Abstract
The discovery of PLCZ1 nearly 20 years ago as the primary Ca2+ oscillation-inducing factor in the sperm of mammals represented a significant breakthrough in our quest to elucidate the molecules and pathways that promote egg activation during fertilization. The advent of the intracytoplasmic sperm injection (ICSI) technique, which made fertilization possible without sperm capacitation, acrosome reaction, and gamete fusion, strengthened the research that led to the discovery of PLCZ1 and became an essential clinical tool for humans. The use of ICSI combined with the detection of PLCZ1 expression and mutations in infertile patients established the fundamental role of PLCZ1 in human fertility while leading to the discovery of novel components of the perinuclear theca, the site of the residence of PLCZ1 in sperm before fertilization. Remarkably, the more extensive use of ICSI in species other than humans and mice revealed poor success and exposed gaps in our understanding of PLCZ1 release and/or activation. Similarly, fertilization using sperm from mouse models lacking Plcz1 has produced striking results whose true implications are yet to be determined. Nevertheless, answers to these unresolved questions will produce a complete picture of the adaptations and molecular players that mammalian species employ to ensure the success of the triggering event of embryo development that has linked generations since the beginning of times.
Introduction
The study of gametes and fertilization
A few thousand years passed between the first known written document addressing the origin of the human fetus, the Garbha Upanishad (~1400 BC), or ‘Esoteric Doctrine over the Embryo’, and the identification and characterization of the gametes, and the cells responsible for fertilization and embryo development. The document written in Sanskrit narrates how upon the ‘unison of the male and female reproductive fluids, male semen and female blood, the embryo develops’. It also provides a timeline of the formation of the different body parts, including the transition of spiritual states that the fetus undergoes until birth. Expectedly, it is devoid of cellular and molecular details. Further, despite continuous interest in human conception and careful anatomical descriptions, it would not be until the middle of the seventeenth century when groups working at different research centers in the Netherlands produced the scientific breakthroughs that opened the field of reproduction to laboratory experimentation that continues until today. The Leyden group first led by Jan Van Horne and Jan Swammerdam and then by Reiner DeGraff honed in on the role of the female organ, adopted the name ovary for it, and went on to describe the production of follicles, ovulation of these vesicles, and the formation of the corpus luteum (reviewed in Lopata & Lehrer 2009, Thiery 2009, Cobb 2012). Antony van Leeuwenhoek, a linen businessman working in the town of Delf, discovered the spermatozoa in 1667 (van Leeuwenhoek 1667, Cobb 2012), although it was the work of Dumas and Prescott in 1824 that first intimated that the role of these cells was to penetrate the egg (reviewed in Lillie 1913). Incidentally, the term ‘egg’ or ‘ovum’ was commonly used around this time even though its identification as we know it today occurred in 1827 by Von Baer (Betteridge 1981, Cobb 2012). Experiments then commenced to characterize the fertilization process in vitro, and a successful description of the complete process was independently reported in 1875 by Hertwig and Fol (reviewed in Lillie 1913) in the sea urchin system. Another century would elapse until in vitro fertilization (IVF) was accomplished in mammals and slightly longer until the first offspring following intracytoplasmic sperm injection (ICSI) would be obtained (Iritani et al. 1988, Palermo et al. 1992). Nevertheless, this nearly 100-years gap was not devoid of findings, with evidence emerging to fill many physiological gaps in the process of fertilization. This time also coincided with the launching of the age of cellular and molecular reproduction that endures to date. In the last 30 years, the available cellular, molecular, and genetic tools have resulted in significant inroads in understanding the molecular underpinning of all events of fertilization, which is required for the continuity of generations. This special issue celebrates the twentieth anniversary of the discovery of PLCZ1 (Saunders et al. 2002), the sperm factor involved in egg activation. This review focuses on how fertilization by ICSI aided in its discovery, and how their combined application has led to new knowledge and unanswered questions.
Egg activation, ions, and Ca2+ oscillations
The feasibility of replicating fertilization in vitro and the emergence of research tools triggered a stampede of research to elucidate the molecules that underpin the multiple steps that underlie fertilization in all species, including the mechanisms of egg activation, our focus here. The question framing this search was the ancestral desire to understand where we come from, or more specifically, how the now identified male gamete induces the development of the female gamete rendering it into an embryo and eventually into an offspring. Three pivotal discoveries guided the search for the sperm’s egg activating factor, namely, the observation that ionic changes underlie egg activation, the discovery that Ca2+ is the physiological ion, and the finding that in mammals the Ca2+ changes are periodical, separated by dozens of minutes and persists for hours. Early in the 19th century, Jacques Loeb produced the foundational observation by recognizing the fundamental role that ionic changes play in the initiation of embryo development. He said it best, ‘It appeared to me that nothing would more clearly demonstrate the sovereign role that electrolytes play in the phenomena of life than by causing, if possible, with their help, unfertilized eggs to develop into larvae’. By changing the sea water’s composition, Loeb accomplished the parthenogenesis of sea urchin eggs, promoting development to the larvae stage (Loeb 1913). With the role of ions firmly planted, the focus changed to what ion(s) might be mediating egg activation, and whether their role in fertilization extended to other species. Once again, sea urchin eggs will be the cellular model, and Daniel Mazia under the tutelage of Victor Heilbrunn demonstrated that the intracellular concentration of free Ca2+ increased after fertilization, and therefore it was the pivotal ion (Mazia 1937, Rall 2019). The universal role of Ca2+ in egg activation was demonstrated by a succession of studies using novel reagents to modulate and monitor intracellular Ca2+. Ca2+ ionophores were used to show that a rapid increase in the intracellular levels of Ca2+ faithfully induced egg activation in marine, amphibian and mammalian eggs (Steinhardt et al. 1974). Soon after, the Ca2+-sensitive protein aequorin became available, and it was used to document the magnitude and duration of the rises induced by ionophores and by fertilization (Ridgway et al. 1977), including the demonstration that in mammals the sperm induces periodical and long-lasting Ca2+ responses, known as oscillations (Cuthbertson et al. 1981, Miyazaki et al. 1986). Therefore, almost a century after the initial observations, Ca2+ emerged as the universal factor of egg activation, although with distinct species-specific spatiotemporal patterns.
Models of egg activation, the sperm factor, and ICSI
The widespread participation of Ca2+ in invertebrate and vertebrate fertilization and the near-universal involvement of the phosphoinositide pathway and 1,4,5-inositol trisphosphate (IP3), raised expectations for a one size-fits-all mechanism of egg activation (Stricker 1999). Nevertheless, experimental evidence showed that none of the proposed models at the time could replicate the whole gamut of Ca2+ responses during fertilization. We will not discuss the main features of each model, for that, we refer the reader to comprehensive published works on the subject (Jones 2007, Kashir et al. 2014). We will focus instead on the work that led to the elucidation of the signaling mechanism(s) responsible for the Ca2+ oscillations in mammals, driven from the outset by widely divergent hypotheses (Runft et al. 2002). The mechanistic chasm arose in no small part because it was conceptually difficult to envisage how the ephemeral interaction of the gametes at the plasma membrane could induce Ca2+ oscillations that lasted for hours. Further, this ‘receptor hypothesis’ was primarily based on findings from hormone and neurotransmitter signaling, which were the most studied and best understood signaling models at the time, but that displayed a short lifespan. An alternative model advanced the view that after gamete fusion, a sperm component(s), the sperm factor, SFs, induced the persistent Ca2+ oscillations, the ‘fusion hypothesis’. The initial supporting evidence showed that injection of sperm extracts, SFs, into eggs induced activation and embryo development. These results were first reported in sea urchin and ascidians (Dale et al. 1985, Dale 1988) and later in hamster and rabbit eggs (Stice & Robl 1990, Swann 1990), including the demonstration in hamster eggs that SFs initiated Ca2+ oscillations indistinguishable from those at fertilization (Swann 1990). Despite the physiological nature of the SF-triggered oscillations, the specificity of its responses was challenged, and it was not until the success of the intracytoplasmic sperm injection technique (Iritani et al. 1988) highlighted by the birth of offspring in humans (Palermo et al. 1992), that this hypothesis became consolidated. It is remarkable, however, that the results of Uehara and Yanagimachi in 1976 (Uehara & Yanagimachi 1976) showing that injection of mammalian sperm heads into hamster eggs induced complete egg activation did not receive more scrutiny and stimulated immediate replication, despite the confounding effects of the injection procedure (Uehara & Yanagimachi 1977), as this data foreshadowed the true mechanism of egg activation a decade ahead of the SF results and the birth of offspring by ICSI.
The realization of the role of SF in egg activation prompted the search for its molecular identity. Biochemical and Ca2+ monitoring studies uncovered many unique features of the SF including its ability to induce production of IP3 (Galione et al. 1997, Wu et al. 1998, Jones et al. 2000, Kurokawa et al. 2005), but identification of PLCZ1 was accomplished after BLAST searches of a mouse EST testis database using mammalian PLC sequences (Saunders et al. 2002). Multiple studies confirmed the role of PLCZ1 in the initiation of Ca2+ oscillations in mammalian species. Furthermore, more detailed studies followed, including the elucidation of the functional role of each of its domains, species-specific differences in activity and localization, and its implications on infertility. Most of these subjects are addressed in other chapters of this issue. Nevertheless, unanswered questions remain, such as the mechanism of PLCZ1 release during fertilization and the site of action in the egg. Remarkably, the use of ICSI and sperm from genetic models (Hachem et al. 2017, Nozawa et al. 2018) have confirmed the role of PLCZ1 as the pivotal SF in mammals while at the same time raising some outstanding questions, the answers to which will provide valuable insights about fertilization in mammals.
ICSI, the magic wand of fertilization research
ICSI-induced Ca2+ oscillations in mouse eggs
Besides being invaluable for overcoming infertility in the clinic (Palermo et al. 1992), ICSI has proven a remarkable research tool to untangle the signaling mechanism(s) of egg activation. The aforementioned pioneer studies of Uehara and Yanagimachi and those that followed by others (Markert 1983) used the technique to learn about the mechanism(s) of sperm head remodeling, pronucleus (PN) formation, and early gene expression. The first reports inquiring about how sperm injection might recreate the Ca2+ responses of fertilization were in human eggs (Tesarik et al. 1994), which was understandable given the routine application and success of the technique in the clinic and the fact that piezo-actuated microinjection was not yet available. However, the reported responses did not match those induced byIVF in humans (Taylor et al. 1993), as the Ca2+ rises were fewer and delayed (Tesarik & Sousa 1994, Tesarik et al. 1994). A more complete and careful examination of the Ca2+ responses induced by this technique followed, especially using mouse gametes, which with the introduction of piezo-driven microinjections rendered ICSI into a consistent and efficient method in this species (Kimura & Yanagimachi 1995). Subsequent results showed that decapitated sperm were sufficient to give rise to normal embryo development (Kuretake et al. 1996). From there onwards, the majority of studies used the injection of sperm heads.
In the mouse, fertilization by ICSI closely replicated the oscillations induced byIVF, although a few deviations were noted (Nakano et al. 1997) (Figs 1 and 2). For example, the first rise following IVF has a higher amplitude and duration, which is also the case after ICSI. However, unlike IVF, the first Ca2+ rise after ICSI unfolds as a uniform increase across the egg rather than as a wave emanating from the site of sperm entry, which is the case after IVF. Furthermore, Nakano et al. (1997) failed to show the super-imposed Ca2+ rises that accompany the first peak following ICSI, although a more recent study applying higher resolution imaging detected them (Ozil et al. 2017). The subsequent oscillations that develop at intervals of 6–15 min and for 3–4 h (Deguchi et al. 2000) and whose termination occurs at around the time of PN formation (Jones et al. 1995) are all well conserved between ICSI and IVF. Further, this pattern is closely matched to those induced by sperm extracts in mouse eggs (Oda et al. 1999). These results show that ICSI in the mouse closely approaches the Ca2+ responses of IVF, and the oscillation patterns may become indistinguishable as gamete preparation and tools continue to improve.
Schematic of the molecular players and Ca2+ oscillations induced by fertilization in mice and bovine oocytes following IVF or ICSI using homologous sperm. Ca2+ profiles are representative profiles, although following bovine ICSI only 50% of fertilized oocytes display oscillations while the rest failed to mount any responses. Two representative ICSI profiles are shown to reflect the large proportion of bovine oocytes unable to mount responses post-ICSI. Ca2+ influx channels in the mouse have been identified and are important to sustain oscillations, although for most other species including the bovine, we remain unaware of their identity and contributions (Wakai et al. 2019). We believe these oocytes express similar family of channels, but this is currently unknown (denoted by a ? symbol). Cond, condensed sperm.
Citation: Reproduction 164, 1; 10.1530/REP-21-0487
Ca2+ oscillations in mouse oocytes induced by fertilization or mRNA injection. (A) Ca2+ oscillations after injection of mPlcz1 mRNA injection. (B) Ca2+ oscillations after IVF with WT or Plcz1-KO sperm (n = 10 oocytes, two replicates; unpublished observations). (C) Ca2+ oscillations after ICSI fertilization with WT or Plcz1-KO sperm (n = 7). Data for this figure are all new from our laboratory and unpublished. ICSI and IVF fertilization procedures were carried out as described in two previous manuscripts by our laboratory (Kurokawa & Fissore 2003, Hachem et al. 2017). Sp, sperm.
Citation: Reproduction 164, 1; 10.1530/REP-21-0487
SF release, PLCZ1 PN homing, and nuclear positioning in the sperm
While identification of the active component of the SF demanded a few years, the advent of ICSI firmly established the notion that the diffusion of a sperm factor into the ooplasm underpinned the events of activation in mammalian eggs. A series of studies extended these results by demonstrating that following ICSI, the sperm’s active component(s) diffuse into the ooplasm within 15–30 min post-injection, and release appears to be complete by 120 min. The conclusions were obtained by re-injecting sperm heads into unfertilized eggs and monitoring Ca2+ rises. Sperm heads that have resided in the ooplasm for less than 1 h initiated oscillations when injected into MII eggs but failed when re-injected after 120 min despite continuing rises in the donor egg (Knott et al. 2003). These studies also showed that the M-phase state of the MII ooplasm was not required for the release of SF and that exposure and solubilization but not the disintegration of the perinuclear theca (PT), the presumed site of the residence of PLCZ1 in the mature sperm, are needed for its release (Knott et al. 2003). A follow-up study demonstrated that the loss of the sperm heads’ Ca2+ oscillation-inducing activity coincided with the loss of PLCZ1 reactivity (Yoon & Fissore 2007). Unfortunately, the ooplasm’s high fluorescent background has prevented thus far to track the distribution of PLCZ1 in the ooplasm, although PLCZ1 reappears and its detection raises well above background in the PNs, which are a recognized site of PLCZ1 accumulation in mouse zygotes (Larman et al. 2004, Sone et al. 2005, Ito et al. 2008). Remarkably, it is this property of mouse PLCZ1, its nuclear localization signal and concentration in the PN, which can explain results that remained unexplained for years. Those observations noted that injections of PNs from fertilized mouse zygotes but not from parthenogenetically activated eggs triggered oscillations in fresh mouse eggs (Kono et al. 1995), which were likely due to the accumulation of PLCZ1 in the fertilized PNs. These results are among the most robust demonstrations that PLCZ1 represents the primary and possibly sole active SF in mammals during natural fertilization.
The molecules involved in determining the location of PLCZ1 in mature sperm are presently unknown but using ICSI has provided essential clues that have led to the discovery of previously unknown proteins required for PLCZ1 to find or be anchored to the PT, its final destination before fertilization. In non-capacitated mouse sperm, a fraction of PLCZ1 is detected overlaying the acrosome, although the highest concentration centers on the post-acrosome region where it appears as a continuous band (Yoon & Fissore 2007, Grasa et al. 2008). Following the acrosome reaction, the post-acrosomal localization becomes predominant (Grasa et al. 2008), which is appropriate given the need to rapidly access the ooplasm after fusion. ICSI’s importance in these studies emanates from its routine application to overcome total fertilization failure (TFF) cases following IVF or for patients with abnormally shaped sperm heads and/or acrosomes. The inability in some patients’ sperm to induce egg activation in conjunction with the absence or mislocalization of PLCZ1 led to genomic analysis that identified essential determinants in PT organization. One of these proteins is ACTL9, an Actin-Like 9 protein, for which homozygous mutations underlie the abnormal organization of the PT, mislocalization of PLCZ1, and inability to trigger steady oscillations (Dai et al. 2021). Another protein uncovered following TTF after ICSI is associated with ~70% of the type I cases of human globozoospermia (Harbuz et al. 2011). An initial study showed that the repeated TFF commonly observed in globozoospermic patients after ICSI was associated with the absence of PLCZ1 expression in these patient’s sperm and inability to trigger Ca2+ oscillations (Yoon et al. 2008). Genomics studies of these men led to research demonstrating that most of these patients had a homozygous deletion of DPY19L2, a gene highly expressed in testes and of unknown function until then. The induced deletion of the homolog gene in mice also caused globozoospermic and activation failure, later demonstrated to be due to the absence of PLCZ1 in mature sperm (Yassine et al. 2015). Dpy19l2 expression was found to be restricted to the inner nuclear membrane facing the acrosomal vesicle, and its absence destabilized the nuclear dense lamina and caused the elimination of the unbound acrosomal vesicle presumably with the associated loss of PLCZ1. Therefore, the ability to inject whole nuclei, including sperm into eggs, has been leveraged to gain fundamental insights that are proving indispensable as we aim to understand the mechanisms that underlie mammalian fertilization.
ICSI-induced responses in rats and how Plcz 1 mRNAs phenocopy sperm-induced Ca2+ responses in rodent species
The use of ICSI in rats has not been as straightforward as in the mouse and humans. Different methods were tested to improve the technique’s success in this species, and the piezo-actuated method emerged as the procedure with the highest survival and developmental rates (Dozortsev et al. 1998). Remarkably, despite the use of this technology, the reported production of offspring in the rat remains lower than other species, with ~10% of embryos transferred surviving to term (Hirohashi & Yanagimachi 2018). A degree of improvement was observed following injection of acrosome-reacted sperm heads, suggesting that both the rat’s large sperm head and large acrosome might be undermining the success of ICSI in this species (Seita et al. 2009).
The low success of ICSI in rats might have discouraged studies reporting ICSI-induced Ca2+ responses in these species. Earlier studies showed that following IVF rat gametes initiate Ca2+ oscillations like in all mammals. However, these responses were of higher frequency and lower amplitude than in other species and terminated abruptly before PN formation (Ben-Yosef et al. 1993, Ito et al. 2008). As documented in numerous studies in the mouse, the injection of PlcζPlcz1 mRNA or recombinant protein phenocopied the responses induced by fertilization (Kurokawa & Fissore 2003, Kouchi et al. 2004, Ito et al. 2008, Cooney et al. 2010). The rat was no exception, and injection of rPlcζPlcz1 mRNA displayed many of the features of the fertilization-induced Ca2+ responses in this species, especially the low amplitude oscillations and their abrupt termination well ahead of PN formation (Ito et al. 2008). Remarkably, the mRNA studies revealed unique features of rat PLCZ1, including its significantly lower specific activity than m- or h-PLCZ1 evidenced by the several-fold higher concentrations required to initiate the oscillations and by delayed initiation of the oscillations (Ito et al. 2008). Given the lack of reports documenting Ca2+ oscillations after rat ICSI, we examined these responses. Rat sperm induced Ca2+ oscillations in all mouse eggs (n = 9) but with fewer rises than those induced by mouse sperm and with lower amplitude just as those observed following rat IVF (Fig. 3A, B, and C). We extended these results by injecting in mouse eggs both m- or r-Plcz1 mRNAs. We confirmed the significantly lower specific activity of rPlcz1 vs mPlcz1 mRNAs characterized by the higher concentrations of mRNA needed to induce responses, the gap to the initiation of the oscillations, and the low amplitude of the rises (Fig. 3D and E). These results confirm that the fertilization-induced Ca2+ oscillations can be replicated by ICSI and by expression of the species’ specific PLCZ1 mRNA. The findings that mRNAinjection can single-handedly phenocopy the most exquisite details of the fertilization initiated Ca2+ responses within a particular species argue for PLCZ1 to be the pivotal and possibly sole factor in most mammals. There are nevertheless glaring exceptions to this correspondence between IVF, ICSI, and mRNA injection induced Ca2+ responses, such as the abnormal Ca2+ oscillations induced by ICSI in the bovine (Malcuit et al. 2006), discussed below, and PLCZ1 KO sperm’s capacity to induce subdued Ca2+ responses after IVF but not after ICSI in the mouse (Fig. 2) (Hachem et al. 2017, Satouh et al. 2017, Nozawa et al. 2018) (see also Satouh in this issue). Whether these are real exceptions to the rule or are artifacts introduced by the techniques used to probe them will require future studies that will undoubtedly advance our understanding of mammalian fertilization.
Rat ICSI and mouse and rat mPlcz1 induced Ca2+ oscillations in mouse oocytes. (A) ICSI using mouse sperm, control. (B) ICSI using rat sperm. (C) Merged Ca2+ oscillations demonstrating differences in amplitude and persistence. (D and E). Considerably different concentrations of m- or r-Plcz1 mRNAs are needed to induce oscillations in mouse oocytes. Data for this figure are all original, from our laboratory, and unpublished. Experimental methods were as noted in previous figures.
Citation: Reproduction 164, 1; 10.1530/REP-21-0487
The underwhelming success of bovine ICSI and long-term research opportunities
Bull sperm and incomplete egg activation after ICSI
The potential benefits of ICSI for the production of livestock are apparent, but unlike the widespread use in other large animal species such as horses, its use in cattle has been limited by low success rates (Coy & Romar 2002, Hinrichs 2005, Unnikrishnan et al. 2021a). In mice and humans, where in vivo matured oocytes are readily available, the use of ICSI results in similar rates of egg activation and embryo development to those obtained after IVF (Westphal et al. 2003, Yoshida & Perry 2007). In cattle, however, ICSI induces lower cleavage rates, ~15%, which supports low rates of development to the blastocyst stage (Chung et al. 2000, Briski & Salamone 2022). Remarkably, despite efforts over the last few decades, the improvement in the success of bovine ICSI from the first successful report in this species (Goto et al. 1990) has not been consistent or significant. Multiple factors conspire to prevent embryo development in species where ICSI is less successful, including oocyte quality and suboptimal culture conditions (Gomez et al. 2000, Nakai et al. 2016). In cattle, the developmental failure occurs early, preventing the formation of a proper zygote. Two pivotal steps are seemingly impaired. First, bovine sperm fail to initiate robust Ca2+ oscillations, a hallmark of fertilization in all mammals (Fissore et al. 1992, Malcuit et al. 2006). Secondly, the remodeling of the sperm nucleus, characterized by swelling of the sperm head, a step that precedes the formation of the male PN, is delayed and in many cases fails to form a PN (Rho et al. 1998, Águila et al. 2017). Perplexingly, bovine zygotes obtained by IVF undergo the highest rates of in vitro pre-implantation embryonic development among all large livestock species with ~80% of the fertilized zygotes proceeding to two cells and ~40% reaching the blastocyst stage (Ferre et al. 2020). Therefore, the ICSI defect in the bovine system appears due to a remodeling failure, which sequentially compromises the release and function of the SF, bPLCZ1, required for MII exit and the transformation of the sperm nucleus into the male PN. The underlying reasons why these nuclei are resistant to remodeling are beyond the scope of this review, but biochemical and morphological changes associated with events before fertilization bypassed during ICSI may render these nuclei prone to remodeling. Alternatively, the release of a remodeling factor provided by the sperm may only be fully accomplished after gamete fusion. Despite the underwhelming ICSI results, bPLCZ1 mRNA initiates robust oscillations in bovine eggs and eggs of other species, suggesting that the limitation of ICSI in this species in triggering fertilization-like oscillations centers on the inability of the delivered sperm to release or activate bPLCZ1. Below, we discuss the Ca2+ responses by ICSI in this species along with the limitations that need resolving.
Bovine ICSI and [Ca2+]i oscillations
The initial low success of ICSI in the bovine could be attributed to a variety of factors, including the possibility that bull sperm was devoid of SF or its SF had lower specific activity, as the active component of SF was not known then. Evidence that bovine sperm expresses an active SF was obtained by injecting bull sperm heads into mouse oocytes, which immediately triggered robust oscillations in nearly all injected eggs (Malcuit et al. 2006). Remarkably, in simultaneous studies, the same cohort of bull sperm induced oscillations in only a low percentage of bovine oocytes, identifying the absence of the Ca2+ activating signal as a reason underpinning ICSI’s failure in this species. Equally noticeable was the evidence that bovine oocytes that mounted oscillations after ICSI were unable to sustain them, in contrast to the same cohort of oocytes fertilized by IVF (Malcuit et al. 2006). These results suggested that an SF is present in bovine sperm but cannot be activated or released to the full extent required to initiate and sustain Ca2+ oscillations when the sperm is injected rather than gaining access through fusion (Fig. 1). The findings that bull sperm trigger immediate oscillations following injection into mouse eggs suggests that the failure to mount complete Ca2+ responses is due to inadequate release of bPLCZ1 into the ooplasm of in vitro matured bovine eggs (Yoon & Fissore 2007).
bPLCZ1 mRNA-induced oscillations and bPLCZ1 localization
Given that bovine sperm induced Ca2+ oscillations after IVF, and to a lesser extent after ICSI, it was necessary to establish the molecular identity, expression levels, and specific activity of the active SF in this species. That bPLCZ1 may be the active SF was suggested by the finding that homologous sequences of PLCZ1 were found in numerous mammalian species, including the bovine (Swann et al. 2006). Western blotting and recombinant bPLCZ1 were used to examine and estimate the expression of bPLCZ1 in bull sperm. It was determined that each bovine sperm expresses ~110–160 fg/sperm of PLCZ1, which is significantly more than that 20–50 fg/sperm expressed by mouse sperm (Saunders et al. 2002) but less than the reported expression of this enzyme in porcine sperm (Kurokawa et al. 2005). The higher expression of PLCZ1 in large mammals may be related simply to the volume of their eggs, or to the longer duration of the oscillations that they experience (Fissore et al. 1992, Malcuit et al. 2006), or to the lower sensitivity of the inositol 1,4,5-trisphosphate receptor (IP3R1- ITPR1) in eggs of these species (He et al. 1999), which mediates intracellular Ca2+ release during fertilization. The specific activity of bPLCZ1 was estimated as in previous studies by injecting its mRNA into bovine and mouse eggs followed by Ca2+ imaging and comparison of the responses. The expression of bPLCZ1 mRNA triggered long-lasting oscillations similar to those observed at fertilization in these species (Ross et al. 2008). The oscillations started approximately within 1-h post-injection and continued for over ~5 h. with a frequency proportional to the injected mRNA concentrations, higher concentrations producing higher frequency oscillations. Notably, in bovine eggs, regardless of the concentration, bPLCZ1 mRNA-induced oscillations transition into a disorganized pattern ~6 h. post-injection, although the transition is faster when using higher mRNA concentrations. It is worth noting that mPlcz1 mRNA injection induces oscillations in bovine eggs, although the mPlcz1 mRNA concentrations needed in this species to induce oscillations are higher than those employed in mouse eggs and higher than b PLCZ1 mRNA concentrations. These results suggest that, at least in these two species, the homologous enzyme functions more efficiently than the heterologous counterpart, a finding confirmed by greater IP3R1-downregulation following injection of the homologous enzyme (Ross et al. 2008). The success of bPLCZ1 mRNA injections in bovine eggs suggests that inefficient bPLCZ1 release might cause the atypical Ca2+ oscillations after ICSI in cattle (Fig. 1).
The evidence that the release of bPLCZ1 might be compromised in bovine ICSI and the knowledge that extensive shedding of sperm membranes takes place during natural fertilization (Ickowicz et al. 2012, Hirohashi & Yanagimachi 2018), which are bypassed during ICSI, led to myriad approaches to aid in the disassembly of the sperm head following ICSI. The proposed methods included exposing the sperm to chemical detergents, enzymes, and physical disruption of the plasma membrane (Tesarik et al. 1994, Ajduk et al. 2006, Morozumi et al. 2006). Some of these attempts improved the efficiency of egg activation in mouse ICSI (Ajduk et al. 2006, Morozumi et al. 2006), but similar approaches produced minor and variable effects in cattle even though some of these approaches effectively disintegrated the sperm membranes (Rho et al. 1998, Wei & Fukui 1999, Suttner et al. 2000, Arias et al. 2014). These results point to a gap in our knowledge of how PLCZ1 is released upon interacting with the ooplasm and suggest that besides relying on the delivery of a complete activating Ca2+ signal, the success of ICSI fertilization depends on the ability of the ooplasm to integrate and remodel the delivered sperm head. The capacity of the ooplasm to effectively remodel the sperm head depends on the oocytes’ maturation conditions. Nearly all in vivo matured mouse oocytes could consistently mount long-lasting and frequent oscillations after injection of a bovine sperm, whereas only a third of in vitro matured oocytes could, and the rest displayed sluggish or failed to mount oscillations (Águila et al. 2017). Remarkably, the amplitude of the Ca2+ oscillations in all in vitro matured oocytes that mounted oscillations was lower, whether after injection of a mouse or bovine sperm (Águila et al. 2017). This may also explain, at least in part, the lower developmental and implantation rates following conventional IVF or ICSI of IVM human oocytes (Smitz et al. 2011).
The location of bPLCZ1 in sperm was also of interest because if it were markedly different than in other species this might affect its release and compromise the success of ICSI. Bovine and human sperm display a similar flat and round or paddled head shape, and expectedly in these species, PLCZ1 localizes to similar regions in a narrow band on the equatorial region of the head (Yoon & Fissore 2007, Grasa et al. 2008), although a recent study in the bovine shows localization to this region only after capacitation (Unnikrishnan et al. 2021b). Regardless, this equatorial position of PLCZ1 favors its rapid release after gamete fusion, as this region has been suggested to be the site where the initiation of gamete fusion takes place in mammals (Sutovsky et al. 2003). Remarkably, after PLCZ1 leaks out into the ooplasm, we are unaware of its precise whereabouts in all species, and the bovine is not an exception. As noted above, whereas mPLCZ1 accumulates in the PN, bPLCZ1 remains in the ooplasm. This partitioning was demonstrated by injection of YFP-tagged bPLCZ1 mRNA, which resulted in a homogeneous distribution of bPLCZ1 without noticeable accumulation in the PNs in bovine zygotes, in contrast to mPlcz 1 that showed PN buildup in bovine zygotes (Cooney et al. 2010). These results are consistent with the dissimilar nuclear localization signal within the linker region of the enzyme between bovine and mice, which is missing three basic residues in the bPLCZ1 sequence (Ito et al. 2008). Collectively, ICSI in the bovine has confirmed the expression and role of PLCZ1 as the main SF in mammals. However, its low success in this species underlines gaps in our knowledge in the early stages of fertilization. For example, what are the signaling and biochemical mechanism(s) that promote the release of PLCZ1 from the sperm head into the ooplasm, and what are the ooplasm’s epitope(s) recognized by the enzyme that guides its distribution to attain its active location. The answers to these questions should aid in the more efficient use of ICSI in species where its success in promoting embryo development remains well below IVF.
Conclusions
The discovery of Plcz1 following the screen of an EST-library representing the transcriptome of a mouse testis culminated, at least in mammals, a quest initiated over 100 years ago when Jacques Loeb identified the role of ions as the vital mechanism underlying egg activation. The PLCZ1 breakthrough similarly triggered the next generation of studies to elucidate the array of molecular mechanisms necessary to induce the sustained Ca2+ oscillations that ensure successful embryonic development in mammals. The simultaneous development of ICSI strengthened the research that identified PLCZ1, and their combined use provided insights that laid the groundwork for significant discoveries, such as the realization that repeated fertilization failure after ICSI is most commonly associated with PLCZ1 deficiency or mutations. Furthermore, these studies were the basis for the isolation and identification of the functional role of novel components of the PT, the site of the residence of PLCZ1, a complex structure whose composition is hard to resolve.
Along with solving important questions, breakthrough findings bring to the fore previously unrealized questions, the unknown unknowns. In our case, and partly explored here, we are unaware of how PLCZ1 gets to the PT and its release from it. Similarly, once in the ooplasm, how PLCZ1 finds its substrate and what organelle(s) hosts the substrate remain to be determined. Lastly, and possibly most importantly, is whether PLCZ1 is the only factor required for egg activation in mammals. Two sets of findings lead to profoundly different interpretations. First, the ever-growing list of PLCZ1 mutations found underlying cases of human infertility despite these patients’ normal sperm parameters and otherwise normal genome profiles strongly supports the view that in humans, PLCZ1 is ‘the sole SF’. Against this line of thought is the evidence by several laboratories that Plcz1 KO mice sire offspring (Hachem et al. 2017, Nozawa et al. 2018), albeit the litters are of drastically reduced size. The interpretation of these results is that a ‘backup mechanism’ exists, at least in mice, and the implications are it might be capable of supporting ‘basal levels’ of egg activation and offspring development to ensure reproduction. A caveat to the interpretation of all genetics studies is that elimination or inactivation of a gene in a target organ or cells from the outset can produce significant changes in the gene expression of others, which might obscure the full impact of the function of the deleted gene. Therefore, future proteomic studies are needed to demonstrate whether the deletion/inactivation of Plcz1 alters the expression of other enzymes that can “make up” for the loss of PLCZ1. Further, the deletion of PLCZ1 should be extended to other mammals to determine how widespread is the reliance of mammals on PLCZ1. In other words, does a backup mechanism exist in other mammals? Regardless, studies over the last twenty years have established the preeminent role of PLCZ1 in egg activation in mammals. With this knowledge at hand, the available Plcz1 KO models, and the increased ability to generate new ones, coupled with the capacity to induce timely fertilization by ICSI, all the tools are at our disposal to answer the remaining questions and continue uncovering the molecules and mechanisms of fertilization.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of this review.
Funding
Part of the work presented here was supported by a grant from the NIH RO1 HD092499 to R A F. H A is funded by a fellowship from the JSPS Overseas Research Fellowships.
Author contribution statement
N G contributed to the design and performed some experiments presented in Figs 2 and 3. N G also wrote the initial draft of the rodent portion of the manuscript and prepared all figures. H A contributed to completing for Figs 2 and 3, wrote the initial draft for the bovine section of the manuscript, and prepared initial draft of Fig. 3. H C L performed all experiments for rat fertilization in Fig. 3, and contributed to the preparation of Fig. 3. R A F wrote the introduction and conclusions and revised the drafts to put together a coherent manuscript. He also obtained the funding to support these experiments.
Acknowledgements
The authors want to thank all past and present members of the Fissore lab whose work in one way or another has contributed to our progress in understanding the features of the Ca2+ responses in mammalian fertilization. Especial tribute to Ms Changli He whose dedicated effort for nearly thirty years contributed to sustain the laboratory.
References
Águila L, Felmer R, Arias ME, Navarrete F, Martin-Hidalgo D, Lee HC, Visconti P & Fissore R 2017 Defective sperm head decondensation undermines the success of ICSI in the bovine. Reproduction 154 307–318. (https://doi.org/10.1530/REP-17-0270)
Ajduk A, Yamauchi Y & Ward MA 2006 Sperm chromatin remodeling after intracytoplasmic sperm injection differs from that of in vitro fertilization. Biology of Reproduction 75 442–451. (https://doi.org/10.1095/biolreprod.106.053223)
Arias ME, Sanchez R, Risopatron J, Perez L & Felmer R 2014 Effect of sperm pretreatment with sodium hydroxide and dithiothreitol on the efficiency of bovine intracytoplasmic sperm injection. Reproduction, Fertility, and Development 26 847–854. (https://doi.org/10.1071/RD13009)
Ben-Yosef D, Oron Y & Shalgi R 1993 Prolonged, repetitive calcium transients in rat oocytes fertilized in vitro and in vivo. FEBS Letters 331 239–242. (https://doi.org/10.1016/0014-5793(9380344-t)
Betteridge KJ 1981 An historical look at embryo transfer. Journal of Reproduction and Fertility 62 1–13. (https://doi.org/10.1530/jrf.0.0620001)
Briski O & Salamone DF 2022 Past, present and future of ICSI in livestock species. Animal Reproduction Science In press. (https://doi.org/10.1016/j.anireprosci.2022.106925)
Chung JT, Keefer CL & Downey BR 2000 Activation of bovine oocytes following intracytoplasmic sperm injection (ICSI). Theriogenology 53 1273–1284. (https://doi.org/10.1016/S0093-691X(0000271-5)
Cobb M 2012 An amazing 10 years: the discovery of egg and sperm in the 17th century. Reproduction in Domestic Animals 47 (Supplement 4) 2–6. (https://doi.org/10.1111/j.1439-0531.2012.02105.x)
Cooney MA, Malcuit C, Cheon B, Holland MK, Fissore RA & D’Cruz NT 2010 Species-specific differences in the activity and nuclear localization of murine and bovine phospholipase C zeta 1. Biology of Reproduction 83 92–101. (https://doi.org/10.1095/biolreprod.109.079814)
Coy P & Romar R 2002 In vitro production of pig embryos: a point of view. Reproduction, Fertility, and Development 14 275–286. (https://doi.org/10.1071/rd01102)
Cuthbertson KS, Whittingham DG & Cobbold PH 1981 Free Ca2+ increases in exponential phases during mouse oocyte activation. Nature 294 754–757. (https://doi.org/10.1038/294754a0)
Dai J, Zhang T, Guo J, Zhou Q, Gu Y, Zhang J, Hu L, Zong Y, Song J & Zhang S et al.2021 Homozygous pathogenic variants in ACTL9 cause fertilization failure and male infertility in humans and mice. American Journal of Human Genetics 108 469–481. (https://doi.org/10.1016/j.ajhg.2021.02.004)
Dale B 1988 Primary and secondary messengers in the activation of ascidian eggs. Experimental Cell Research 177 205–211. (https://doi.org/10.1016/0014-4827(8890038-9)
Dale B, Defelice LJ & Ehrenstein G 1985 Injection of a soluble sperm fraction into sea-urchin eggs triggers the cortical reaction. Experientia 41 1068–1070. (https://doi.org/10.1007/BF01952148)
Deguchi R, Shirakawa H, Oda S, Mohri T & Miyazaki S 2000 Spatiotemporal analysis of Ca(2+) waves in relation to the sperm entry site and animal-vegetal axis during Ca(2+) oscillations in fertilized mouse eggs. Developmental Biology 218 299–313. (https://doi.org/10.1006/dbio.1999.9573)
Dozortsev D, Wakaiama T, Ermilov A & Yanagimachi R 1998 Intracytoplasmic sperm injection in the rat. Zygote 6 143–147. (https://doi.org/10.1017/s0967199498000069)
Ferre LB, Kjelland ME, Taiyeb AM, Campos-Chillon F & Ross PJ 2020 Recent progress in bovine in vitro-derived embryo cryotolerance: impact of in vitro culture systems, advances in cryopreservation and future considerations. Reproduction in Domestic Animals 55 659–676. (https://doi.org/10.1111/rda.13667)
Fissore RA, Dobrinsky JR, Balise JJ, Duby RT & Robl JM 1992 Patterns of intracellular Ca2+ concentrations in fertilized bovine eggs. Biology of Reproduction 47 960–969. (https://doi.org/10.1095/biolreprod47.6.960)
Galione A, Jones KT, Lai FA & Swann K 1997 A cytosolic sperm protein factor mobilizes Ca2+ from intracellular stores by activating multiple Ca2+ release mechanisms independently of low molecular weight messengers. Journal of Biological Chemistry 272 28901–28905. (https://doi.org/10.1074/jbc.272.46.28901)
Gomez E, Perez-Cano I, Amorocho B, Landeras J, Ballesteros A & Pellicer A 2000 Effect of injected spermatozoa morphology on the outcome of intracytoplasmic sperm injection in humans. Fertility and Sterility 74 842–843. (https://doi.org/10.1016/s0015-0282(0001520-x)
Goto K, Kinoshita A, Takuma Y & Ogawa K 1990 Fertilisation of bovine oocytes by the injection of immobilised, killed spermatozoa. Veterinary Record 127 517–520.
Grasa P, Coward K, Young C & Parrington J 2008 The pattern of localization of the putative oocyte activation factor, phospholipase Czeta, in uncapacitated, capacitated, and ionophore-treated human spermatozoa. Human Reproduction 23 2513–2522. (https://doi.org/10.1093/humrep/den280)
Hachem A, Godwin J, Ruas M, Lee HC, Ferrer Buitrago M, Ardestani G, Bassett A, Fox S, Navarrete F & De Sutter P et al.2017 PLCzeta is the physiological trigger of the Ca(2+) oscillations that induce embryogenesis in mammals but conception can occur in its absence. Development 144 2914–2924. (https://doi.org/10.1242/dev.150227)
Harbuz R, Zouari R, Pierre V, Ben Khelifa M, Kharouf M, Coutton C, Merdassi G, Abada F, Escoffier J & Nikas Y et al.2011 A recurrent deletion of DPY19L2 causes infertility in man by blocking sperm head elongation and acrosome formation. American Journal of Human Genetics 88 351–361. (https://doi.org/10.1016/j.ajhg.2011.02.007)
He CL, Damiani P, Ducibella T, Takahashi M, Tanzawa K, Parys JB & Fissore RA 1999 Isoforms of the inositol 1,4,5-trisphosphate receptor are expressed in bovine oocytes and ovaries: the type-1 isoform is down-regulated by fertilization and by injection of adenophostin A. Biology of Reproduction 61 935–943. (https://doi.org/10.1095/biolreprod61.4.935)
Hinrichs K 2005 Update on equine ICSI and cloning. Theriogenology 64 535–541. (https://doi.org/10.1016/j.theriogenology.2005.05.010)
Hirohashi N & Yanagimachi R 2018 Sperm acrosome reaction: its site and role in fertilization. Biology of Reproduction 99 127–133. (https://doi.org/10.1093/biolre/ioy045)
Ickowicz D, Finkelstein M & Breitbart H 2012 Mechanism of sperm capacitation and the acrosome reaction: role of protein kinases. Asian Journal of Andrology 14 816–821. (https://doi.org/10.1038/aja.2012.81)
Iritani A, Utsumi K, Miyake M, Hosoi Y & Saeki K 1988 In vitro fertilization by a routine method and by micromanipulation. Annals of the New York Academy of Sciences 541 583–590. (https://doi.org/10.1111/j.1749-6632.1988.tb22295.x)
Ito M, Shikano T, Kuroda K & Miyazaki S 2008 Relationship between nuclear sequestration of PLCzeta and termination of PLCzeta-induced Ca2+ oscillations in mouse eggs. Cell Calcium 44 400–410. (https://doi.org/10.1016/j.ceca.2008.02.003)
Jones KT 2007 Intracellular calcium in the fertilization and development of mammalian eggs. Clinical and Experimental Pharmacology and Physiology 34 1084–1089. (https://doi.org/10.1111/j.1440-1681.2007.04726.x)
Jones KT, Carroll J, Merriman JA, Whittingham DG & Kono T 1995 Repetitive sperm-induced Ca2+ transients in mouse oocytes are cell cycle dependent. Development 121 3259–3266. (https://doi.org/10.1242/dev.121.10.3259)
Jones KT, Matsuda M, Parrington J, Katan M & Swann K 2000 Different Ca2+-releasing abilities of sperm extracts compared with tissue extracts and phospholipase C isoforms in sea urchin egg homogenate and mouse eggs. Biochemical Journal 346 743–749. (https://doi.org/10.1042/bj3460743)
Kashir J, Nomikos M, Lai FA & Swann K 2014 Sperm-induced Ca2+ release during egg activation in mammals. Biochemical and Biophysical Research Communications 450 1204–1211. (https://doi.org/10.1016/j.bbrc.2014.04.078)
Kimura Y & Yanagimachi R 1995 Development of normal mice from oocytes injected with secondary spermatocyte nuclei. Biology of Reproduction 53 855–862. (https://doi.org/10.1095/biolreprod53.4.855)
Knott JG, Kurokawa M & Fissore RA 2003 Release of the Ca(2+) oscillation-inducing sperm factor during mouse fertilization. Developmental Biology 260 536–547. (https://doi.org/10.1016/s0012-1606(0300251-3)
Kono T, Carroll J, Swann K & Whittingham DG 1995 Nuclei from fertilized mouse embryos have calcium-releasing activity. Development 121 1123–1128. (https://doi.org/10.1242/dev.121.4.1123)
Kouchi Z, Fukami K, Shikano T, Oda S, Nakamura Y, Takenawa T & Miyazaki S 2004 Recombinant phospholipase Czeta has high Ca2+ sensitivity and induces Ca2+ oscillations in mouse eggs. Journal of Biological Chemistry 279 10408–10412. (https://doi.org/10.1074/jbc.M313801200)
Kuretake S, Kimura Y, Hoshi K & Yanagimachi R 1996 Fertilization and development of mouse oocytes injected with isolated sperm heads. Biology of Reproduction 55 789–795. (https://doi.org/10.1095/biolreprod55.4.789)
Kurokawa M & Fissore RA 2003 ICSI-generated mouse zygotes exhibit altered calcium oscillations, inositol 1,4,5-trisphosphate receptor-1 down-regulation, and embryo development. Molecular Human Reproduction 9 523–533. (https://doi.org/10.1093/molehr/gag072)
Kurokawa M, Sato K, Wu H, He C, Malcuit C, Black SJ, Fukami K & Fissore RA 2005 Functional, biochemical, and chromatographic characterization of the complete [Ca2+]I oscillation-inducing activity of porcine sperm. Developmental Biology 285 376–392. (https://doi.org/10.1016/j.ydbio.2005.06.029)
Larman MG, Saunders CM, Carroll J, Lai FA & Swann K 2004 Cell cycle-dependent Ca2+ oscillations in mouse embryos are regulated by nuclear targeting of PLCzeta. Journal of Cell Science 117 2513–2521. (https://doi.org/10.1242/jcs.01109)
Lillie FR 1913 The mechanism of fertilization. Science 38 524–528. (https://doi.org/10.1126/science.38.980.524)
Loeb J 1913 Artificial Parthenogenesis and Fertilization. University of Chicago Press.
Lopata AL & Lehrer SB 2009 New insights into seafood allergy. Current Opinion in Allergy and Clinical Immunology 9 270–277. (https://doi.org/10.1097/ACI.0b013e32832b3e6f)
Malcuit C, Maserati M, Takahashi Y, Page R & Fissore RA 2006 Intracytoplasmic sperm injection in the bovine induces abnormal [Ca2+]I responses and oocyte activation. Reproduction, Fertility, and Development 18 39–51. (https://doi.org/10.1071/rd05131)
Markert CL 1983 Fertilization of mammalian eggs by sperm injection. Journal of Experimental Zoology 228 195–201. (https://doi.org/10.1002/jez.1402280205)
Mazia D 1937 The release of calcium in Arbacia eggs on fertilization. Journal of Cellular and Comparative Physiology 10 291–304. (https://doi.org/10.1002/jcp.1030100304)
Miyazaki S, Hashimoto N, Yoshimoto Y, Kishimoto T, Igusa Y & Hiramoto Y 1986 Temporal and spatial dynamics of the periodic increase in intracellular free calcium at fertilization of golden hamster eggs. Developmental Biology 118 259–267. (https://doi.org/10.1016/0012-1606(8690093-x)
Morozumi K, Shikano T, Miyazaki S & Yanagimachi R 2006 Simultaneous removal of sperm plasma membrane and acrosome before intracytoplasmic sperm injection improves oocyte activation/embryonic development. PNAS 103 17661–17666. (https://doi.org/10.1073/pnas.0608183103)
Nakai M, Ito J, Suzuki SI, Fuchimoto DI, Sembon S, Suzuki M, Noguchi J, Kaneko H, Onishi A & Kashiwazaki N et al.2016 Lack of calcium oscillation causes failure of oocyte activation after intracytoplasmic sperm injection in pigs. Journal of Reproduction and Development 62 615–621. (https://doi.org/10.1262/jrd.2016-113)
Nakano Y, Shirakawa H, Mitsuhashi N, Kuwabara Y & Miyazaki S 1997 Spatiotemporal dynamics of intracellular calcium in the mouse egg injected with a spermatozoon. Molecular Human Reproduction 3 1087–1093. (https://doi.org/10.1093/molehr/3.12.1087)
Nozawa K, Satouh Y, Fujimoto T, Oji A & Ikawa M 2018 Sperm-borne phospholipase C zeta-1 ensures monospermic fertilization in mice. Scientific Reports 8 1315. (https://doi.org/10.1038/s41598-018-19497-6)
Oda S, Deguchi R, Mohri T, Shikano T, Nakanishi S & Miyazaki S 1999 Spatiotemporal dynamics of the [Ca2+]I rise induced by microinjection of sperm extract into mouse eggs: preferential induction of a Ca2+ wave from the cortex mediated by the inositol 1,4,5-trisphosphate receptor. Developmental Biology 209 172–185. (https://doi.org/10.1006/dbio.1999.9233)
Ozil JP, Sainte-Beuve T & Banrezes B 2017 Mg(2+)]o/[Ca(2+)]o determines Ca(2+) response at fertilization: tuning of adult phenotype? Reproduction 154 675–693. (https://doi.org/10.1530/REP-16-0057)
Palermo G, Joris H, Devroey P & Van Steirteghem AC 1992 Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet 340 17–18. (https://doi.org/10.1016/0140-6736(9292425-f)
Rall JA 2019 Calcium and muscle contraction: the triumph and tragedy of Lewis Victor Heilbrunn. Advances in Physiology Education 43 476–485. (https://doi.org/10.1152/advan.00094.2019)
Rho GJ, Kawarsky S, Johnson WH, Kochhar K & Betteridge KJ 1998 Sperm and oocyte treatments to improve the formation of male and female pronuclei and subsequent development following intracytoplasmic sperm injection into bovine oocytes. Biology of Reproduction 59 918–924. (https://doi.org/10.1095/biolreprod59.4.918)
Ridgway EB, Gilkey JC & Jaffe LF 1977 Free calcium increases explosively in activating medaka eggs. PNAS 74 623–627. (https://doi.org/10.1073/pnas.74.2.623)
Ross PJ, Beyhan Z, Iager AE, Yoon SY, Malcuit C, Schellander K, Fissore RA & Cibelli JB 2008 Parthenogenetic activation of bovine oocytes using bovine and murine phospholipase C zeta. BMC Developmental Biology 8 16. (https://doi.org/10.1186/1471-213X-8-16)
Runft LL, Jaffe LA & Mehlmann LM 2002 Egg activation at fertilization: where it all begins. Developmental Biology 245 237–254. (https://doi.org/10.1006/dbio.2002.0600)
Satouh Y, Nozawa K, Yamagata K, Fujimoto T & Ikawa M 2017 Viable offspring after imaging of Ca2+ oscillations and visualization of the cortical reaction in mouse eggs. Biology of Reproduction 96 563–575. (https://doi.org/10.1093/biolre/iox002)
Saunders CM, Larman MG, Parrington J, Cox LJ, Royse J, Blayney LM, Swann K & Lai FA 2002 PLC zeta: a sperm-specific trigger of Ca(2+) oscillations in eggs and embryo development. Development 129 3533–3544. (https://doi.org/10.1242/dev.129.15.3533)
Seita Y, Ito J & Kashiwazaki N 2009 Removal of acrosomal membrane from sperm head improves development of rat zygotes derived from intracytoplasmic sperm injection. Journal of Reproduction and Development 55 475–479. (https://doi.org/10.1262/jrd.20216)
Smitz JE, Thompson JG & Gilchrist RB 2011 The promise of in vitro maturation in assisted reproduction and fertility preservation. Seminars in Reproductive Medicine 29 24–37. (https://doi.org/10.1055/s-0030-1268701)
Sone Y, Ito M, Shirakawa H, Shikano T, Takeuchi H, Kinoshita K & Miyazaki S 2005 Nuclear translocation of phospholipase C-zeta, an egg-activating factor, during early embryonic development. Biochemical and Biophysical Research Communications 330 690–694. (https://doi.org/10.1016/j.bbrc.2005.03.032)
Steinhardt RA, Epel D, Carroll Jr EJ & Yanagimachi R 1974 Is calcium ionophore a universal activator for unfertilised eggs? Nature 252 41–43. (https://doi.org/10.1038/252041a0)
Stice SL & Robl JM 1990 Activation of mammalian oocytes by a factor obtained from rabbit sperm. Molecular Reproduction and Development 25 272–280. (https://doi.org/10.1002/mrd.1080250309)
Stricker SA 1999 Comparative biology of calcium signaling during fertilization and egg activation in animals. Developmental Biology 211 157–176. (https://doi.org/10.1006/dbio.1999.9340)
Sutovsky P, Manandhar G, Wu A & Oko R 2003 Interactions of sperm perinuclear theca with the oocyte: implications for oocyte activation, anti-polyspermy defense, and assisted reproduction. Microscopy Research and Technique 61 362–378. (https://doi.org/10.1002/jemt.10350)
Suttner R, Zakhartchenko V, Stojkovic P, Muller S, Alberio R, Medjugorac I, Brem G, Wolf E & Stojkovic M 2000 Intracytoplasmic sperm injection in bovine: effects of oocyte activation, sperm pretreatment and injection technique. Theriogenology 54 935–948. (https://doi.org/10.1016/S0093-691X(0000403-9)
Swann K 1990 A cytosolic sperm factor stimulates repetitive calcium increases and mimics fertilization in hamster eggs. Development 110 1295–1302. (https://doi.org/10.1242/dev.110.4.1295)
Swann K, Saunders CM, Rogers NT & Lai FA 2006 PLCzeta(zeta): a sperm protein that triggers Ca2+ oscillations and egg activation in mammals. Seminars in Cell and Developmental Biology 17 264–273. (https://doi.org/10.1016/j.semcdb.2006.03.009)
Taylor CT, Lawrence YM, Kingsland CR, Biljan MM & Cuthbertson KS 1993 Oscillations in intracellular free calcium induced by spermatozoa in human oocytes at fertilization. Human Reproduction 8 2174–2179. (https://doi.org/10.1093/oxfordjournals.humrep.a137999)
Tesarik J & Sousa M 1994 Comparison of Ca2+ responses in human oocytes fertilized by subzonal insemination and by intracytoplasmic sperm injection. Fertility and Sterility 62 1197–1204. (https://doi.org/10.1016/s0015-0282(1657185-4)
Tesarik J, Sousa M & Testart J 1994 Human oocyte activation after intracytoplasmic sperm injection. Human Reproduction 9 511–518. (https://doi.org/10.1093/oxfordjournals.humrep.a138537)
Thiery M 2009 Reinier De Graaf (1641–1673) and the Graafian follicle. Gynecological Surgery 6 189–191. (https://doi.org/10.1007/s10397-009-0466-6)
Uehara T & Yanagimachi R 1976 Microsurgical injection of spermatozoa into hamster eggs with subsequent transformation of sperm nuclei into male pronuclei. Biology of Reproduction 15 467–470. (https://doi.org/10.1095/biolreprod15.4.467)
Uehara T & Yanagimachi R 1977 Activation of hamster eggs by pricking. Journal of Experimental Zoology 199 269–274. (https://doi.org/10.1002/jez.1401990211)
Unnikrishnan V, Kastelic J & Thundathil J 2021a Intracytoplasmic sperm injection in cattle. Genes 12 198. (https://doi.org/10.3390/genes12020198)
Unnikrishnan V, Kastelic JP & Thundathil JC 2021b Ouabain-induced activation of phospholipase C zeta and its contributions to bovine sperm capacitation. Cell and Tissue Research 385 785–801. (https://doi.org/10.1007/s00441-021-03455-2)
van Leeuwenhoek A 1667 Observationes D. Anthonii Lewenhoeck, de natis e’ semini genitali animalcules. Philosophical Transactions of the Royal Society B 12 1040–1046. (https://doi.org/10.1016/0002-9378(7890424-6)
Wakai T, Mehregan A & Fissore RA 2019 Ca(2+) signaling and homeostasis in mammalian oocytes and eggs. Cold Spring Harbor Perspectives in Biology 11 a035162. (https://doi.org/10.1101/cshperspect.a035162)
Wei H & Fukui Y 1999 Effects of bull, sperm type and sperm pretreatment on male pronuclear formation after intracytoplasmic sperm injection in cattle. Reproduction, Fertility, and Development 11 59–65. (https://doi.org/10.1071/rd98106)
Westphal LM, Hinckley MD, Behr B & Milki AA 2003 Effect of ICSI on subsequent blastocyst development and pregnancy rates. Journal of Assisted Reproduction and Genetics 20 113–116. (https://doi.org/10.1023/a:1022678807398)
Wu H, He CL, Jehn B, Black SJ & Fissore RA 1998 Partial characterization of the calcium-releasing activity of porcine sperm cytosolic extracts. Developmental Biology 203 369–381. (https://doi.org/10.1006/dbio.1998.9070)
Yassine S, Escoffier J, Martinez G, Coutton C, Karaouzene T, Zouari R, Ravanat JL, Metzler-Guillemain C, Lee HC & Fissore R et al.2015 Dpy19l2-deficient globozoospermic sperm display altered genome packaging and DNA damage that compromises the initiation of embryo development. Molecular Human Reproduction 21 169–185. (https://doi.org/10.1093/molehr/gau099)
Yoon SY & Fissore RA 2007 Release of phospholipase C zetaand [Ca2+]I oscillation-inducing activity during mammalian fertilization. Reproduction 134 695–704. (https://doi.org/10.1530/REP-07-0259)
Yoon SY, Jellerette T, Salicioni AM, Lee HC, Yoo MS, Coward K, Parrington J, Grow D, Cibelli JB & Visconti PE et al.2008 Human sperm devoid of PLC, zeta 1 fail to induce Ca(2+) release and are unable to initiate the first step of embryo development. Journal of Clinical Investigation 118 3671–3681. (https://doi.org/10.1172/JCI36942)
Yoshida N & Perry AC 2007 Piezo-actuated mouse intracytoplasmic sperm injection (ICSI). Nature Protocols 2 296–304. (https://doi.org/10.1038/nprot.2007.7)
