The soluble sperm factor that activates the egg: PLCzeta and beyond

Egg activation is caused by increases in cytosolic Ca²⁺, and in mammalian eggs (MII oocytes) the sperm triggers a prolonged series of repetitive transients, or oscillations, in the cytosolic free Ca²⁺ concentration (Sanders & Swann 2016, Swann & Lai 2016). These Ca²⁺ oscillations are driven by increased inositol 1,4,5-trisphosphate (InsP₃) production which causes cycles of Ca²⁺ release from the InsP₃-receptor (IP₃R). Since the 1990s we have known that mammalian sperm contain a soluble protein ‘sperm factor’ (or sperm-borne oocyte-activating-factor), that can trigger Ca²⁺ oscillations after gamete fusion (Swann & Lai 2016). Its existence inside the sperm can explain why intracytoplasmic sperm injection (ICSI) mimics fertilization in causing Ca²⁺ oscillations in mouse and human eggs (Kurokawa & Fissore 2003, Jones 2018). It is now widely acknowledged that this sperm factor in mammals is the sperm-specific protein phospholipase Czeta (PLCζ) (Swann & Lai 2016, Jones 2018). Key evidence includes the finding that microinjection of PLCζ cRNA or protein causes prolonged sperm-like Ca²⁺ oscillations in all mammalian eggs studied (Swann & Lai 2016) and that functionally disruptive mutations in PLCζ alone lead to male factor infertility and egg activation failure in humans in ICSI (Escoffier et al. 2016).

Recently two groups have reported the phenotype of PLCζ-knockout (KO) mice. They both found that injecting PLCζ KO mouse sperm into eggs (hence ICSI) fails to trigger any Ca²⁺ oscillations (Hachem et al. 2017, Nozawa et al. 2018). This shows that PLCζ accounts for the Ca²⁺ signals and egg activation after ICSI. However, during in vitro fertilization (IVF) and mating with PLCζ KO males some eggs are activated at fertilization and embryo development still occurs (Hachem et al. 2017, Nozawa et al. 2018). Success rates of IVF are lower and litter sizes are smaller with PLCζ KO males, but the result contrasts with what happens with ICSI. The reason why IVF leads some eggs to activate with PLCζ KO sperm is because there are ~3 large Ca²⁺ oscillations that occur about 40 min later than expected when compared to wild-type sperm (Nozawa et al. 2018). The existence of these delayed Ca²⁺ oscillations with PLCζ KO sperm has been reproduced in my lab (Fluks, Parrington and Swann unpublished). The late Ca²⁺ oscillations with PLCζ KO sperm lead to delayed egg activation, including cortical granule exocytosis which is required to block extra sperm entry (Nozawa et al. 2018). This means that many such zygotes fail to develop because they are polyspermic. Overall, the data suggest that PLCζ initiates the Ca²⁺ oscillations at fertilization, accounting for most of the Ca²⁺ spikes, but that during IVF the sperm has another mechanism for promoting later Ca²⁺ oscillations in the mouse (Jones 2018). Two characteristics of this secondary mechanism is that it is delayed after gamete fusion, and that it is active in IVF and not with ICSI.

In looking for PLCζ-independent mechanisms for Ca²⁺ oscillations we need to consider previous data gather from mammalian zygotes. First, all previous studies have shown that without sperm-egg membrane fusion in IVF, there are no Ca²⁺ oscillations (Swann & Lai 2016). So, it is reasonable to assume that a second mechanism for Ca²⁺ release involves a sperm factor that is either soluble and enters the egg by cytosolic diffusion or that it is introduced by the sperm membrane into the egg plasma membrane by two-dimensional diffusion. For either option I will describe it as a sperm factor. It has been suggested that the PLCζ-independent sperm factor may be ‘cryptic’ because it is only apparent when PLCζ is absent (Jones 2018). Whilst this is true from an observational point of view, it does not mean it is inactive during normal fertilization. In fact, it is difficult to see how a second factor could only arise when PLCζ was not present. As far as we know PLCζ is only active in eggs, so a lack of PLCζ would not be evident until after gametes have fused. Clearly, gene expression in spermatogenesis cannot compensate for future events, hence the second factor should operate in IVF with wild-type sperm. In hindsight we can see evidence of a secondary mechanism because it was previously found that ICSI causes a shorter duration of Ca²⁺ oscillations than IVF in mouse zygotes (Kurokawa & Fissore 2003). If the secondary factor operates in normal IVF, it also gives it a selective advantage for it to persist in the presence of PLCζ. One attractive idea is that this factor is a ‘primitive’ factor from a role in egg activation in species earlier in the vertebrate lineage (Nozawa et al. 2018).

Previous studies restrict the options for how any factor can trigger Ca²⁺ oscillations in the absence of PLCζ. For example, one could propose that the second factor promotes Ca²⁺ influx into the egg, perhaps by the insertion of sperm derived Ca²⁺ channels into the egg membrane. However, there are many ways to increase Ca²⁺ influx into unfertilized mammalian eggs and none of them trigger Ca²⁺ oscillations without PLCζ. An updated version of the ‘Ca²⁺ conduit’ idea remains implausible (Swann & Lai 2016). The second factor cannot work via messengers such as NAADP, or cADPR, since these also fail to trigger Ca²⁺ oscillations in mouse eggs (Swann & Lai 2016). A sperm protein called PAWP has been suggested to trigger Ca²⁺ oscillations in eggs, but the key data on PAWP is not reproducible (Sanders & Swann 2016). Furthermore, PAWP is supposed to cause Ca²⁺ oscillations during ICSI, but we now know that PLCζ accounts for these Ca²⁺ oscillations. Another study has suggested that extramitochondrial citrate synthase is the second sperm factor in mammals (Kang et al. 2020). However, the phenotype of extramitochondrial citrate synthase KO sperm at fertilization is apparently the same as PLCζ KO sperm, with delayed Ca²⁺ oscillations (Kang et al. 2020). This result is difficult to rationalize because these citrate synthase KO sperm still contained PLCζ and the initial Ca²⁺ oscillations should not be delayed. In addition, we have found that citrate synthase protein injection into mouse eggs does not trigger Ca²⁺ release (Sanders and Swann, unpublished observations). From what we know about how to cause Ca²⁺ oscillations in mouse eggs, we can conclude that the second factor is either making InsP₃ or else directly stimulating the IP₃Rs.

If the second sperm factor generates InsP_3, this implicates another PLC. There are many other PLC isoforms in mammalian sperm (Parrington et al. 2002). However, the other PLCs are about two or three orders of magnitude less active in causing Ca²⁺ release than PLCζ in eggs (Mehlmann et al. 2001, Swann & Lai 2016). To be active in eggs they would have to be expressed at >2 pg per sperm, and yet there is only 40 pg of total protein in a mouse sperm (Mehlmann et al. 2001). The second sperm factor could stimulate an egg membrane-derived PLC, but this is not consistent with some previous findings. For example, if eggs are imaged using GFP-tagged C1 domains, there is no measurable diacylglycerol increase in the plasma membrane for at least 2 h of sperm-induced Ca²⁺ oscillations in mouse eggs, despite the ability of this probe to respond to other stimuli (Yu et al. 2008). Hence, it appears that a plasma membrane derived PLC activity is not stimulated in fertilizing eggs. This is not an issue for PLCζ which is the only mammalian PLC without a PH domain and it binds to PIP₂ in intracellular vesicles (Fig. 1) and not the plasma membrane (Swann & Lai 2016). However, conventional PLCs (β, γ or δ1 class) locate to the plasma membrane with a PH domain, and one would expect some diacylglycerol increase to occur if they were active at fertilization. If the second sperm factor makes InsP₃ then it probably needs to stimulate another unconventional PLC that is localized on vesicles in the egg. It is not clear whether any other PLCs would match the unusual localization pattern of PLCζ, but it might be worth investigating the localization of the epsilon or eta class of PLCs in eggs.

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A schematic representation of the hypothesis for PLCζ and a second factor may act to cause Ca²⁺ oscillations in fertilizing mouse eggs.

Citation: Reproduction 160, 1; 10.1530/REP-20-0079

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In the absence of data on other PLCs I can suggest an alternative idea, that the second sperm factor acts to sensitize the IP₃R. Strontium ions or thimerosal both stimulate Ca²⁺ oscillations in mouse eggs, and they both appear to act directly to sensitize the IP₃R to release Ca²⁺ (Swann & Lai 2016). The schematic in Fig. 1 shows the second factor affecting IP₃R-induced Ca²⁺ release following PLCζ entry. If the target is the IP₃R, or vesicular PIP₂, the protein factor is likely to be soluble and diffuse into the cytosol. To explain why PLCζ-independent Ca²⁺ release is not evident with ICSI, it is possible that the second factor is released from the sperm during their preparation when the sperm is damaged, or when the head is removed. Damaging the sperm membrane is standard practice before ICSI. Plasma membrane damage during cryopreservation may also lead to the loss of the second factor from sperm, which could explain why there was a lack of Ca²⁺ oscillations with most cryopreserved PLCζ KO sperm in IVF (Hachem et al. 2017). The other feature of the second sperm factor is a delayed action. It could be that the synthesis of a second sperm factor protein from RNA in the sperm could account for the >40-min delay before Ca²⁺ transients (Jones 2018). However, the total amount of RNA in a single mouse sperm (0.1 pg) is similar to the amount of PLCζ RNA injected into an egg to cause Ca²⁺ oscillations, and yet, sperm RNA is made up of several hundred varieties. Any protein synthesized from sperm RNA would have to be >100 times more potent than PLCζ which is active at concentrations of less than 10 nM. A more realistic idea is that secondary factor is another protein delivered by the sperm. The delay could be because this protein needs to first diffuse and equilibrate throughout the egg and then act indirectly to sensitize IP₃Rs. The second factor may not be active in human fertilization since human eggs are less sensitive to Ca²⁺ release, and for example do not oscillate in response to strontium medium (Lu et al. 2018). This could explain why inactivating mutations in human PLCζ lead to male factor infertility with both normal conception and ICSI (Escoffier et al. 2016). The second factor may only be evident in mouse and rat eggs or possibly in some non-mammalian species that do not appear to use PLCζ to activate the egg (Swann & Lai 2016).