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Phospholipase C causes Ca²⁺ oscillations and parthenogenetic activation of human oocytes

¹ Department of Anatomy and Developmental Biology, University College London, Gower Street, London WC1E 6BT, ² Assisted Conception Unit and GKT Department of Women’s Health, Guy’s and St Thomas’s NHS Trust, Guy’s Hospital, London SE1 9RT, ³ Wales Heart Research Institute, Wales College of Medicine, Heath Park, Cardiff University, Cardiff CF14 4XN and ⁴ Department of Obstetrics and Gynaecology, Wales College of Medicine, Heath Park, Cardiff University, Cardiff CF14 4XN, UK

Correspondence should be addressed to K Swann; Email: SwannK1{at}cf.ac.uk

	Abstract

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At fertilization in mammals the sperm activates development of the oocyte by inducing a prolonged series of oscillations in the cytosolic free Ca²⁺ concentration. One theory of signal transduction at fertilization suggests that the sperm cause the Ca²⁺ oscillations by introducing a protein factor into the oocyte after gamete membrane fusion. We recently identified this sperm-specific protein as phospholipase C (PLC), and we showed that PLC triggers Ca²⁺ oscillations in unfertilized mouse oocytes. Here we report that microinjection of the complementary RNA for human PLC causes prolonged Ca²⁺ oscillations in aged human oocytes that had failed to fertilize during in vitro fertilization or intracytoplasmic sperm injection. The frequency of Ca²⁺ oscillations was related to the concentration of complementary RNA injected. At low concentrations, PLC stimulated parthenogenetic activation of oocytes. These embryos underwent cleavage divisions and some formed blastocysts. These data show that PLC is a novel parthenogenetic stimulus for human oocytes and that it is unique in its ability to mimic the repetitive nature of the Ca²⁺ stimulus provided by the sperm during human fertilization.

	Introduction

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Mammalian oocytes are ovulated in a state of arrest at metaphase of the second meiotic division. During fertilization the sperm releases the oocyte from metaphase arrest and initiates development by inducing a series of repeated rises in the cytosolic free Ca²⁺ concentration (Whittingham 1980, Cuthbertson & Cobbold 1985, Stricker 1999). Such repeated rises in Ca²⁺, or Ca²⁺ oscillations, occur at intervals of several minutes for the first few hours after sperm–oocyte interaction. These Ca²⁺ oscillations have been observed during in vitro fertilization (IVF) in a range of mammalian species including oocytes from humans (Taylor et al. 1993). In addition, in human and mouse oocytes prolonged Ca²⁺ oscillations have also been demonstrated following intracytoplasmic sperm injection (ICSI; Tesarik & Sousa 1994, Nakano et al. 1997). The Ca²⁺ oscillations seen in mammals appear to be both necessary and sufficient for the activation of development. In the mouse, for example, it has been shown that the Ca²⁺ oscillations are necessary for second-polar-body emission and pronuclear formation (Kline & Kline 1992). The importance of Ca²⁺ is also underlined by the finding that most of the treatments that cause the parthenogenetic activation of development in mammals are effective because they cause a marked rise in the cytosolic Ca²⁺ concentration (Whittingham 1980, Swann & Ozil 1994). In mouse and domestic animal oocytes parthenogenetic activating agents such as ethanol, Ca²⁺ ionophores and electrical-field stimulation cause a single and prolonged rise in Ca²⁺ (Swann & Ozil 1994). In mouse, and some other species, parthenogenetic activating agents such as Sr²⁺ ions or thimerosal have been shown to cause Ca²⁺ oscillations that are similar, but not identical, to those seen at fertilization (Kline & Kline 1992, Cheek et al. 1993).

The mechanism by which the sperm stimulates the Ca²⁺ oscillations at fertilization is not fully resolved (Stricker 1999). However, one theory is that the sperm introduces a protein factor into the egg cytosol after gamete fusion, and that this sperm factor protein initiates Ca²⁺ release in the oocyte via the generation of inositol 1,4,5-trisphosphate (InsP₃; Swann et al. 2004). This theory is supported by the finding that injecting cytosolic extracts from sperm can cause Ca²⁺ oscillations in a range of different mammalian oocytes, including those from humans (Swann 1990, Homa & Swann 1994, Wu et al. 1997). We recently demonstrated that mouse sperm possess a novel and specific isoform of phospholipase C (PLC) referred to as PLC (Saunders et al. 2002). PLC was shown to be the protein present in the sperm extracts that is responsible for generating Ca²⁺ release and InsP₃ production (Saunders et al. 2002). Most critically it was demonstrated that PLC is an effective mimic of the sperm because microinjection of complementary RNA (cRNA) encoding for PLC into mouse oocytes causes Ca²⁺ oscillations identical to those seen during fertilization. Injection of cRNA for PLC also leads to egg activation and development to the blastocyst stage in the mouse (Saunders et al. 2002). As well as mice, both humans and monkeys have been shown to possess a sperm-specific PLC (Cox et al. 2002). Previous work suggested that the sperm factor protein was not species specific and, consistent with this, we found that injection of cRNA for the human or monkey isoforms of PLC is able to cause Ca²⁺ oscillations in mouse oocytes and stimulate embryo development up to the blastocyst stage at similar rates to those seen after in vivo fertilization (Cox et al. 2002). However, the effect of PLC in human oocytes has not previously been reported.

Like other mammals human oocytes can also be parthenogenetically activated by stimuli that elevate Ca²⁺ levels. Calcium ionophores such A23187 [GenBank] have been shown to cause both fresh and aged oocytes to undergo pronuclear formation and begin early cleavage divisions (Winston et al. 1991). However, some studies have found that Ca²⁺ ionophore alone does not reliably activate human oocytes (Balakier & Casper 1993, Rinaudo et al. 1997). So the most common current activation protocols combine Ca²⁺ ionophore with protein synthesis or protein kinase inhibitors such as 6-dimethyl aminopurine (6-DMAP) or puromycin (Cibelli et al. 2001, Nakagawa et al. 2001, Lin et al. 2003). These combination protocols have proved effective in stimulating human oocytes to form pronuclei, but the success rates of subsequent preimplantation development are still poor compared with embryo development after fertilization. In order to try and improve activation and development after parthenogenesis, attempts have been made to use stimuli that mimic IVF in causing repetitive Ca²⁺ increases. Data in mouse and rabbit oocytes that were exposed to repeated electrical-field pulses have suggested that repeated rises in Ca²⁺ can improve activation rates, and subsequent development, compared with stimuli that cause a single Ca²⁺ increase (Ozil 1990, Ozil & Huneau 2001). Limited application of this technology has suggested that the repetitive stimuli may also be useful in activating human oocytes (Zhang et al. 1999). In this study we demonstrate that microinjection of cRNA encoding human PLC protein can cause a prolonged series of Ca²⁺ oscillations in aged human oocytes that have failed to fertilize during IVF or ICSI. The induction of Ca²⁺ oscillations by PLC can also lead to parthenogenetic activation up to the blastocyst stage.

	Materials and Methods

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Obtaining and handling of human oocytes
Human oocytes were obtained from patients whose gametes had failed to fertilize following conventional IVF or ICSI. Ethical approval for the project was obtained from St Thomas’s Hospital Local Research Ethics Committee and from the Human Fertilization and Embryology Authority who issued a licence for the work (R0147). Consent for the use of unfertilized oocytes was obtained from patients before starting their treatment. For treatment the patients underwent pituitary downregulation and controlled ovarian hyperstimulation using gonadotrophins. Ovarian stimulation was achieved using a daily dose of 150–450 IU of recombinant follicle-stimulating hormone (Gonal F; Serono Laboratories, Welwyn Garden City, Herts, UK; or Puregon; Organon, Cambridge, UK). Human chorionic gonadotrophin, 10 000 IU (Profasi from Serono Laboratories or Pregnyl from Organon), was administered when at least three follicles had reached a mean diameter of 18 mm or more. Transvaginal follicular aspiration was performed 34–36 h after human chorionic gonadotrophin injection and 3–6 h later oocytes were prepared for IVF or ICSI dependent upon earlier semen analysis. Following IVF insemination or ICSI oocytes were cultured overnight and checked for signs of fertilization 19–20 h later. Only those oocytes that appeared to be at metaphase II or I and that showed no signs of fertilization were used for the project. Such unfertilized oocytes were transferred from the Assisted Conception Unit at Guy’s Hospital to laboratories at University College London and microinjected within the next 1–2 h. Unless otherwise stated, all manipulations in the laboratory were carried out on ooyctes in Hepes/KSOM (HKSOM) medium (Saunders et al. 2002).

In experiments where Ca²⁺ was monitored oocytes were injected with solutions containing various concentrations of PLC cRNA and 1 mM Oregon Green BAPTA dextran (Molecular Probes, Eugene, OR, USA) in a buffered salt solution (120 mM KCl/20 mM Hepes, pH 7.4) that had been treated with Chelex 100 beads (Sigma) to remove divalent cations. In developmental experiments where Ca²⁺ was not monitored the Oregon Green BAPTA dextran was omitted from the injection buffer. cRNA encoding the 608-amino-acid sequence of the human form of PLC was prepared as described previously (Saunders et al. 2002). The cRNA was stored in aliquots at –80°C until being thawed immediately prior to injection. For injection the oocytes were placed on a Nikon Diaphot stage and micro-injected by application of brief pressure pulses as described previously (Swann 1990). The injection was between 3 and 5% of the oocyte’s total volume. For Ca²⁺ measurements oocytes were immediately transferred to a chamber of approximately 1 ml containing HKSOM medium and fluorescence monitored as described below. For separate developmental studies the oocytes were placed in 2 µM cytochalasin D (Sigma) for approximately 2 h to prevent second-polar-body extrusion and then cultured in 20 µl drops of Sydney IVF cleavage medium (COOK) under mineral oil (Sigma) at 37°C in a 6% CO₂ incubator from days 1 to 3 (up to the eight-cell stage). Embryos were transferred to Sydney IVF blastocyst medium (COOK) for the remaining culture time. Each day embryos were removed from the incubator briefly and their developmental stage was noted.

Measurements of intracellular Ca²⁺
Fluorescence measurements were carried out on oocytes in drops of media in a heated chamber on the stage of a Zeiss Axiovert microscope equipped with epifluorescence optics and a 20 x 0.75NA objective lens. Low-level fluorescence excitation was used to minimize oocyte damage. The light from a halogen lamp passed through a 490 nm bandpass filter and emission collected with a 510 nm long-pass filter. Fluorescence light (100–1000 photons/s) was measured from each oocyte with an imaging photon detector (Photek Ltd, East Sussex, UK) using software and a system designed by Science Wares (Falmouth, MA, USA).

	Results

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Microinjection of human PLC cRNA into aged human oocytes caused a series of Ca²⁺ oscillations as indicated by the repetitive increases in the fluorescence of Oregon Green BAPTA dextran. Fig. 1 shows examples of the pattern of Ca²⁺ oscillations triggered in different oocytes by injecting different concentrations of PLC cRNA. The Ca²⁺ oscillations consisted of series of sharp rises in Ca²⁺, followed by a fairly abrupt return to baseline Ca²⁺ levels. There was then a very gradual increase in Ca²⁺ before the next Ca²⁺ rise suggesting the existence of a pacemaker that leads to another Ca²⁺ increase every 10 min to 2 h (Fig. 1a). This general pattern of Ca²⁺ oscillations is similar to those reported after IVF or ICSI in human oocytes (Tesarik & Sousa 1994), and broadly similar to the pattern of Ca²⁺ oscillations seen in other mammals during fertilization (Swann & Ozil 1994). The main difference between the patterns of oscillation was in regard to the frequency of Ca²⁺ oscillations. In particular high concentrations of PLC caused the highest-frequency oscillations. In addition we found that pipette concentrations of 1 or 10 µg/ml cRNA lead to oscillations that started with a relatively low frequency, but oscillations tended to show an increase in frequency with time such that the final frequency tended to match that seen with higher concentrations of PLC cRNA (Fig. 1b). A sustained low frequency of Ca²⁺ oscillations was only seen when we injected pipette concentrations of approximately 0.1 µg/ml PLC cRNA (Fig. 1c). This concentration of PLC cRNA also appeared to be the minimally effective concentration because only six out of 13 oocytes showed Ca²⁺ responses. With pipette concentrations of 10 µg/ml or greater all the oocytes we injected underwent Ca²⁺ oscillations. Table 1 shows the intervals of Ca²⁺ oscillations seen after injecting different amounts of PLC into human oocytes. Despite some changes in frequency with time at intermediate concentrations, there is a trend towards greater intervals between Ca²⁺ increases with lower concentrations of injected PLC cRNA. Since the amount of PLC protein synthesized in mouse oocytes was shown to be proportional to the concentration of PLC cRNA injected (Saunders et al. 2002), these data suggest that the concentration of PLC protein affects the frequency of Ca²⁺ oscillations.

View larger version (21K): [in this window] [in a new window]   Figure 1 Injection of different concentrations of human PLC cRNA causes Ca2+ oscillations in aged human oocytes. Shown are three sample traces of the intracellular Ca2+ concentration, as measured by Oregon Green BAPTA dextran fluorescence (Fl.; in arbitrary units). Recordings were made from aged human oocytes after injection of PLC cRNA. In each panel the oocytes were injected 10–20 min before the start of the recording. The concentrations to the right of the traces refer to those in the injection pipette (2–5% oocyte volume injected). The repetitive rises in Ca2+ are seen as distinct spike-like increases in fluorescence.

View this table: [in this window] [in a new window]   Table 1 The pattern of Ca2+ oscillations induced by PLC in aged human oocytes. The mean interval of Ca2+ transients was only scored for oocytes that showed Ca2+ oscillations. For concentrations of 10 µg/ml PLC cRNA and less the data are taken from oocytes where prolonged recording were made (at least 10 h). The final frequency of Ca2+ transients was scored in the last 4 h of a 12 h (*) or 10 h () recording. PLC cRNA concentration refers to the concentration in the pipette.

View this table: [in this window] [in a new window]   Table 2 In vitro development of human oocytes following injection of PLC cRNA. PLC cRNA concentration refers to the concentration in the pipette.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

Our work also represents the first clear demonstration of a method for generating prolonged repetitive Ca²⁺ signals in human oocytes. One study has reported the activation of aged human oocytes by applying three electrical-field pulses rather than one (Zhang et al. 1999). However, the fertilized human oocyte clearly displays more than three Ca²⁺ rises and extending this methodology requires rapid washing techniques that are technically demanding (Ozil 1990). Strontium-containing medium has been used to stimulate repetitive Ca²⁺ increases in mouse eggs and might offer another simpler means of stimulating oscillations in human oocytes. However, we failed to observe any Ca²⁺ transients when we incubated human oocytes in 10 mM Sr²⁺ media (N Rodgers & K Swann, unpublished observations), and it remains uncertain whether Sr²⁺ can be used for this purpose. Thimerosal is reported to cause Ca²⁺ oscillation in human oocytes (Homa & Swann 1994), but as a thiol oxidizing agent it also effects the cytoskeleton and may not be compatible with human embryo development (Cheek et al. 1993). Our current data with PLC, therefore, provide perhaps one of the only relatively simple means of stimulating multiple and long-lasting Ca²⁺ increases in human oocytes.

Previous work on mouse oocytes demonstrated that injecting cRNA encoding the human, mouse or monkey isoforms of PLC could initiate Ca²⁺ oscillations and activation of mouse oocytes (Cox et al. 2002, Saunders et al. 2002). The injected cRNA is converted into PLC protein in proportion to the amount of cRNA injected (Saunders et al. 2002). The amount of human PLC that caused Ca²⁺ oscillations in aged human oocytes in this study is within a similar range to that which causes Ca²⁺ oscillations in mouse oocytes (Cox et al. 2002). However, mouse oocytes were induced to undergo Ca²⁺ oscillations by injection of final concentrations of approximately 1 ng/ml human PLC cRNA, which is about 10 times lower than the amount that we used to generate Ca²⁺ oscillations in aged human oocytes. This could mean that there are differences in sensitivity to PLC between mouse and human oocytes. Nevertheless, it is also likely that aged human oocytes do not translate the cRNA into protein as efficiently as the recently ovulated mouse oocytes. We used the method of injecting cRNA, rather than PLC protein, because of the unstable nature of the recombinant protein. Injecting the cRNA also provides a way of introducing PLC into an oocyte without any contamination from other proteins. Nevertheless, injecting cRNA rather than recombinant protein leads to a gradual increase in PLC with time. This probably accounts for the increase in frequency of Ca²⁺ oscillations seen in oocytes injected with intermediate concentrations of PLC.

There appears to be a narrow concentration range of PLC that can be used to activate oocytes. The lowest concentrations of PLC cRNA are not effective in all oocytes, but too high a concentration can lead to high-frequency Ca²⁺ oscillations that appear to be detrimental to development beyond the two-cell stage (Cox et al. 2002). The low frequency of Ca²⁺ oscillations that was consistent with reasonable rates of implantation development is similar to the low frequency of oscillations that were first reported using aequorin to measure Ca²⁺ levels during human fertilization (Taylor et al. 1993). Such low-frequency Ca²⁺ oscillations during fertilization are also seen in the mouse when aequorin is used to measure Ca²⁺ (Cuthbertson & Cobbold 1985). In parallel experiments we found that a slightly greater proportion of oocytes activated (75%) than showed Ca²⁺ oscillations (approximately 50%) when 0.1 µg/ml PLC cRNA was injected. This could reflect the fact that Oregon Green BAPTA dextran buffers the Ca²⁺ levels to some extent, and so a higher proportion of oocytes may actually undergo oscillations when Ca²⁺ is not measured. In future it would be useful to measure Ca²⁺ with less-invasive probes so that the same oocytes can be observed for Ca²⁺ oscillations and development.

Whereas the overall numbers are small, our data are noteworthy in that development to the blastocyst stage was seen after artificial activation of aged human oocytes. Previous studies have reported the same parthenogenetic development to the blastocyst stage with freshly ovulated human oocytes (Cibelli et al. 2001, Lin et al. 2003). However, when using aged human oocytes the Ca²⁺-iono-phore-based protocols have stimulated early cleavage stage development, but blastocyst formation has not been reported (Winston et al. 1991, Balakier & Casper 1993, Nakagawa et al. 2001). It is possible that we obtained blastocysts in some cases because oocytes were stimulated with a repetitive Ca²⁺ signal. However, more-extensive studies are required to make direct developmental comparisons between oocytes activated by PLC compared with those stimulated by ionophore. Whatever the case our data do suggest that PLC could be used to generate human parthenogenetic embryos and this in itself has clinical implications. First, there are clearly some cases where failed fertilization after ICSI is due to failed oocyte activation (Rawe et al. 2000). In some cases embryo development and live births have been achieved after sperm injection by providing an activation stimulus in the form of Ca²⁺ ionophore (Eldar-Geva et al. 2003, Murase et al. 2004). PLC offers an alternative means by which failed activation may be restored with a more physiological stimulus than ionophore. Secondly, the generation of parthenogenetic blastocysts from oocytes can provide a source of embryos for the creation of stem cells (Lin et al. 2003, Vrana et al. 2003). The use of such parthenogentic embryos may be more ethically acceptable than using embryos from fertilized zygotes.

	Acknowledgements

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
We thank Mark Larman and Christopher Saunders for advice and preparation of cRNA and Nazar Amso for discussion about the manuscript. We also thank the patients of the Assisted Conception Unit at Guy’s Hospital who so willingly took part in this study to advance the understanding of fertilization. This work was supported by a Wellcome Trust grant (K S), an MRC studentship (N T R) and KEF funds (F A L). The imaging equipment was from a JIF fund to the Anatomy Department at University College London.

	Footnotes

 
Received 7 September 2004
First decision 17 September 2004
Accepted 20 September 2004

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