the acrosome reaction [121].

In mammals, contact of the spermatozoon with the zona pellucida induces the acrosome reaction causing an increase in both Ca2+ and pH and also a change in the membrane potential [74,122-123]. Ion channels responsible for this calcium entry and increase include, low and high voltage-activated channels, receptor-operated Ca2+ channels, storeoperated Ca2+ channels [85] and T-type voltage-gated Ca2+ channels [73,123]. These channels may also be activated by progesterone or mannose [84,122,124-126]. Although voltage-gated Ca2+ channels have been shown in human sperm [127], their function in the induction of the acrosome reaction has not yet been elucidated [for review see 128]. A capacitating Ca2+ entry mechanism has been proposed as a possible mechanism for gating plasma membrane Ca2+ channels. In mouse sperm, the existence of a Ca2+ influx dependent on depletion of Ca2+ stores has recently been shown [129]. Rossato and collaborators [130] demonstrated that depletion of Ca2+ stores activated gating of Ca2+-activated K+ channels, with a K<sup>+</sup> efflux causing a hyperpolarization, and the capacitative gating of voltagegated Ca2+ channels, with a subsequent depolarization of the plasma membrane. The mammalian sperm head also contains several type of Cl channel [131], while the cytoplasm is rich in Cl ions [132].

#### **PLASMA MEMBRANE EVENTS AT FERTILIZATION**

The importance of ion fluxes across the plasma membrane in the process of oocyte activation has been recognized for over 50 years [133-136]. Direct measurement of electrical events during oocyte activation using intracellular micro-electrodes have been carried out in ctenophores, echinoderms, annelids, teleosts, amphibians and mammals [137-147]. Not surprisingly, most data has been generated in the sea urchin, where it was noted to be a biphasic depolarization [138].

#### **The Initial Step Depolarization and the Latent Period**

Close observation of the initial phase of the activation potential in sea urchin oocytes showed it to be composed of discrete step-like events. Each spermatozoon that entered the egg induced a small, l-2mV, step-like depolarization [148]. A composite shoulder phase indicated polyspermy. In a monospermic situation, of the hundreds of attached spermatozoa, only the fertilizing spermatozoon is capable of reacting with the oocyte, thereby inducing a single step-like depolarization (Fig. **1**) [149]. This report was the first to show how the fertilizing spermatozoon differed from the supernumerary spermatozoa in its capacity to generate a discrete electrical event. This step, which is the earliest detectable event in the egg at fertilization is not seen in parthenogenetically activated oocytes [18]. Voltageclamp studies confirmed this sperm induced electrical event in the sea urchin [150-151], and a comparable event has been identified both in the ascidian [152,153] and in the anuran *Discoglossus pictus* [146]. Fig. **1** shows that in all these deuterostomes the step event lasts about 5-10 seconds at room temperature.

Owing to the rapid succession of change in the oocyte during activation it is difficult to dissect out the cause of the initial step with the subsequent activation events. Germinal vesicle stage oocytes may also be fertilized. However, since they are immature they do not give rise to the cortical reaction, the fertilization potential, or other autocatalytic events seen in the mature oocyte. When a GV oocyte is fertilized not all of the spermatozoa are capable of penetrating the cell or of producing a fertilization cone. Over a thousand spermatozoa may attach to the GV oocyte, but usually not more than 10 enter, as demonstrated by the formation of fertilization cones and histological sections. If we record electrically from immature oocytes, successful spermatozoa give rise to an electrical depolarization and conductance increase and induce a fertilization cone some 50 sec later [154]. Other sperm were not capable of inducing either an electrical event or a cone, whilst a third category induced a step depolarization that after several seconds spontaneously reversed. These sperm did not enter the oocyte, nor did they induce the formation of a fertilization cone. These experiments raised the possibility that the step depolarization was the direct result of spermegg fusion, the conductance increase being due to the appearance of sperm channels in the newly formed syncytium (Fig. **2**). If fusion is inhibited by the ATPase inhibitor Quercetin [155], or by removing Mg2+ [156], spermatozoa are unable to generate electrical changes in the oocyte. Furthermore, the step event may be experimentally reversed by adding a spermicide to inseminated oocytes [154]. Spontaneously or experimentally induced reversible sperm steps have since been seen in a variety of circumstances, [see 151 for review].

Longo and colleagues voltage clamped sea urchin oocytes, fertilized them and then serially sectioned them to locate the fertilizing spermatozoon. Since the authors did not detect gamete fusion until 5 sec after the step depolarization they suggested that the step is a pre-fusion event, suggesting that factors in the sperm plasma membrane cause channel openings in the oocyte through a second messenger mechanism [157-158]. These second messenger systems could be cytoplasmic such as inositol trisphosphate or ADP ribose, or membrane linked such as G-proteins. Since Gprotein activators closely mimicked events of activation, these authors proposed a G-protein linked mechanism for the generation of these depolarisations [159]. McCulloh and Chambers [143,160] have data that show the onset of the step event is coincident with an increase in capacitance. Since, in biological membranes capacitance is proportional to surface area, an increase in capacitance at this moment indicates gamete fusion. To estimate the elementary conductance change underlying the step depolarization in sea urchins the ratio of the change in voltage noise variance to the change in potential was calculated. Knowing the cell membrane resistance during this change the single channel conductance was estimated to be about 30-90pS [149]. It is still not clear if the step event is the result of sperm-oocyte fusion, or the release of a channel gating factor into the oocyte cytoplasm, however since the input resistance of the oocyte decreases from 25 MOhms to 15 MOhms at the step and, assuming the sperm membrane is in parallel with the oocyte membrane, then the sperm would have a conductance of 15nS, or in other words, it would have to contain 40 channels of 400pS each to induce the step event.

**Figure 1:** The fertilization potential in an ascidian oocyte (a), a frog oocyte (b) and a sea urchin oocyte(c). Note in all cases the potential is preceded by a small step depolarization, thought to indicate sperm-oocyte fusion, that is accompanied by an increase in voltage noise in (a) and (c) and an increase in conductance in (b). These voltage recordings were made with intracellular micro-electrodes.

In most animals there is a time delay from the moment the spermatozoon attaches to the oocyte surface until cortical exocytosis. The delay between the two corresponds more or less to what has been described in sea urchins as the latent period [see 161 for a review], during which there is no obvious morphological change in the egg surface. Rothschild and Swann in 1952 suggested that a fast propagated change traverses the oocyte surface during the latent period [162]. The sea urchin oocyte is a useful model to study the latent period for two reasons; first because the latent period is relatively long and is temperature dependent [18,149,161], and second because the cortical reaction may be reversibly interrupted by a mild heat shock [163,164], giving rise to "partially fertilized oocytes". In these oocytes, 50% of the surface may be activated, while the rest is undistinguishable from a virgin oocyte. Upon reinsemination spermatozoa are able to interact with the " virgin surface " of such oocytes and therefore it appears unlikely that any major change has traversed this area during the latent period [165]. When fertilization occurs in the presence of the microfilament inhibiting agents cytochalasin B or D, the latent period is increased by up to 100% [166]. In contrast, there is no change if the gametes are pre-exposed to these agents and subsequently fertilized in natural sea water. Together these experiments suggest that a microfilament dependent stage of sperm-oocyte interaction occurs during the latent period. If the preceding arguments are correct this event is post fusion.

**Figure 2:** One theory for the step-like depolarization is that it represents sperm-oocyte fusion. This electrical event resulting from the ion channels in the sperm plasma membrane now in parallel with those in the oocyte plasma membrane.

#### **The Fertilization Potential**

Mature oocytes are low conductance cells containing a wide variety of both voltage-gated and chemically-gated channels. Approximately 5-10 seconds after the step depolarization there is a much larger depolarization called the 'fertilization potential'. At about the same time there is a massive release of Ca2+ from intracellular stores and shortly after a cortical reaction leading to cortical granule exocytosis in the sea urchin or a surface contraction in the ascidian [18,167-169].

The potential remains at a positive value for several minutes and then gradually returns to its original value [170]. Recording voltage with an intracellular microelectrode allows a qualitative measurement of the electrical event. To quantify and define the actual molecular mechanism underling these voltage changes it is necessary to voltage clamp the membrane with a second intracellular micro-electrode or use the patch clamp technique. Using the patch and whole cell clamp techniques a new population of chemically gated channels called "fertilization channels" were identified in the ascidian oocyte plasma membrane at fertilization.

Fertilization channels in Ciona were found to have a single-channel conductance of 400pS [171]. Since the reversal potential was around 0 mV, it was suggested that these channels were not ion specific. To date these channels are amongst the largest observed in biological membranes. Whole-cell currents in ascidian oocytes also studied during fertilization were shown to peak near -30 mV and approach zero near 0 mV [172], supporting the single-channel data. Knowing the total conductance change at fertilization, the single channel conductance and the probability of a channel being open, we estimated that the fertilizing spermatozoon opens between 200 to 2000 fertilization channels in the oocyte. The fertilization current is a long bell shaped current of about 1000pA over a 60 second period. Since it is inward, and we know that 10<sup>7</sup> ions may flow through an acetycholine channel in one second considerable movement of Na<sup>+</sup> and Ca2+ into the oocyte may occur through these channels.

Nude ascidian oocytes may be cut into small fragments and each fragment has the capability of developing into an embryo. By using the whole cell clamp technique on unfertilized and fertilized fragments and inseminating each fragment, it was found that fertilization channel precursors and voltage-gated ion channels are uniformly distributed around the ascidian oocyte surface [173-175]. Since fertilization currents were similar in whole oocytes or fragments, irrespective of their size and global origin, it was concluded that the fertilizing spermatozoon opens a fixed number of fertilization channels limited to an area around its point of entry (Fig. **3**) [174]. The localized ion current through these channels may regulate movements of the cytoskeleton involved in cytoplasmic segregation.

In the sea urchin, the fertilizing spermatozoon triggers an inward current of about -500pA, while the conductance increases from 20 to 40 nS. The I/V curve for this current and the reversal potential of about +10 mV suggest it is non specific for ions [176]. As we have mentioned in the previous section there are also several types of voltage gated ion specific channels in oocytes and these may also be activated during fertilization, however the reversal potential for Ca2+ and Na+ channels does not coincide with the reversal potential for the fertilization current [140,147,177-180].

In amphibians, a Cl specific channel is responsible for the fertilization potential. For example, the membrane depolarization at fertilization in *Xenopus laevis* is influenced by the external chloride concentration [181,182] and this is a consequence of Cl ion efflux [183]. Similarly, a positive shift in the fertilization potential was shown in *Rana pipiens* associated with an increase of either K+ or Cl- and a decrease in Na+ conductance [29,184]. Membrane potential changes in *Rana cameranoi* oocytes are also based on K+ as well as on Cl conductance [185]. Cl- ions are apparently responsible for the first depolarization phase evoked by sperm, whereas K+ contributes to the repolarizing phase. The role for Ca2+ channels in the amphibian fertilization potential is less clear.

**Figure 3:** Whole-cell voltage clamping allows quantification of the electrical events at fertilization. The current measured is a direct measurement of the ionic flow into the oocytes. In a whole ascidian oocyte the fertilization current is about -700pA (a). Oocytes may be cut into small fragments and inseminated (b). Note that the current is of similar amplitude, indicating that the spermatozoon gates a fixed number of fertilization channels localized to the area of fusion.

In the anuran *Discoglossus pictus* the sperm entry site is a pre-determined specialized area called the animal dimple [186]. Discoglossus oocytes as other frog oocytes may be activated by pricking with a steel needle. (It should be pointed out that oocytes from other animal groups are not activated upon pricking). At fertilization, the spermatozoon initiates a large regenerative depolarization that is Cl dependent [146]; pricking elicits a comparable response. When oocytes were pricked outside the dimple area, we saw a wave of contraction spread from the puncture site to the antipode. Since the activation potential was not generated until the wave reached the dimple, it appears that the channels underlying the depolarization are found in the dimple, and that they are gated by a second messenger liberated in the egg cytoplasm that spreads around the oocyte with the contraction wave.

The fertilization potential in mammals is similar in time course to those in invertebrates and amphibians, however inverted in polarity. The first measurements of membrane potential at fertilization in the hamster and mouse showed a series of hyperpolarizations [187-188], while subsequent studies showed the underlying fertilization current to be an outward Ca2+ activated K+ current in the human [189] and bovine [60]. The pattern in rabbit oocytes is different, in that a preliminary depolarization is followed by a repeated diphasic hyperpolarization/depolarization pattern of membrane activity [190].

#### **Gating of the Fertilization Channels**

Fertilization channels are specialized ligand-gated ion channels To date, two ligands have been identified, Ca2+ and ADP-ribose. In ascidians, the fertilization channels are not Ca2+ gated, but by ADP-ribose. In fact, raising the level of intracellular Ca2+ in ascidian oocytes, by perfusion or by loading the oocyte cortex (>50uM) with Ca2+ through voltage gated channels, did not activate fertilization channels. Alternatively, oocytes exposed to low-Ca2+ sea water, perfused with the Ca2+ chelator K-EGTA or Ca2+ blocking agents to prevent the release of Ca2+ from intracellular organelles, and subsequently inseminated, generated fertilization currents [191]. Oocytes exposed to the Ca2+ ionophore A23187 were found to contract without generating a fertilization current, while microinjection of InsP3 or soluble fractions of homogenized spermatozoa induced both a contraction and a fertilization current [192]. Although elevated Ca2+ does not gate fertilization channels in ascidians, it appears to be involved in the mechanism of cortical contraction [169,192-193]. Measurements with ion selective electrodes show that the intracellular pH of ascidian oocytes ranges from 7.2- 7.4 and does not vary during activation, making pH an unlikely trigger of early activation events [194].

Nitric oxide has been shown to increase in ascidian oocytes at fertilization [195]. Generating cytosolic nitric oxide in ascidian oocytes with the donor sodium nitroprusside triggers a fertilization-like current and the release of intracellular calcium through a ruthenium-red sensitive mechanism [195]. While, micro-injection of soluble extracts of ascidian sperm cause calcium release in ascidian oocytes but not gating of the fertilization channel [196]. With whole cell and single channel recording it has been shown that the fertilization channel is directly gated by ADPribose (Fig. **4**). The channel was shown to be permeable to Ca2+ and Na+ , with a reversal potential of 0 to +20mV, and a unitary conductance of 140pS. BAPTA or antagonists of intracellular calcium release did not inhibit the ADPribose current showing it is activated in a calcium-independent manner. In situ, the fertilization current is blocked by nicotinamide [197]. In conclusion, ascidian sperm trigger the hydrolysis of nicotinamide nucleotides in the oocyte to ADP-ribose which directly gate the fertilization channels and also assists in the release of intracellular Ca2+ (Fig. **5**) [195,197,198]. Since sperm extracts do not trigger all activation events it seems probable that there are multiple pathways at activation, gated by more than one factor from the spermatozoon.

In the sea urchin, the fertilization current may be induce by micro-injection of InsP3 and inhibited by BAPTA and therefore probably Ca2+ gated [176], while in amphibian oocytes there is evidence that intracellular Ca2+ contributes to gating the Cl channels [26]. Glahn and Nuccitelli [199] recorded the fertilization current in Xenopus oocytes and showed it to be generated by Ca2+-activated Cl channels. In mammals, the fertilization channels are Ca2+-activated K+ channels [60, 200-201]. Loading mammalian oocytes with EGTA prevents hyperpolarization, while Ca2+ injection into the oocyte triggers a hyperpolarization. The oscillations in membrane potential coincide with the oscillations in intracellular calcium [for review see 202] that continue up to pronuclear formation [203]. These oscillations decrease in frequency and amplitude during their progression. The oscillations are large in hamster [204] and smaller in mouse [142, 205] and rabbit [190]. After oocyte activation, plasma membrane Ca2+ channels have an increased role in replenishing stores for the continuation of Ca2+ oscillations [206], in fact in hamster, external Ca2+

is required for oocyte activation [188]. In the bovine, a clear relationship between electrical properties of the oocyte plasma membrane and intracellular calcium modifications has also been shown following fertilization, as well as following chemical oocyte activation or after exposure to specific Ca2+ mobilizers [60]. In the human oocyte, the fertilization current is bell-shaped and outward [189] and may be activated by sperm extracts in the mouse [207] or following ionophore exposure in the human [201].

**Figure 4:** Single channel patch-clamp recordings show that the fertilization channel in the ascidian oocyte plasma membrane is directly gated by the second messengers cADPr and ADPr. The top trace is before addition of the reagents. cADPr was used at a concentration of 5uM and ADPr at 10nM. The lower trace is an outside-out configuration after excision of a patch of membrane from the oocyte. The reversal potential for the channel is +20mV.

#

**Figure 5:** A summary of activation events in the ascidian oocyte. The fertilizing spermatozoon fuses to the plasma membrane and releases sperm factor(s) into the oocyte. These factor(s) stimulate both the production of IP3 and ADPr, the latter through the production of NO. ADPr gates the fertilization channels, IP3 gates the release of intracellular Ca2+.

#### **REFERENCES**

<sup>© 2011</sup> The Author(s). Published by Bentham Science Publisher. This is an open access chapter published under CC BY 4.0 https://creativecommons.org/licenses/by/4.0/legalcode

# **CHAPTER 8**

# **Recent Advances in the Understanding of the Molecular Effectors of Mammalian Egg Activation**

# **Christopher Malcuit1 and Rafael A. Fissore2\***

*1 Bioengineering Institute, Worcester Polytechnic Institute, Worcester, Massachusetts, 01605, USA and <sup>2</sup> Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, Massachusetts, 01003, USA*

**Abstract:** Fertilization is the process by which male and female gametes, and their respective haploid genomes, fuse to form a single cell (the zygote) containing a complete diploid complement of genetic material representative of each parent. The male gamete, the sperm, transduces an activation stimulus to the awaiting female gamete, the egg (also, oocyte or ova), initiating a cascade of events collectively referred to as egg activation. The mechanisms of this process are both complex and tightly coordinated in order to impart a high level of fidelity necessary for initiation of embryonic development and propagation of the species. Although mechanistic discrepancies of this event exist even between species of the same phyla, calcium is the predominant driving force of egg activation in all species studied to date, and is responsible for promoting the resumption and exit of meiosis and the initiation of the developmental program. This chapter will focus on recent discoveries of the molecular mechanisms of egg activation with specific regard to the regulation of calcium signaling as it appears in the oscillatory mammalian system.

#### **INTRODUCTION**

In mammals, ovulation occurs after oocytes have reached – and become arrested at – the second meiotic metaphase, or *MII*. Prior to reaching this stage, oocytes undergo a series of processes that last for weeks or months (according to the species) and that entail growth of the oocyte, and transcription / storage of key maternal mRNAs required for cellular metabolism prior to activation of the embryonic genome. Immediately prior to ovulation, fully grown oocytes enter a process termed *oocyte maturation* that spans 12 to 40 hr during which they experience accumulation and activation of metaphase-associated kinases, and a major reorganization of cytoskeletal and key calcium (Ca2+) sensitive elements [1-5]. Such a remodeling of effector molecules and organelles is both necessary and critical for mounting an appropriate response to the fertilizing sperm, as aberrations in maturation events render the oocyte either incapable of fertilization or developmentally incompetent [6].

Shortly after fusion the sperm evokes – in the egg – a series of molecular cascades, collectively referred to as *egg activation* that result in three major groups of events (reviewed in detail in [7-9]): **1.** The release of cortical granule material to create membrane and zona pellucida modifications resulting in a block to polyspermy [10-11], **2.** the destruction of cyclin B [12] and synchronous deactivation of M-phase associated kinases thereby leading to exit from MII arrest and the resumption of meiosis [13-15], and **3.** recruitment of stored maternal mRNAs, formation of the pronuclei, and full initiation of embryonic development [16-17]. These events are made possible by an increase in the concentration of intracellular free Ca2+ ions ([Ca2+]i) within the egg. Although the precise mechanism by which the sperm initiates the Ca2+ release that is responsible for triggering embryonic development is still under scrutiny, in all species studied to date it has been shown to involve the activation of the phosphoinositide (PI) pathway [18, 19]. Activation of the PI pathway following fertilization results in the production of inositol 1,4,5 trisphosphate (IP3) and 1,2-diacylglycerol (DAG) via the hydrolysis of phosphatidyl 4,5-bisphosphate (PIP2) by a phosphoinositide-specific phospholipase C (PLC) isoform [20-23]. A resulting increase in the intracellular concentrations of IP3 is responsible for mediating Ca2+ release by binding and gating its receptor, the type I IP3 receptor (IP3R1, [24]), a tetrameric ligand-gated Ca2+ channel located on the endoplasmic reticulum (ER) membrane, the main Ca2+ store of the cell [25-26]). Additionally, production of DAG may be involved in the regulation of Ca2+ influx [27] either directly [28], or indirectly via activation of protein kinase C (PKC, [29, 30]).

Spatiotemporal dynamics of Ca2+ release differ markedly in eggs of different species. For example, organisms such as the frog and sea urchin, where fertilization takes place in a very accessible, ex vivo, environment, a single, ~10

**<sup>\*</sup>Address correspondence to Rafael A. Fissore:** Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, Massachusetts, ISB, 661 North Pleasant Street 01003, USA. Tel:<PHONE_NUMBER>; Fax:<PHONE_NUMBER>; E-mail<EMAIL_ADDRESS>
minute [Ca2+]i wave is propagated almost immediately following sperm-egg interaction (reviewed in [31]). This mechanism no doubt evolved to achieve formation of the fertilization envelope to block polyspermic fertilization in such a promiscuous environment [32]. Conversely, in mammals, where sperm must traverse the oviduct to reach the egg, the potential rate of polyspermy is much lower, and the mechanism to block polyspermy is a slower process involving exocytosis of cortical granule material [10]. In these animals, Ca2+ efflux from the ER takes the form of long lasting (>5 hours) and persistent (every 15-60 minutes, species-dependent) oscillations beginning shortly after fertilization ([33]). It is the full complement of this regime of [Ca2+]i oscillations that is both necessary and sufficient to achieve complete activation of the developmental program [16-17]. This chapter will highlight recent progress in the understanding of the molecular events underlying egg activation in mammals. For more comprehensive reviews on the subject, readers are referred to excellent recent reviews by Parrington et al ([34]), Horner and Wolfner ([35]) and Ducibella and Fissore ([9]).

# **[CA2+]I OSCILLATIONS, CA2+ EFFECTORS AND EGG ACTIVATION**

The certainty that Ca2+ was the mediator of egg activation led to the search for effectors capable of transducing the elevations in [Ca2+]i into events of egg activation. A kinase that immediately emerged as a candidate in participating in the biochemical changes leading to egg activation was the type II Ca2+/calmodulin-dependent protein kinase (CaMKII). Earlier studies have implicated CaMKII in the exit of MII in *Xenopus* eggs, as studies showed that its activation or injection of a constitutively active (CA) form of the protein induced MPF inactivation and exit from the MII arrest [36-37]. At the time of these findings, the mechanism underlying MII arrest and its abrogation by CaMKII were still unknown. Nonetheless, subsequent studies have shown that active CaMKII is required to promote the activation of the anaphase promoting complex (APC) [38] through destruction of a member of the early mitotic inhibitor 2 (Emi2) protein. The APC, an E3 ubiquitin ligase, targets cell cycle proteins such as cyclin B and securin for proteasomal degradation following their poly-ubiquitination [39-44]. The loss of cylin B, the regulatory subunit of MPF [45] and limiting factor for the maintenance of the metaphase state, results in the exit from M-phase and thus the resumption of meiosis. Therefore, at the MII arrest, the activity of the APC complex is held in check by the action of Emi2, whose destruction depends on a CaMKII priming phosphorylation [46]. Interestingly, this Ca2+ mediated cell-cycle transition is thought to be unique to eggs [42], and requires stable levels of cytostatic factor (CSF) [12, 47], which is itself, unique to eggs [48] and that is responsible for stabilizing MPF.

As previously noted, while it was evident that Ca2+ and CaMKII were indispensable for egg activation, the molecular partners that made possible this cellular transition were not known. Furthermore, an additional puzzling finding was the discovery that polo-like kinase 1 (Plk1), a kinase originally described to be involved in spindle and chromosome organization [49], was also required for egg activation. Descombes and Nigg [50] had shown that elimination of Plk-1 from *Xenopus* egg extracts prevented the ability of Ca2+-CaMKII complex of promoting MII exit. Interestingly, these findings were not reconciled until recently when the functional association between these two kinases was clarified. Research shows that in addition to phosphorylation by CaMKII, Emi2 requires a subsequent phosphorylation by Plk1 [51], after which it is targeted for degradation by the SCF (Skp1-Cullin F-box) ubiquitin ligase complex allowing MII exit. Further, several recent studies have consolidated the primary role of CaMKII as the main Ca2+ effector responsible for MII exit after fertilization in mammals [29]. Using expression of cRNAs encoding for a CA-CaMKII protein these studies showed that this protein alone can promote exit of MII as well as support pre-implantation development to the blastocysts stage [52]. Although the developmental competence of these embryos was not tested further, i.e. development to term, these studies highlight the ability of CaMKII to integrate and translate the majority of the signals encoded by the fertilization-initiated [Ca2+]i oscillations.

Several important questions remain regarding the role of Ca2+ and CaMKII in egg activation including whether [Ca2+]i oscillations are required or offer an advantage over single [Ca2+]i rises for development to term. To this end, elegant studies by Ozil and co-workers initially appear to support this paradigm. In their studies, activation methods that mimicked, more closely, the sperm-like patterns of oscillations resulted in higher rates of implantation and development [17, 53, 54]. In these studies the amplitude, frequency, and number of [Ca2+]i rises were modulated by electroporation of extracellular calcium ([Ca2+]e) into the ooplasm of rabbit and mouse eggs. Notably, each parameter tested had a marked effect on the rates of egg activation as well as post-implantation development. In line with the developmental benefits of Ca2+, more recent studies revealed that different events of egg activation have dissimilar requirements regarding their initiation and completion, with the completion of each event needing a higher number of rises [16-17]. For instance, it was shown that recruitment of maternal RNAs, as evidenced by the detection of new protein synthesis, was initiated by the administration of 8 [Ca2+]e pulses but only became fully apparent, or fertilizationlike, in zygotes receiving a total of 24 [Ca2+]e pulses [16]. While these results imply a correspondence between [Ca2+]i oscillations and protein synthesis, it is important to discriminate whether the observed changes in protein profiles were not simply due to progression into zygotic interphase, which seemed to exhibit similar requirements for [Ca2+]e pulses [16]. Nonetheless, the approach used in this study, followed by identification of the proteins regulated by Ca2+ pulses, could prove invaluable in elucidating yet uncharacterized signaling pathways during egg activation. Follow up studies by the same authors have challenged the initial view that egg activation by [Ca2+]i oscillations offer developmental advantages. In these studies, the authors show that a single, large Ca2+ pulse where [Ca2+]i remains elevated approximately the same amount time than that attained by repeated oscillations accomplishes similar rates of embryo development to that induced by sperm-initiated oscillations [55]. Therefore, these studies raise the prospect that for all events of egg activation to unfold in a manner that is consistent with embryo development, the egg must receive a "threshold Ca2+ signal"; whether it is in the form of a single rise or multiple oscillations is not important for the final outcome [55]. While this interpretation and findings are helpful to gain insight into how eggs "read" or "decode" the Ca2+ input initiated by the sperm, it is worth noting that in those studies [Ca2+]i increases were attained without stimulation of the PI pathway, the activation of which with the subsequent, and possibly persistent, production of DAG and activation of PKC, may have detrimental consequences for development given the persistent Ca2+ influx that might elicit [29]. Therefore, it is possible that under natural conditions of fertilization in mammals, [Ca2+]i oscillations might be the only way for the sperm to deliver the "threshold Ca2+ signal".

An additional benefit of [Ca2+]i oscillations, and the subsequent activation of CaMKII, might be in the regulation of zygotic gene expression. Two recent studies have demonstrated that the gene expression profile of late preimplantation embryos is susceptible to the egg activation procedure. In one study, both premature termination of [Ca2+]i oscillations or excessive Ca2+ stimulation, while it did not affect embryo development to the blastocyst stage, influenced gene expression and pre- and post-implantation development, respectively [17]. Another study found that activation in the absence of [Ca2+]i elevation compromised pre-implantation development and gene expression [56]. It remains to be demonstrated whether the effects of Ca2+ on gene expression are mediated directly by the [Ca2+]i rise acting on transcription/translation regulators [57] or if it may be due to the downstream activation of CaMKII activity. In this regard, it is interesting to note that the activity of CaMKII not only increased in response to augmenting levels of [Ca2+]i, but oscillated in tight coordination with each [Ca2+]i transient after fertilization, as determined in single eggs by simultaneous monitoring of [Ca2+]i levels and kinase activity [58-59]. Importantly, in hippocampal dendrites, CaMKII directly phosphorylates the cytoplasmic polyadenylation element binding protein (CPEB) that is directly responsible for the polyadenylation, recruitment, and translation of mRNAs [60]. A similar mechanism therefore, could potentially function in the egg. This would provide a direct link between the results mentioned above in which repetitive [Ca2+]i pulses modulated protein expression patterns from stored maternal mRNAs [16-17], as some of the early recruited mRNAs may be critical for the activation of the zygotic genome [61].

It is worth noting that other kinases such as PKC [27], or proteins such as actin [62, 63] and calreticulin [64], may also serve as effectors of [Ca2+]i rises during egg activation. For example, conventional PKCs, which are activated by DAG and [Ca2+]i rises may play an important role in regulating Ca2+ influx that is required to maintain oscillations during fertilization [27, 29]. Another kinase that deserves a closer look is the myosin light chain kinase (MYLK2). Recent studies using CA-CaMKII show that of all the egg activation events, the only one not recapitulated by CA-CaMKII was the exocytosis of cortical granules (CG; [52, 65]). Interestingly, inhibition of MYLK2 reduced CG exocytosis and prevented extrusion of the second polar body, suggesting that this kinase is also an effector of Ca2+ during mammalian egg activation [62]. Therefore, while CaMKII may be the master translator of the [Ca2+]i oscillations during mammalian fertilization, other proteins and kinases may act more subtly to impart full developmental competence. The generation of isoform-specific CaMKII null mice should be used to precisely ascertain what events of egg activation are exclusively regulated by CaMKII and how activation with and without [Ca2+]i oscillations impact gene expression and developmental competence1 .

# **INITIATION OF [CA2+]I SIGNALING**

There has been much debate and speculation as to the mechanism(s) that triggers activation of the PI pathway and [Ca2+]i oscillations during mammalian fertilization, and this has led to the proposal of several theories to explain this

<sup>1</sup> Note. A recent manuscript using isoform-specific CaMKII null mice demonstrated that two events of egg activation, cortical granule exocytosis and recruitment of maternal mRNAs, are not under the direct control of CaMKII. Backs J et al. Proc Natl Acad Sci USA 2010; 107:81-6.

phenomenon. One early hypothesis, referred to as the "conduit hypothesis", proposed that sperm fusion allows Ca2+ to passively enter the egg. However, the findings that the initial fertilization Ca2+ responses proceed unaltered in the absence of [Ca2+]e [13, 66], clearly demonstrated that [Ca2+]e is not necessary for the initiation of [Ca2+]i oscillations. The "receptor hypothesis" suggests that upon sperm-egg membrane contact, receptor-ligand interactions on the surface of the gametes relay intracellular signaling events that initiate Ca2+ release in the egg. One of the signaling cascades thought to be engaged by the interaction of gametes is that mediated by protein tyrosine kinases (PTKs). Specifically, the Src-family of PTKs (SFKs) may activate PLC [67-73], thereby triggering Ca2+ release through the production of IP3. In accordance with this notion, PTK and PLC activity are up-regulated shortly following fertilization in echinoderm and fish eggs [21, 74, 75], and fertilization-triggered Ca2+ signaling in egg extracts was reconstituted by adding activated membrane raft fractions [70], which suggests the presence of a receptor-mediated SFK/PLC activation model in Xenopus fertilization. Furthermore, inhibition of PLC activation by a dominant negative approach using over expression of PLC SH2 domains prevented the sperm-induced [Ca2+]i rise in sea urchin [76] and starfish eggs [77]. A recent study demonstrated the presence and participation of additional SFK isoforms in sea urchin fertilization and found direct interaction between SFK1 and PLC in this species [78]. Nonetheless, in mammals, extensive pharmacological studies [79] along with dominant negative approaches [80-81] and injection of recombinant PLC [82] failed to show any involvement of this pathway in evoking/preventing fertilization-like [Ca2+]i responses. This is in spite the fact that PLC and SFK isoforms are expressed in mouse eggs [81, 83], and that stimulation of its activity in these cells by exogenous expression of tr-kit, a sperm tyrosine kinase which activates Fyn, a SFK present in mouse and rat eggs [84], was able to induce exit from meiosis and pronuclear formation [85]. Therefore, available evidence argues against a role for SFKs in the initiation of [Ca2+]i oscillations in mammalian fertilization. Importantly, more recent studies have demonstrated pivotal functions for these kinases in a wide variety of events involving oocyte maturation, MII exit and zygotic progression in the mouse [86-88]. Lastly, extensive studies, which initially relied on the injection of activators and inhibitors of alpha subunits of Gq proteins, implicated the activation of PLCβ isoforms in the initiation of Ca2+ release in mammalian fertilization [89-91]. Although later findings demonstrated that inhibition of Gαq subunits by injection of a function-blocking antibody was without effect on fertilization-induced [Ca2+]i oscillations [92], and mice lacking one of the PLCβ isoforms are fertile [93], more recent studies utilizing RNAi knockdown of PLCβ1 in mouse eggs showed that the amplitude, but not the frequency or duration of [Ca2+]i rises was impacted in these eggs [94].

As the above mentioned experiments could not physiologically reproduce the initiation of [Ca2+]i oscillations in mammals, the need for a novel mechanism to explain the initiation of oscillations in these species was warranted. The hypothesis that emerged, "the fusion hypothesis", proposed that upon gamete fusion the sperm delivers a factor, commonly referred to as the sperm factor (SF), into the ooplasm capable of activating the PI pathway and oscillations [95]. The initial, and sole, experimental support for this hypothesis was the demonstration that injection of sperm extracts into mammalian eggs was able to replicate the pattern of oscillations initiated by the sperm [66, 96-98]. Curiously, the first demonstration of this mechanism was obtained in sea urchin eggs [99], a species in which, paradoxically, the hypothesis under discussion may not account for the mechanism of fertilization (see above). Remarkably, earlier evidence in mammals that could have offered support for the fusion theory seemed to have been overlooked, as injection of human and hamster sperm heads into mouse oocytes resulted in pronuclear formation, implying the occurrence of oocyte activation without sperm-oolema interaction [100], which are the basis of the sperm factor hypothesis. Nonetheless, the subsequent use of the same technique in humans, aptly named intracytoplasmic sperm injection (ICSI), which resulted in the birth of young [101] and was also shown to initiate fertilization-like [Ca2+]i responses [102-104] consolidated the concept that a sperm product was responsible for initiating oscillations in mammalian eggs. While the success of ICSI unquestionably implicates the sperm as the harbor of the factor that triggers oscillations, close examination of the ICSI-induced [Ca2+]i responses underscores the notion that events that take place prior to or during interaction of the gamete membranes are pivotal for normal fertilization. For instance, in mouse eggs, ICSI-initiated oscillations occur less frequently after the first hour and show premature termination [102]. More revealing still are the findings in large domestic species where fertilization by ICSI fails altogether to initiate fertilization-like [Ca2+]i oscillations [105, 106].

#### **IDENTIFICATION OF THE 'SPERM FACTOR'**

While early studies assumed that all SF activity was rapidly released into the ooplasm [96-97], subsequent studies revealed that the Ca2+-inducing activity was also present in detergent-resistant sperm domains, most likely the sperm perinuclear theca [107-111]. Consistent with the concept of SF distribution to several sperm compartments was the demonstration that complete release of SF activity into the ooplasm required 2 hours [108]. Second, consistent with its perinuclear localization in the sperm, *in vitro* fertilization and ICSI studies showed that sperm's Ca2+-releasing activity could be recovered after fertilization, as it associated with the pronuclei of the developing zygotes [108, 112-113]. Third, *in vitro* PLC assays using sperm extracts showed that these extracts possessed high PLC activity, nearly twice as high as the activity present in other tissues known to express several PLC isoforms [22, 114]. Importantly, the PLC activity of sperm extracts is prominent even in the presence of basal [Ca2+]e, which is very relevant given that this molecule is expected to initiate oscillations in mammalian eggs, which at the time of fertilization show [Ca2+]i basal levels of 0.1 M. Therefore, since several PLC isoforms are expressed in mammalian sperm [93, 115-116], these enzymes surfaced as logical candidates to be the SF. Importantly, injection of recombinant proteins representing most of the isoforms expressed in sperm failed to initiate oscillations, or it did so at non physiological concentrations [82]. Furthermore, chromatographic fractionation of sperm extracts revealed that none of the known PLCs were present in the fractions with [Ca2+]i oscillation-inducing activity [116-117]. Hence, if a sperm PLC were to be the SF, it had to be a novel PLC. To this extent, a novel sperm-specific PLC, PLC [23], was identified in a PLC homology screen of mouse testis expressed sequence tags. Initial studies revealed that PLC exhibits [Ca2+]i oscillation-inducing activity ascribed, thus far, only to the sperm or SF.

PLC is to date, the most elementary of PLC isoforms identified. In concurrence with the modular organization of other PLCs [118], PLC consists of 2 Ca2+-binding EF hands, X and Y catalytic domains, and the Ca2+-dependent phospholipid-binding C2 domain [119]. Notably, PLC lacks the typical pleckstrin homology (PH) domain, which has been found in all previously identified PLC isoforms [120]. In support of its purported role as the SF, injection of recombinant PLC [121-122] or PLCζ cRNA has been shown to evoke sperm-like- oscillations in mouse [23, 121-124], rat [125], human [126], bovine [127-128], porcine [129], and equine [130] eggs. In addition, *in vitro* PLC assays using recombinant PLCζ revealed high enzymatic activity at basal [Ca2+]e concentrations [122]. Moreover, PLCζ cRNA-induced oscillations in mouse eggs cease at approximately the time of pronuclear formation, which is comparable to what is observed after natural fertilization [124, 131]. Further, zygotes activated by injection of PLCζ cRNA showed high rates of *in vitro* development to the blastocyst stage [23, 123]. Lastly, a report in mouse sperm has localized PLCζ to the post-acrosomal region of mouse sperm [121] and to the equatorial region of bull and human sperm [132-134], which are the first sperm areas thought to come in contact with the ooplasm after the fusion of gametes, respectively [111]. Collectively, the evidence in support of PLCζ as the mammalian SF is compelling. Nevertheless, important questions remain to be addressed regarding the expression, localization and storage of PLCζ in sperm, its mechanism of release into the ooplasm, and mechanism(s) of activation once in the egg. Additionally, it needs to be demonstrated whether or not PLCζ represents the sole inducer of [Ca2+]i oscillations during fertilization and whether its absence impacts fertility.