Here we extend our previous work in the mouse and nonhuman primate and document, for the first time in humans, that zinc exocytosis is associated with egg activation and that a reduction in zinc is sufficient to trigger cell cycle resumption10,11. Due to legal restrictions on research with human eggs, we were limited to parthenogenetic activation methods to interrogate the zinc spark. Of the methods we used, injection of hPLCζ cRNA is the most physiologically relevant approach as it induces a series of calcium transients similar to sperm-induced activation24,25. This method produced zinc sparks and in mouse we captured the sperm-mediated zinc spark in real time during in vitro fertilization. These results strongly support the physiological significance of this biological event (Zhang et al., Sci Reports, accepted). During our studies in both mouse and human, we noted that there was a meiotic maturation-dependent acquisition of the ability of the gamete to mount a zinc spark, with cells arrested at prophase I having on average a smaller zinc spark compared to those arrested at metaphase II. These results suggest that the machinery that elicits the zinc spark is likely not fully established until just prior to fertilization, which may be an important mechanism to prevent premature egg activation. In the mouse, a significant increase of intracellular zinc occurs during meiotic maturation that is regulated predominantly by the zinc transporters ZIP6 and ZIP1013. Moreover, a prominent polarization of cortical vesicle distribution also occurs in the gamete between prophase I and metaphase II and these vesicles are the source of the zinc spark16. Thus, there appears to be a spatio-temporal regulation of the gamete’s ability to produce a zinc spark, which is likely to be conserved as the human gamete both expresses ZIP6 and ZIP10 and has labile zinc localized to punctate vesicle-like structures13.

This tight coordination between the acquisition of zinc spark capacity and the timing of egg activation parallels what has been observed with cortical granule exocytosis (CGE) and calcium transients. For example, cortical granules are secretory structures that contain proteases that cleave Zona Pellucida Glycoprotein 2 and are involved in establishing the block to polyspermy. Gametes arrested at prophase I are less competent than mature MII eggs to undergo CGE, even when stimulated with treatments that induce calcium transients that mimic what occur during fertilization26. Both CGE and zinc exocytosis are calcium-dependent events, so our results are consistent with the knowledge that the egg’s ability to elicit repetitive calcium transients also increases throughout meiotic progression. GV oocytes, for example, are unable to initiate and maintain calcium transients as efficiently as MII eggs27,28. In response to ionomycin, mouse MII eggs exhibit a significantly greater calcium response compared to GV oocytes and the greatest change was observed between metaphase of meiosis I and MII29. This differential calcium transient response is due to meiotic maturation-associated changes in calcium homeostasis mechanisms, including calcium channel modifications and densities, endoplasmic reticulum (ER) reorganization and increases in ER calcium stores30. We therefore anticipate that the acquisition of zinc spark potential will mirror what has already been demonstrated for calcium29. However, future studies are warranted to define the precise timing during meiotic maturation when the gamete is able to achieve a maximal zinc spark response.

Although both mouse and human GV oocytes had a significantly reduced ability to produce a zinc spark in response to ionomycin activation relative to MII eggs, the response of mouse GV oocytes in general was more uniform and attenuated compared to the human GV oocytes. This discrepancy may reflect species-specific differences in calcium machinery and signaling mechanisms. For example, hamster GV oocytes already contain ~80% of the IP3-sensitive calcium stores present in MII eggs31. Alternatively, we can not discount the possibility that there may be inherent differences in the quality of the mouse and human gametes. For example, the mouse GV oocytes were obtained following gonadotropin stimulation protocols that enrich for a synchronous population of fully-grown oocytes, whereas the human GV oocytes were obtained because they failed to resume meiosis following hyperstimulation and are likely more varied in quality. This possibility underscores the inherent challenges of conducting basic research on human gametes – namely that the majority of the oocytes are those discarded from Assisted Reproduction Technologies (ART) cycles because they did not meet the developmental and/or morphological criteria for clinical use and that the sample size is limited. Despite these challenges, use of similar human oocyte populations for basic research has yielded important knowledge of human egg biology32,33,34.

The work described here, in addition to providing new insight into the fundamental biology of the human egg, also has important translational impact for ART in two distinct ways. First, intracytoplasmic sperm injection (ICSI), the process in which a sperm is microinjected directly into the egg, was originally developed to overcome male factor infertility but now accounts for up to roughly 70% of procedures done in clinical ART (www.sart.org). Although ICSI results in successful fertilization ~70% of the time, complete fertilization failure still occurs in up to 5% of the cases due to the inability of the egg to properly activate25. To improve the success of ICSI in patients with low fertilization potential, methods combining ICSI and egg activation have been implemented clinically. This technique, referred to as Assisted Oocyte Activation (AOA), relies on approaches that mimic or induce the fertilization-induced intracellular calcium rise such as calcium ionophore or PLCζ23,25. Successful pregnancies have been reported using AOA35. We demonstrated that treatment with an intracellular zinc chelator can activate human eggs and thus may have important applications in AOA. In mouse, the fertilization-induced zinc spark, unlike calcium transients, is confined to the first minutes of egg activation (Zhang et al., Sci Reports, accepted). Because of this narrow window, reducing intracellular zinc availability may be a more targeted and specific approach for driving egg activation and may thereby increase the efficacy of AOA. Importantly, TPEN-mediated AOA has resulted in healthy live offspring in murine and porcine models14,17.

Second, advances in understanding the physicochemical roles of zinc in gamete function may have clinical impact because the zinc spark occurs rapidly following egg activation in the human egg and can be detected in the extracellular space. Moreover, there are variations in both the zinc spark and calcium transient profiles between individual eggs suggesting underlying differences in quality. In fact, elegant studies in the human have shown that calcium transient profiles following sperm-induced egg activation are distinct depending on egg quality parameters4. For example, processes known to diminish gamete quality, including in vitro maturation, extended culture and vitrification and thawing, all result in alterations of the frequency and amplitude of calcium transients during fertilization4. In the human, establishing the direct relationship between zinc dynamics during fertilization and further embryonic development has not been possible. To address this important knowledge gap, we demonstrated in the mouse that parameters of the first zinc spark (amplitude and total zinc release) are highly associated with blastocysts of increased quality (Zhang et al., Sci Reports, accepted). Efforts are currently underway to develop platforms to detect and quantify the human zinc spark in a non-invasive, safe and efficacious manner. Specifically, methods by which exocytosed zinc in the media can be measured following fertilization are needed to limit the exposure of the zygote to both dyes and damaging photons required for imaging. The zinc spark technology possesses several advantages over existing embryo selection methods as this biological phenomenon occurs extracellularly within minutes of fertilization and specific zinc spark profiles strongly correlate with and are predictive of preimplantation embryo development (Zhang et al., Sci Reports, accepted). Thus, selecting embryos based on their fertilization-induced zinc spark profile would minimize the need for extended embryo culture and multiple embryo transfer – both of which have measureable risks36,37,38,39. Such changes would improve reproductive outcomes for ART, which now account for millions of births worldwide.