Abstract
In 2002, a report suggested that oocyte activation is induced by Plcz1 in mouse oocytes, which prompted great interest in exploring the role of sperm PLCZ1. Thus, PLCZ1 loss-of-function experiments became a crucial tool for addressing this subject. Although the only option to completely delete a target protein in fully functional spermatozoa is to use gene-deficient animals, Plcz1-deficient mice were not reported until 2017. Challenges to obtain suitable in vivo models have been related to altered expression of Capza3, a neighbor gene to Plcz1 locus in mammalian genomes that is required for spermatogenesis. With the advancement of genome-editing technologies, two groups independently and simultaneously produced Plcz1 mutant mouse lines, which were the first animal models to be artificially and reliably deficient for sperm PLCZ1. All Plcz1 mutant mouse lines display normal spermatogenesis and, surprisingly, subfertility rather than complete infertility. Moreover, analysis of oocyte Ca2+ dynamics indicates that mouse PLCζ1 is an essential sperm-derived oocyte activation factor via intracytoplasmic sperm injection, as PLCZ1 deficiency causes a complete lack of Ca2+ oscillations. This seemingly contradictory phenotype can be explained by atypical Ca2+ oscillations that are provoked slowly and less frequently in the case of fertilization accompanied by physiological sperm–egg fusion. These findings not only raise new questions concerning the sperm basic biology, by clearly demonstrating the existence of a PLCZ1-independent oocyte activation mechanism in mice, but also have implications for the treatment and phenotypic interpretation of patients presenting oocyte activation failure.
Introduction
Mammalian oocytes arrested at metaphase II stage while they are spawned into the oviduct and wait for the spermatozoon to resume their cell cycle. This resumption, as part of oocyte activation processes, is triggered by serial increases (and decreases) of cytosolic-free calcium ion, named Ca2+ oscillations (Sanders & Swann 2016, Swann & Lai 2016). The success of intracytoplasmic sperm injection (ICSI) accompanied with the Ca2+ oscillations in mammalian oocytes suggests the existence of a sperm-borne oocyte activation factor (SOAF) in the sperm head (Kurokawa & Fissore 2003). Moreover, SOAF identification as a proteinaceous factor has been explored by researchers in this field (Perry et al. 1999, 2000). Parrington et al. also suggested the involvement of glucosamine-6-phosphate isomerase in this process in mice, also called Oscillin in hamsters (Parrington et al. 1996); however, Wolny et al. denied the involvement of Oscillin in mammalian oocyte activation, as determined by the expression of a recombinant Oscillin homolog (Wolny et al. 1999). Moreover, the postacrosomal sheath WW domain-binding protein (PAWP), which is a WBP2 N-terminal-like protein that localizes in the cytosolic portion of the sperm head, has also been suggested to be involved in the mammalian oocyte activation (Wu et al. 2007). Nevertheless, PAWP was shown to be dispensable for oocyte activation in mice, as PAWP-null sperm is still able to trigger Ca2+ oscillations identical to those of WT sperm and PAWP knockout male mice can originate healthy litters (Satouh et al. 2015).
As demonstrated in the PAWP case, analyzing cells lacking the gene that encodes the factor of interest can greatly help in understanding how important the factor is. Although significant technological innovations have been made to assess spermatogenesis in vitro, no strategies are currently available to produce fully functional and swimming spermatozoa in vitro and allow an accurate assessment of sperm proteins (Sato et al. 2011, Komeya et al. 2016). Therefore, genetically modified animals are currently necessary for investigating the molecular and cellular features of spermatozoa.
In 2002, Saunders et al.reported the generation of an almost physiological pattern of oocyte Ca2+ oscillations upon Plcz1 cRNA microinjection, which opened new knowledge avenues regarding the oocyte-activation mechanism by spermatozoon and paved the way for the hypothesis that PLCZ1 is a SOAF (Saunders et al. 2002). Therefore, obtaining Plcz1-deficient animals has been regarded as a key for answering the question, ‘do sperm from Plcz1 deficient animals lose the ability to activate oocytes?’ However, for a long time after the seminal report on PLCZ1 role on Ca2+ oscillation induction, only one model of Plcz1-deficient mice was described in conference proceedings (Ito 2010), and no peer-reviewed report on the subject was published. Nevertheless, the lack of sperm PLCZ1 was observed in mutant animals, such as wobbler mice that exhibit malformed sperm and infertility (Heytens et al. 2010). Moreover, almost complete loss of human PLCZ1 was observed in the spermatozoa of infertile men (Yoon et al. 2008, Heytens et al. 2009, Taylor et al. 2010, Chithiwala et al. 2015, Yelumalai et al. 2015), and changes in the coding region of PLCZ1, such as loss-of-function H398P and less functional H233L mutations, were also reported in human patients (Kashir et al. 2011, 2012).
Although these experimental approaches strongly suggest that PLCZ1 is essential for oocyte activation, they do not supplant the use of a targeted gene deletion model due to the following reasons. First, given the nature of spermatozoa, heterogeneity in cellular quality may cause a discrepancy between experimental assays and the real phenotype, as minor populations of spermatozoa with superior fertilizing ability may determine the assay outcome. In particular, human spermatozoa have higher heterogeneity than those of murine origin (Sousa et al. 2011, Yaniz et al. 2016). Secondly, if the type of genetic change is a point or small indel mutation, it is often difficult to guarantee the complete loss of gene expression, and a small amount of residual or truncated protein expression cannot be disregarded. Finally, if Plcz1 is not specifically targeted, it is difficult to accurately discuss its importance, since unspecific loss of proteins other than PLCZ1 cannot be prevented. It should be noted that what is expected for Plcz1-deficient mice is to obtain spermatozoa that were absolutely null for PLCZ1.
Plcz1 locus and spermatogenesis failure
The spermatogenesis phenotype derived from target gene deletion affects significantly the subsequent experimental procedures. If the gene deletion causes complete failure of spermatogenesis, so that no elongated sperm is obtained, the options to analyze the target protein function in mature sperm are largely limited. Moreover, the more the target gene deletion affects the sperm morphology and/or motility the more difficult it becomes to accurately interpret the experimental outcomes using a population of mature live spermatozoa; for example, in vitro fertilization (IVF). The common phenotype among the Plcz1-edited mouse lines (Hachem et al. 2017, Nozawa et al. 2018), in which Plcz1 depletion itself does not cause significant problems on spermatogenesis, allows to analyze PLCZ1-deficient mature spermatozoa through various types of assays.
Before the establishment of the CRISPR/Cas9 technology to produce gene-deficient models (Cong et al. 2013, Wang et al. 2013), the homologous recombination method using embryonic stem cells was the mainstream method to achieve in vivo models of the targeted gene deletion. Noteworthy, a preliminary report in 2010 indicated that Plcz1 knockout causes spermatogenesis failure (Ito 2010), which is consistent with the previous findings in patients and the wobbler mice phenotype (Heytens et al. 2009, 2010, Taylor et al. 2010, Chithiwala et al. 2015, Yelumalai et al. 2015). However, a mutant mouse line with whole exon deletion of Plcz1 generated using the CRISPR/Cas9 system does not exhibit spermatogenesis failure. In addition, spermatogenesis failure was avoided when point mutations equivalent to those reported in human diseases were introduced (Nozawa et al. 2018). In agreement, small indel mutant mouse lines (17 or 22 bp deletions), which were generated by other groups using the CRISPR/Cas9 approach and were carefully examined for the loss of PLCZ1 expression, did not present spermatogenesis failure (Hachem et al. 2017). These discrepancies can be explained by the lower rate of unspecific effects on neighbor genes caused by the CRISPR/Cas9 technology.
In the conserved mammalian genomic region in the vicinity of Plcz1 is located (in the opposite direction, and with a gap of 100 bp in the mouse genome and 19 bp in the human genome) the gene that encodes the capping actin protein of the muscle Z-line subunit alpha 3 (Capza3) (Fig. 1). CAPZA3 is specifically expressed in the testis and is located in the post-acrosomal region of the mouse sperm head (Tokuhiro et al. 2008). Moreover, it was reported that a single nucleotide missense mutation (T/A transversion changing an ATG (M) to an AAG (K) codon in the open reading frame) in Capza3 leads to severe globozoospermia in mice (Geyer et al. 2009), contrary to Plcz1 mutant mice generated with CRISPR/Cas9 technology (Hachem et al. 2017, Nozawa et al. 2018). Furthermore, Capza3 expression was reported to be associated to that of Plcz1 (Javadian-Elyaderani et al. 2016). The homologous recombination method often involves embedding vector components in the vicinity of the genomic region of the target gene; thus, it may lead to reduced or absent expression of Capza3 when targeting Plcz1.
The Plcz1 gene locus and Capza3 gene. Red arrowheads indicate targeted sites for CRISPR/Cas9 genome editing. Black or gray solid box indicates coding or non-coding region in exons, respectively.
Citation: Reproduction 164, 1; 10.1530/REP-21-0438
The Plcz1 gene locus and Capza3 gene. Red arrowheads indicate targeted sites for CRISPR/Cas9 genome editing. Black or gray solid box indicates coding or non-coding region in exons, respectively.
Citation: Reproduction 164, 1; 10.1530/REP-21-0438
The Plcz1 gene locus and Capza3 gene. Red arrowheads indicate targeted sites for CRISPR/Cas9 genome editing. Black or gray solid box indicates coding or non-coding region in exons, respectively.
Citation: Reproduction 164, 1; 10.1530/REP-21-0438
There are a number of reports regarding the localization of PLCZ1. In the spermatozoon of WT mouse, it is localized in a perinuclear cytosolic structure near the post-acrosomal sheath, which is close to where CAPZA3 is located. In humans, PLCZ1 was also reported to be localized in the cytosolic portion surrounding the acrosome and equatorial segment (Escoffier et al. 2015). Recently, it was reported that the actin-like protein 9 (ACTL9), another actin-related protein that interacts with ACTL7, is located in the vicinity of the post-acrosomal sheath and that its attenuation causes damage to the perinuclear theca, resulting in decreased levels of PLCZ1 (Dai et al. 2021). Although the direct interaction of PLCZ1 with CAPZA3 or ACTL9 remains unclear, these observations suggest that PLCZ1 localization in the head cytoplasmic fraction of mature sperm is associated with its actin-based structure, which may contribute to PLCZ1 persistence in mature mammalian spermatozoa. It has also been reported that the development of globozoospermia is correlated with the loss or abnormal expression of DPY19L2, and that the severity of the sperm morphological abnormalities correlates with the degree of PLCZ1 loss or abnormal localization (Escoffier et al. 2015). Therefore, considering the process of spermatogenesis and the pattern of PLCZ1 localization, it is expected that PLCZ1 localizes close to actin filament structures in the head cytoplasmic fraction of mature sperm during the later stages of spermatogenesis (Pleuger et al. 2020). Hence, when sperm are malformed due to reasons other than Plcz1 mutations, their abundance is reduced via destabilization of actin filament-dependent structures, suggesting that PLCZ1 can be a biomarker of sperm quality.
Importantly, the phenotype of Plcz1-deficient mice suggests that abnormalities in the genomic regions near Plcz1 can cause sperm malformation, and, conversely, sperm malformation (from mutations in other than Plcz1 locus) can occur first, resulting in PLCZ1 reduction or loss. These two phenomena may occur independently (Fig. 2), a duality that is suggested to underlie the challenges in interpreting the relationship between PLCZ1 abundance and sperm morphology. Moreover, these events may also lead to sperm functional heterogeneity, such as in the case of ICSI treatment of human patients with abnormal sperm morphology.
Relationship of spermatogenesis failure and PLCZ1 deficiency. Note that PLCZ1 has an aspect as a sperm quality marker, and that relatively higher heterogeneity exists in human spermatozoa.
Citation: Reproduction 164, 1; 10.1530/REP-21-0438
Relationship of spermatogenesis failure and PLCZ1 deficiency. Note that PLCZ1 has an aspect as a sperm quality marker, and that relatively higher heterogeneity exists in human spermatozoa.
Citation: Reproduction 164, 1; 10.1530/REP-21-0438
Relationship of spermatogenesis failure and PLCZ1 deficiency. Note that PLCZ1 has an aspect as a sperm quality marker, and that relatively higher heterogeneity exists in human spermatozoa.
Citation: Reproduction 164, 1; 10.1530/REP-21-0438
Reproductive phenotype of Plcz1 deleted mice
Two different research groups have generated several mutant mouse lines using the CRISPR/Cas9 system (Hachem et al. 2017, Nozawa et al. 2018). Among them, homozygous male mice without all the coding exons (exon 2–14) of Plcz1 (named Plcz1 em1Osb ) and mutant mice harboring a homozygous 22 bp deletion in exon 3 (named Plcz1 em1Jparr ) showed complete loss of PLCZ1 expression (Fig. 1). These male mice showed no problems regarding the testis weight, spermatogenesis, and sperm swimming ability. Moreover, ICSI using their spermatozoon did not result in the formation of pronuclear (PN) in any of the mutants, which clearly demonstrated that PLCZ1 is the SOAF in ICSI. However, surprisingly, these mutant males showed subfertility in crosses with WT females – the detailed litter size was 2.3 ± 0.50 for Plcz1 em1Osb (heterozygous control: 8.9 ± 0.26) and 4.2 ± 0.6 for Plcz1 em1Jparr (WT control: 7.8 ± 0.8) (Hachem et al. 2017, Nozawa et al. 2018). When fertilized oocytes were collected from the oviducts after crossing with these mutant males, polyspermic fertilization was found in about 20% of the oocytes, at, which was a significantly higher rate than that obtained with WT males. In addition, the number of oocytes that exhibited 0PN with no PN but with fertilization cone(s), and 1PN oocytes with only one PN also increased. Although, 1PN is often considered to result from parthenogenetic activation, it was noted that swollen sperm chromosomes united with oocyte chromosomes in the oocyte cytoplasm after IVF using spermatozoa from Plcz1em1Osb (unpublished observation by Satouh and Nozawa). IVF with spermatozoa from both Plcz1em1Osb and Plcz1em1Jparr male mice showed a higher rate of polyspermic fertilization than upon mating (Hachem et al. 2017, Nozawa et al. 2018), an effect retained even when sperm concentration in the IVF was lowered. The intracellular Ca2+ pattern in ICSI was the same in both mutants, and no Ca2+ spike was observed. Noteworthy, no Ca2+ spike nor PN was observed upon injection of three sperm heads of Plcz1em1Osb spermatozoa (at the same time) or whole sperm (Nozawa et al. 2018).
As described above, most of the results from the two mutant models are equivalent, confirming the solidity of the phenotypes of PLCZ1 deficiency in IVF, ICSI, and in vivo fertility. Nevertheless, different results regarding the occurrence of Ca2+ oscillations in IVF were reported. In particular, all oocytes fused with Plcz1em1Osb sperm exhibited Ca2+ spikes delayed by several tens of minutes in their onset and were reduced in number compared with those fused with WT spermatozoon (2.75 ± 0.65 spikes generated vs 12.0 ± 5.68 spikes generated) (Fig. 3) (Nozawa et al. 2018). Moreover, Hachem et al. reported that almost all oocytes inseminated with Plcz1em1Jparr spermatozoa fail to generate Ca2+ spikes (Hachem et al. 2017).
Calcium oscillation outcomes in different fertilization modes. Atypical, or PLCZ1-independent, Ca2+ oscillations are evoked only in the IVF and exhibit delayed onset and less number of spikes.
Citation: Reproduction 164, 1; 10.1530/REP-21-0438
Calcium oscillation outcomes in different fertilization modes. Atypical, or PLCZ1-independent, Ca2+ oscillations are evoked only in the IVF and exhibit delayed onset and less number of spikes.
Citation: Reproduction 164, 1; 10.1530/REP-21-0438
Calcium oscillation outcomes in different fertilization modes. Atypical, or PLCZ1-independent, Ca2+ oscillations are evoked only in the IVF and exhibit delayed onset and less number of spikes.
Citation: Reproduction 164, 1; 10.1530/REP-21-0438
This apparent disparity was solved by the discovery of a positive correlation between the number of Ca2+ spikes detected in the atypical Ca2+ oscillations and PN formation rate (discussed in the following section), indicating that these atypical Ca2+ oscillations have physiological significance. Furthermore, in support of this, the atypical Ca2+ oscillation was also observed by Swann and colleagues (reviewed in Swann 2020). It is reasonable to assume that the different interpretations are results of the imaging system used, especially owing to its toxicity and sensitivity. Nozawa et al. employed the low-invasive directed system which supports healthy birth of mouse pups even after Ca2+ recording for 5 h by using protein-based indicator GECO (genetically-encoded Ca2+ indicators for optical imagings) series (Zhao et al. 2011), a spinning-disk confocal microscope, and a highly sensitive camera (Satouh et al. 2017, Nozawa et al. 2018). On the other hand, Hachem et al. employed the imaging system using Fura-2 acetoxymethyl ester, a chemical indicator excitable at 340 and 380 nm UV light (Hachem et al. 2017). Although it could be argued that differences in sperm condition can also contribute to give the difference between two groups (fresh in Nozawa et al. (2018) and cryopreserved in Hachem et al. (2017)), identical waveforms were observed using cryopreserved Plcz1em1Osb spermatozoa (unpublished observation by Satouh and Nozawa). Therefore, the PLCζ1-independent atypical Ca2+ oscillation may be more sensitive to the observation method than the typical Ca2+ oscillations and easily disappear.
Findings by applied use of PLCZ1-deficient spermatozoa
The number of the atypical Ca2+ oscillations elicited by PLCZ1-deficient spermatozoa in IVF was approximately three and varied among oocytes. The number of Ca2+ spikes is suggested to affect the developmental ability of mammalian embryos, as indicated by the correlation between the attenuated number of Ca2+ spikes and infertility (Yoon et al. 2012, Yeste et al. 2016). Indeed, Hoechst staining of oocytes collected from the oviducts of female mice after mating with Plcz1-deficient males indicated that the success rate of oocyte activation varies even for oocytes that are fertilized by a single sperm (Nozawa et al. 2018). Therefore, it is possible that a single PLCZ1-deficient spermatozoon can elicit either an insufficient or sufficient number of Ca2+ spikes required for oocyte activation.
Using the abovementioned observation method that does not interfere with the activation process of embryos while simultaneously recording oocyte intracytoplasmic Ca2+, it was possible to record and retrospectively match the number of Ca2+ spikes and fused sperm, as well as the rate and number of PN formed in individual eggs during IVF. Overall, a significant difference in the number of Ca2+ spikes was observed between monospermic-fertilized oocytes that were able to form PN and those that failed to do so, and that they were activated by three or more Ca2+ spikes (Nozawa et al. 2018). This finding resolves the long-debated question of ‘how many Ca2+ spikes are minimally required to initiate the initial development?’ Interestingly, Perry and colleagues succeeded in achieving very efficient parthenogenesis of mouse oocytes by removing Zn2+ using a chelator (Suzuki et al. 2010). Zn2+ ions in eggs are stored in vesicles just below the plasma membrane (Que et al. 2015) and are released synchronously with the first one or two Ca2+ spikes when oocytes are artificially activated (Duncan et al. 2016). These suggest the importance of the first few spikes to activate the oocyte. However, with respect to the number of spikes that occur in the mouse, which is more than a dozen, Ducibella et al. suggested that the involved mechanisms are progressively turned on to ensure not only the resumption of the cell cycle but also the early development and birth of the litter (Ducibella & Fissore 2008); therefore, the importance of the remaining Ca2+ spikes (e.g. in the epigenetic state of the progeny) should be analyzed in the future.
Addressing why polyspermy occurs frequently in PLCZ1-independent atypical Ca2+ waveforms, focusing on the cases of atypical Ca2+ oscillations displaying delayed onset is also important. In particular, the relationship between the two polyspermy block systems (zona pellucida block to polyspermy (ZPBP) and plasma membrane block to polyspermy (PMBP)), and their correlations to Ca2+ waveform were explored. In vitro analyses suggest that PLCZ1-independent atypical Ca2+ waveforms induce retarded establishment of both ZPBP and PMBP, resulting in polyspermic fertilization (Nozawa et al. 2018). Using female mice lacking astacin-like metalloendopeptidase (ASTL), which completely lack ZPBP (Burkart et al. 2012), showed that all ASTL-deficient oocytes present 2PN as a result of 1:1 fertilization between the oocyte and WT spermatozoon, indicating that the PMBP system can solely ensure monospermy in mouse fertilization (Nozawa et al. 2018). Thus, raised frequency of polyspermy in PLCZ1-independent fertilization results from the delay of PMBP establishment, not ZPBP. This result suggests a superiority of PMBP in the establishment of the mammalian polyspermy block and may affect even on the comprehension of in vivo fertilization studies indicating that mouse fertilization occurs slowly and in a very small number of gametes (La Spina et al. 2016, Muro et al. 2016).
Human disease and mutant mice phenotypes
It may be difficult to relate the phenotype of complete loss of PLCZ1, which does not cause infertility in mice, to human symptoms, unless it is carefully ensured that PLCZ1 expression is fully suppressed in humans. Moreover, there are limited approaches to replicate human symptoms in mice. Generation of point mutant mice that harbor similar amino acid substitution to those identified in human patients may be a useful strategy.
Plcz1 mutant mouse lines harboring the H435P mutation, which corresponds to the human H398P variant (Heytens et al. 2009), and the D210R mutation, which leads to almost complete loss of Plcz1 ability to induce Ca2+ oscillations (Saunders et al. 2002), were generated using the CRISPR/Cas9 system (Nozawa et al. 2018). In both point mutants, male mice were phenocopies of the Plcz1em1Osb model, with no spermatogenesis abnormalities. However, PLCZ1 expression was almost completely lost in spermatozoa of the D210R mutant, whereas a low molecular weight protein was detected in the spermatozoa of the H398P mutant, which may be derived from a truncated protein form. The spermatozoa from H398P, even with the truncated form, or that from the D210R mutant, showed complete loss of Ca2+ oscillations in ICSI, and atypical and slowly initiated oscillations were produced in IVF, similar to Plcz1em1Osb spermatozoa (Nozawa et al. 2018). However, detection of the truncated form by antibodies is an important point to note when testing protein levels using antibodies without considering molecular weight, such as ELISA or immunostaining.
Spermatozoa from all Plcz1 mutants exhibits high incidence of polyspermic fertilization in IVF (up to approximately 80%) per fertilized oocytes. Thus, H435P and D210R mutant spermatozoa were examined regarding the ICSI outcome along with artificial oocyte activation by Plcz1 mRNA injection to ensure both monospermic fertilization and reliable oocyte activation. The success of this approach was confirmed when healthy pups were obtained at identical birth rate to that of ICSI using WT oocyte and spermatozoa (Nozawa et al. 2018). As for the linkage with sperm malformation, since sperm heterogeneity in morphology is relatively high in humans (Sousa et al. 2011, Yaniz et al. 2016), it should be particularly considered that PLCZ1 behaves similarly to a sperm quality biomarker. Recently, several reports have examined the correlation between infertility, sperm malformation, and the presence of PLCZ1 mutations in humans (Escoffier et al. 2016, Dai et al. 2020, Mu et al. 2020, Wang et al. 2020, Yan et al. 2020, Yuan et al. 2020a, b). Additional details on other PLCZ1 mutations will be addressed in other sections herein. Nevertheless, the analysis of these pathologies with abnormal sperm morphologies may require attention to the presence of mutations in the neighbor of PLCZ1, and the combination of ICSI with artificial oocyte activation is suggested to be effective for investigating PLCZ1 deficiency.
Summary and future perspectives
The PLCZ1-independent, minor, and atypical Ca2+ oscillations could not be discovered without the analysis of animals in which PLCZ1 expression is completely prevented. Conversely, as PLCZ1-containing spermatozoon can ensure a sufficient number of spikes in a single sperm clearly highlight that PLCZ1 is an essential SOAF to ensure single sperm fertilization in mammalian fertilization. Noteworthily, investigations using PLCZ1-deficient mice proved that PLCZ1 contents can be an absolute parameter to predict the success of oocyte activation, especially in the ICSI, which is the most applied treatment in artificial reproductive therapy. Furthermore, the phenotype of PLCZ1-deficient mice, as subfertile rather than fully infertile, also suggests the possibility that human patients with PLCZ1 mutations are less likely to be detected as potentially infertile. Thus, diagnosis based on PLCZ1 sequencing may be useful; however, considering that point mutations can exhibit different expression patterns (Nozawa et al. 2018), additional analysis strategies may be warranted, such as the use of recombinant proteins to determine the precise effect of each mutation on the oocyte-activating potency of the spermatozoon.
In the PLCZ1-independent mechanism, the number of Ca2+ spikes increases with the number of sperm to be fused, and consequently, the PN formation rate is higher when multiple spermatozoa are fused to one oocyte (Nozawa et al. 2018). These findings resemble those reported in birds, reptiles, and amphibians, called physiological polyspermy (Iwao 2012). Since PLCZ1-dependent full and typical Ca2+ oscillation is obtained in the ICSI using WT mouse sperm heads, at least some soluble content in sperm heads is transmitted during the ICSI. Perry et al. suggested that multiple SOAFs exist in the mouse sperm head, as heat-sensitive and -stable factors participating in oocyte activation (Perry et al. 2000). However, the Ca2+ waveform was completely lost in the ICSI of PLCZ1-deficient spermatozoa even when multiple sperm heads were injected.
In avian oocyte, litters were successfully obtained via ICSI by injecting not only PLCZ1 but also aconitate hydratase and citrate synthase, which may be of mitochondrial origin, implying a synergic effect between these proteins (Mizushima et al. 2014). Diffusion of organelle-contained soluble content into the egg cytoplasm by fertilization is expected to take longer than that of the soluble content in the sperm cytoplasm, considering the complexity of the membrane structure and the directionality of degradation. This is also in common with the delayed occurrence of PLCZ1-independent atypical Ca2+ waves. However, in mice, this contradicts the no Ca2+ spike nor PN formation phenotype obtained by the injection of the whole (with intact tail) PLCZ1-deficient spermatozoon by ICSI (Nozawa et al. 2018). Kang et al. recently reported that extramitochondrial citrate synthase (eCS) is the second SOAF in mouse spermatozoon (Kang et al. 2020). However, the phenotype of eCS-deficient spermatozoon elicits a series of Ca2+ oscillations with a similar pattern to that caused by PLCZ1-deficient spermatozoon, whereas eCS deficient spermatozoa retain PLCZ1 content.
Considering that ICSI, compared with IVF or in vivo fertilization, promotes the loss of the interaction and fusion between plasma membranes of spermatozoon and oocyte, it is possible that membranous activation factors from the sperm plasma membrane diffuse into the oocyte plasma membrane by sperm–egg fusion, as observed in Caenorhabditiselegans (Takayama & Onami 2016). In addition, the Ca2+ bomb theory, which was postulated before the SOAF theory dominated, may also be reconsidered (Machaty 2016). PLCZ1-deficient mouse spermatozoon introduced in this review is currently the only material that does not generate PLCZ1-dependent Ca2+ oscillation, preserving swimming and fusion abilities, and it would be an essential tool for analyzing PLCZ1-independent oocyte activation. Studies using Plcz1-deleted mice have addressed various questions in the field of basic biology and assisted reproduction, as well as have provided clues for further questions or enigmas. Elucidation of PLCZ1-related underlying mechanisms, including those that explain these multiple contradictions, remains to be addressed in the future.
Declaration of interest
The author declares that there is no conflict of interest that could be perceived as prejudicing the impartiality of this review.
Funding
This study was supported by the Japan Society for the Promotion of Science KAKENHI (grants 17K15126, 19K06686) and Kanzawa Medical Research Foundation.
Acknowledgements
The author thank Kaori Nozawa and the staff of Animal Resource Center for Infectious Diseases, Research Institute for Microbial Diseases, Osaka University for technical assistance of generation, maintenance, and analysis of Plcz1 mutant mice.
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