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

in Reproduction
Author:
Karl SwannSchool of Biosciences, Cardiff University, Cardiff, UK

Search for other papers by Karl Swann in
Current site
Google Scholar
PubMed
Close

Correspondence should be addressed to K Swann; Email: Swannk1@cardiff.ac.uk
Free access

PLCzeta(ζ) initiates Ca2+ oscillations and egg activation at fertilization in mammals, but studies in mouse eggs fertilized by PLCζ knockout (KO) sperm imply that there is another slow acting factor causing Ca2+ release. Here, I propose a hypothesis for how this second sperm factor might cause Ca2+ oscillations in mouse eggs.

Abstract

PLCzeta(ζ) initiates Ca2+ oscillations and egg activation at fertilization in mammals, but studies in mouse eggs fertilized by PLCζ knockout (KO) sperm imply that there is another slow acting factor causing Ca2+ release. Here, I propose a hypothesis for how this second sperm factor might cause Ca2+ oscillations in mouse eggs.

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 InsP3, 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.

Figure 1
Figure 1

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

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).

Declaration of interest

The author declares that there is no conflict of interest that could be perceived as prejudicing the impartiality of this article.

Funding

This research did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.

Author contribution statement

K S conceived the ideas and wrote the paper.

Acknowledgements

The author is grateful to Jessica Sanders and Monika Fluks who helped generate the unpublished data that was referred to and to John Parrington for sending me the PLCζ KO male mice.

References

  • Escoffier J, Lee HC, Yassine S, Zougari R, Martinez G, Karaouzene T, Coutton C, Kherraf ZE, Halouni L & Triki C et al.2016 Homozygous mutation of PLCz1 leads to defective human oocyte activation and infertility that is not rescued by the WW-binding protein PAWP. Human Molecular Genetics 25 878891. (https://doi.org/10.1093/hmg/ddv617)

    • Search Google Scholar
    • Export Citation
  • Hachem A, Godwin J, Ruas M, Lee HC, Ferrer-Buitrago M, Ardestani G, Bassett A, Fox S, Naverrete F & De Sutter P et al.2017 PLCζ is the physiological trigger of the Ca2+ oscillations that induced embryogeneis in mammals but conception can occur in its absence. Development 144 29142924. (https://doi.org/10.1242/dev.150227)

    • Search Google Scholar
    • Export Citation
  • Jones KT 2018 Mammalian sperm contain two factors for calcium release and egg activation: phospholipase C zeta and a cryptic activating factor. Molecular Human Reproduction 24 465468. (https://doi.org/10.1093/molehr/gay038)

    • Search Google Scholar
    • Export Citation
  • Kang W, Harada Y, Yamatoya K, Kawano N, Kanai S, Miyamoto Y, Nakamura A, Miyado M, Hayashi Y & Kuoki Y et al.2020 Extra-mitochondrial citrate synthase initiates calcium oscillation and supresses age-dependent sperm dysfunction. Laboratory Investigation 100 583595. (https://doi.org/10.1038/s41374-019-0353-3)

    • Search Google Scholar
    • Export Citation
  • Kurokawa M & Fissore RA 2003 ICSI-generated mouse zygotes exhibit altered calcium oscillations, inositol 1,4,5-trisphohate receptor 1 downregulation, and embryo development. Molecular Human Reproduction 9 523533. (https://doi.org/10.1093/molehr/gag072)

    • Search Google Scholar
    • Export Citation
  • Lu Y, Reffy R, Ferrer Buitrago M, Vander Jeught M, Neupane J, De Vos WH, Van der Abbeel E, Lierman S, De Sutter P & Heindryckx B 2018 Strontium fails to trigger Ca2+ release and activation in human oocytes despite the presence of functional TRPV3 channels. Human Reproduction Open 3 hoy005. (https://doi.org/10.1093/hropen/hoy005)

    • Search Google Scholar
    • Export Citation
  • Mehlmann LM, Chattopadhyay A, Carpenter G & Jaffe LA 2001 Evidence that phospholipase C from the sperm is not responsible for initiating Ca2+ release at fertilization in mouse eggs. Development al Biology236 492501. (https://doi.org/10.1006/dbio.2001.0329)

    • Search Google Scholar
    • Export Citation
  • Nozawa K, Satouh Y, Fujimoto T, Oji A & Ikawa M 2018 Sperm borne phospholipase C zeta1 ensures monospermic fertilization in mice. Scientific Reports 8 1315. (https://doi.org/10.1038/s41598-018-19497-6)

    • Search Google Scholar
    • Export Citation
  • Parrington J, Jones ML, Tunwell R, Devader C, Katan M & Swann K 2002 Phospholipase C isoforms in mammalian spermatozoa: potential components of the sperm factor that causes Ca2+ release in eggs. Reproduction 123 3139. (https://doi.org/10.1530/rep.0.1230031)

    • Search Google Scholar
    • Export Citation
  • Sanders JR & Swann K 2016 Molecular triggers of egg activation at fertilization in mammals. Reproduction 152 R41R50. (https://doi.org/10.1530/REP-16-0123)

    • Search Google Scholar
    • Export Citation
  • Swann K & Lai FA 2016 Egg activation by a soluble sperm protein. Physiological Reviews 96 127149. (https://doi.org/10.1152/physrev.00012.2015)

    • Search Google Scholar
    • Export Citation
  • Yu Y, Halet G, Lai FA & Swann K 2008 Regulation of diacylglycerol production and protein kinase C stimulation during sperm- and PLCζ-mediated mouse egg activation. Biology of the Cell 100 633643. (https://doi.org/10.1042/BC20080033)

    • Search Google Scholar
    • Export Citation

 

  • Collapse
  • Expand

     An official journal of

    Society for Reproduction and Fertility

 

  • View in gallery
    Figure 1

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

  • Escoffier J, Lee HC, Yassine S, Zougari R, Martinez G, Karaouzene T, Coutton C, Kherraf ZE, Halouni L & Triki C et al.2016 Homozygous mutation of PLCz1 leads to defective human oocyte activation and infertility that is not rescued by the WW-binding protein PAWP. Human Molecular Genetics 25 878891. (https://doi.org/10.1093/hmg/ddv617)

    • Search Google Scholar
    • Export Citation
  • Hachem A, Godwin J, Ruas M, Lee HC, Ferrer-Buitrago M, Ardestani G, Bassett A, Fox S, Naverrete F & De Sutter P et al.2017 PLCζ is the physiological trigger of the Ca2+ oscillations that induced embryogeneis in mammals but conception can occur in its absence. Development 144 29142924. (https://doi.org/10.1242/dev.150227)

    • Search Google Scholar
    • Export Citation
  • Jones KT 2018 Mammalian sperm contain two factors for calcium release and egg activation: phospholipase C zeta and a cryptic activating factor. Molecular Human Reproduction 24 465468. (https://doi.org/10.1093/molehr/gay038)

    • Search Google Scholar
    • Export Citation
  • Kang W, Harada Y, Yamatoya K, Kawano N, Kanai S, Miyamoto Y, Nakamura A, Miyado M, Hayashi Y & Kuoki Y et al.2020 Extra-mitochondrial citrate synthase initiates calcium oscillation and supresses age-dependent sperm dysfunction. Laboratory Investigation 100 583595. (https://doi.org/10.1038/s41374-019-0353-3)

    • Search Google Scholar
    • Export Citation
  • Kurokawa M & Fissore RA 2003 ICSI-generated mouse zygotes exhibit altered calcium oscillations, inositol 1,4,5-trisphohate receptor 1 downregulation, and embryo development. Molecular Human Reproduction 9 523533. (https://doi.org/10.1093/molehr/gag072)

    • Search Google Scholar
    • Export Citation
  • Lu Y, Reffy R, Ferrer Buitrago M, Vander Jeught M, Neupane J, De Vos WH, Van der Abbeel E, Lierman S, De Sutter P & Heindryckx B 2018 Strontium fails to trigger Ca2+ release and activation in human oocytes despite the presence of functional TRPV3 channels. Human Reproduction Open 3 hoy005. (https://doi.org/10.1093/hropen/hoy005)

    • Search Google Scholar
    • Export Citation
  • Mehlmann LM, Chattopadhyay A, Carpenter G & Jaffe LA 2001 Evidence that phospholipase C from the sperm is not responsible for initiating Ca2+ release at fertilization in mouse eggs. Development al Biology236 492501. (https://doi.org/10.1006/dbio.2001.0329)

    • Search Google Scholar
    • Export Citation
  • Nozawa K, Satouh Y, Fujimoto T, Oji A & Ikawa M 2018 Sperm borne phospholipase C zeta1 ensures monospermic fertilization in mice. Scientific Reports 8 1315. (https://doi.org/10.1038/s41598-018-19497-6)

    • Search Google Scholar
    • Export Citation
  • Parrington J, Jones ML, Tunwell R, Devader C, Katan M & Swann K 2002 Phospholipase C isoforms in mammalian spermatozoa: potential components of the sperm factor that causes Ca2+ release in eggs. Reproduction 123 3139. (https://doi.org/10.1530/rep.0.1230031)

    • Search Google Scholar
    • Export Citation
  • Sanders JR & Swann K 2016 Molecular triggers of egg activation at fertilization in mammals. Reproduction 152 R41R50. (https://doi.org/10.1530/REP-16-0123)

    • Search Google Scholar
    • Export Citation
  • Swann K & Lai FA 2016 Egg activation by a soluble sperm protein. Physiological Reviews 96 127149. (https://doi.org/10.1152/physrev.00012.2015)

    • Search Google Scholar
    • Export Citation
  • Yu Y, Halet G, Lai FA & Swann K 2008 Regulation of diacylglycerol production and protein kinase C stimulation during sperm- and PLCζ-mediated mouse egg activation. Biology of the Cell 100 633643. (https://doi.org/10.1042/BC20080033)

    • Search Google Scholar
    • Export Citation