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Mature amphibian eggs arrested at meiotic metaphase II must undergo activation to initiate embryonic development soon after fertilization. Fertilizing sperm provide eggs with a signal that induces egg activation, and an increase in intracellular Ca²⁺ concentration in the egg cytoplasm (a Ca²⁺ rise) is the most important signal for this initiation. The sperm transmits the signal for the Ca²⁺ rise, known as the sperm factor, which is divergent between anurans and urodeles. In monospermic anurans, the sperm transmits the signal through a receptor on the egg membrane, causing a single rapid Ca²⁺ rise. Sperm matrix metalloproteinase-2 is a potential candidate for the receptor-mediated sperm factor in anurans. In physiologically polyspermic urodeles, multiple slower Ca²⁺ rises are caused by a soluble sperm factor (sperm-specific citrate synthase) which is transferred to the egg cytoplasm after sperm–egg fusion. We discuss the molecular mechanisms of egg activation in amphibian fertilization, focusing on recent progress in characterizing these sperm factors and their divergence during the evolution of tetrapod vertebrates.

Two decades have passed since the discovery of phospholipase C zeta (PLCZ1) as the sperm oocyte-activating factor. At present, there is a general consensus that PLCZ1 is responsible for triggering the calcium (Ca²⁺) oscillations necessary to start the oocyte activation process in mammals. One proof is that abnormal, reduced, or absent PLCZ1 in human spermatozoa leads to fertilization failure (FF) after intracytoplasmic sperm injection (ICSI). ICSI is the most effective assisted reproduction technique and enables overcoming almost all male infertility conditions. Despite fertilization rates of up to 80%, FF does occur in 1–3% of ICSI cycles, which leaves these patients with few options for obtaining genetically related offspring. Assisted oocyte activation (AOA) using Ca⁺² ionophores has emerged as a useful treatment option for these patients. While AOA has been proven very beneficial for the treatment of sperm-related FF, some cases of female-related FF cannot be overcome by AOA. Therefore, the development of appropriate diagnostic tests that predict the prognosis of AOA treatment would be advantageous to improve the clinical management of these patients and shorten the time to pregnancy. The aim of this review is to provide an up-to-date overview of the genetic causes of FF after ICSI and to discuss the advantages and disadvantages of using PLCZ1 as a diagnostic marker or therapeutic molecule in comparison with currently available diagnostic tests and treatments.

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.

Oocyte activation deficiency (OAD) remains the predominant cause of total/low fertilization rate in assisted reproductive technology. Phospholipase C zeta (PLCZ1) is the dominant sperm-specific factor responsible for triggering oocyte activation in mammals. OAD has been linked to numerous PLCZ1 abnormalities in patients experiencing failed in vitro fertilization or intracytoplasmic sperm injection cycles. While significant efforts have enhanced our understanding of the clinical relevance of PLCZ1, and the potential effects of genetic variants upon functionality, our ability to apply PLCZ1 in a diagnostic or therapeutic role remains limited. Artificial oocyte activation is the only option for patients experiencing OAD but lacks a reliable diagnostic approach. Immunofluorescence analysis has revealed that the levels and localization patterns of PLCZ1 within sperm can help us to indirectly diagnose a patient’s ability to induce oocyte activation. Screening of the gene encoding PLCZ1 protein is also critical if we are to fully determine the extent to which genetic factors might play a role in the aberrant expression and/or localization patterns observed in infertile patients. Collectively, these findings highlight the clinical potential of PLCZ1, both as a prognostic indicator of OAD and eventually as a therapeutic agent. In this review, we focus on our understanding of the association between OAD and PLCZ1 by discussing the localization and expression of this key protein in human sperm, the potential genetic causes of OAD, and the diagnostic tools that are currently available to us to identify PLCZ1 deficiency and select patients that would benefit from targeted therapy.

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.

In 2002, sperm-specific phospholipase C zeta1 (PLCZ1) was discovered and through these 20 years, it has been established as the predominant sperm oocyte-activating factor. PLCZ1 cRNA expression or direct protein microinjection into mammalian oocytes triggers calcium (Ca²⁺) oscillations indistinguishable from those observed at fertilization. The imperative role of PLCZ1 in oocyte activation is revealed by the vast number of human mutations throughout the PLCZ1 gene that have been identified and directly linked with certain forms of male infertility due to oocyte activation deficiency. PLCZ1 is the smallest PLC in size, comprising four N-terminal EF-hand domains, followed by X and Y catalytic domains, which are separated by the XY-linker, and ending with a C-terminal C2 domain. The EF hands are responsible for the high Ca²⁺ sensitivity of PLCZ1. The X and Y catalytic domains are responsible for the catalysis of the phosphatidylinositol-4,5-bisphosphate [PI(4,5)P₂] substrate to produce the Ca²⁺-mobilising messenger, inositol 1,4,5-trisphosphate (IP₃), while the XY-linker plays multiple roles in the unique mode of PLCZ1 action. Finally, the C2 domain has been proposed to facilitate the anchoring of PLCZ1 to intracellular vesicles through its direct interactions with specific phosphoinositides. This review discusses recent advances in the structure and function relationship of PLCZ1 and the potential binding partners of this important sperm-specific protein in the sperm and oocyte. The unravelling of all the remaining hidden secrets of sperm PLCZ1 should help us to understand the precise mechanism of fertilization, as well as enabling the diagnosis and treatment of currently unknown forms of PLCZ1 -linked human infertility.