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Hum. Reprod. Advance Access published online on November 14, 2007
Human Reproduction, doi:10.1093/humrep/dem350
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© The Author 2007. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Preimplantation development of mouse oocytes activated by different levels of human phospholipase C zeta

Y. Yu1, C.M. Saunders2, F.A. Lai2 and K. Swann1,3

1 Department of Obstetrics and Gynaecology, School of Medicine, Cardiff University, Heath Park, Cardiff CF14 4XN, UK 2 Wales Heart Research Institute, School of Medicine, Cardiff University, Heath Park, Cardiff CF14 4XN, UK

3Correspondence address. Tel: +44 2920 742039; Fax: +44 2920 744399; E-mail: swannk1{at}cardiff.ac.uk


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Funding
 References
 
BACKGROUND: A sperm-specific phospholipase C zeta (PLC{zeta}) has been shown to trigger Ca2+ oscillations in mouse and human oocytes and appears to be the sperm factor responsible for activation at fertilization. Previously, complementary RNA (cRNA) injection was used to introduce PLC{zeta} into oocytes, but it was unclear how much PLC{zeta} protein is required for development. Here we have injected cRNA encoding luciferase-tagged human PLC{zeta} (hPLC{zeta}-luc) into mouse oocytes and established the relationship between hPLC{zeta}-luc expression, Ca2+ oscillations and development.

METHODS: Mouse oocytes were injected with hPLC{zeta}-luc cRNA and a fluorescent Ca2+dye to monitor hPLC{zeta}-luc expression and Ca2+ oscillations, respectively. After inducing diploidy, development in vitro was monitored in hPLC{zeta}-luc cRNA microinjected oocytes and compared with parallel oocytes activated by incubation in Sr2+.

RESULTS: Repetitive Ca2+ oscillations and oocyte activation were triggered by hPLC{zeta} over a wide range of luciferase expression levels. However, subsequent development of embryos to the blastocyst stage was observed only when expression of hPLC{zeta}-luc was optimized within a specific range. The blastocyst cell number was also affected by the level of hPLC{zeta} expression.

CONCLUSIONS: Human PLC{zeta} can readily activate mouse oocytes, however, effective development to blastocyst stages is only achieved within a specific window of hPLC{zeta}-luc protein expression levels.

Key words: calcium/fertilization/oocyte/activation/human phospholipase C zeta


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Funding
 References
 
During fertilization of human oocytes, the sperm induces a long-lasting series of repetitive intracellular Ca2+ transients (Ca2+ oscillations) (Miyazaki et al., 1993Go; Taylor et al., 1993Go; Malcuit et al., 2006Go). Such Ca2+ oscillations are also observed in human or mouse oocytes injected with human sperm during ICSI (Tesarik and Sousa, 1995Go; Nakano et al., 1997Go; Sakurai et al., 1999Go; Yanagida et al., 2000Go). These Ca2+ oscillations are a conserved feature of oocyte activation in all mammals and have been extensively studied in mouse and hamster oocytes (Miyazaki et al., 1993Go; Stricker, 1999Go; Malcuit et al., 2006Go). Oscillations in Ca2+ are responsible for causing the major events of oocyte activation such as cortical granule exocytosis, zona hardening and polyspermy block, resumption of second meiotic division and initiation of the first mitotic cell cycle (Kline and Kline, 1992Go; Malcuit et al., 2006Go). Furthermore, in mouse and rabbit oocytes the number, frequency, as well as the amplitude and duration of Ca2+ pulses influences the efficiency of activation (Vitullo and Ozil, 1992Go; Ozil and Huneau, 2001Go), and may regulate later developmental events, such as the ratio of cell types in the blastocyst, or the embryo size and morphology after implantation (Mikich-Bos et al., 1997Go; Ozil and Huneau, 2001Go; Ozil et al., 2006Go).

Artificial, or parthenogenetic, activation of mammalian oocytes can be achieved by a range of stimuli that cause a rise in intracellular Ca2+ levels, such as ethanol, Ca2+ ionophores, electroporation in the presence of Ca2+, or incubation in Sr2+ media (Swann and Ozil, 1994Go). Ca2+ elevating agents, in combination with drugs such as cycloheximide or 6-DMAP, are also used to trigger oocyte activation after nuclear transfer procedures designed to generate cloned embryos (Cibelli et al., 2001Go; Hall et al., 2007Go). Such parthenogenetic activating agents have been used to stimulate development after failed fertilization in some cases of human ICSI (Tesarik and Sousa, 1995Go; Battaglia et al., 1997Go; Chi et al., 2004Go). It has been suggested that this approach could be employed to overcome some cases of failed fertilization due to male factors (Moaz et al., 2006Go). There have been several reports of pregnancies and live births after chemical activation of oocytes that had previously failed to fertilize during ICSI (Eldar-Geva et al., 2003Go; Chi et al. 2004Go; Murase et al., 2004Go; Heindryckx et al., 2005Go; Yanagida et al., 2006Go). However, the success rate of such procedures is unclear. In addition, the stimulation by Ca2+ ionophore does not mimic the type of stimulation that occurs during normal fertilization since the characteristic sperm-induced Ca2+ oscillations are not observed with artificial activation protocols. The application of ionophores, ethanol or single electric pulses all generates a single large Ca2+ increase in the mammalian oocyte (Loi et al., 1998; Lee et al., 2004Go; Mizutani et al., 2004Go; Alexander et al., 2006Go). Incubation in media containing Sr2+ ions does cause repetitive Ca2+ oscillation in rodent oocytes, and is the parthenogenetic agent of choice for nuclear transfer work (Wakayama et al., 1998Go). However, the pattern of Sr2+-induced Ca2+ oscillations is different from that at fertilization (Kline and Kline, 1992Go). Furthermore, although one report suggests that Sr2+ can activate human oocytes (Yanagida et al., 2006Go), Sr2+ is generally not reported to be effective in activating oocytes from other non-rodent species, and there are no reports or indications that it can cause Ca2+ oscillations in human oocytes (Rogers et al., 2004Go). Hence, it would be useful to apply a stimulus that mimics the endogenous series of Ca2+ oscillations observed at fertilization. Preliminary experimental studies have found that using electrical field pulses to impose at least three, as opposed to one, transient Ca2+ increase is a more effective way of inducing development after failed ICSI in human oocytes (Zhang et al., 1999Go). These failed ICSI oocytes would probably contain a sperm and so would not necessarily have been parthenogenetic when activated, but the data does suggest that stimuli that begin to mimic a repetitive Ca2+ signal may be a more effective way to stimulate embryo development.

Substantial evidence now suggests that during normal fertilization, and after ICSI, the sperm causes Ca2+ oscillations by the introduction of a soluble protein factor into the ooplasm (Swann, 1990Go; Homa and Swann, 1994Go; Malcuit et al., 2006Go; Swann et al., 2006Go). We recently demonstrated that the soluble protein factor in sperm extracts is a novel type of phospholipase C (PLC) called PLC{zeta} (Saunders et al., 2002Go). PLC{zeta} is sperm-specific and injecting this protein, or the cognate complementary RNA (cRNA), can produce Ca2+ oscillations similar to those generated by the sperm at fertilization in the mouse (Saunders et al., 2002Go; KouChi et al., 2004Go). PLC{zeta} has also been shown to be the protein responsible for oocyte activation during ICSI in the mouse (Fujimoto et al., 2004Go). Furthermore, a reduction in the level of PLC{zeta} protein in sperm via RNA interference can lead to a decrease in the number of Ca2+ oscillations, and sperm carrying transgenic RNA inhibitor for PLC{zeta} appear to be unable to support development to term (Knott et al., 2005Go). These data all suggest that PLC{zeta} is the sperm protein that triggers mammalian development.

PLC{zeta} is found in a wide range of mammalian species and has been demonstrated to be present in human sperm (Cox et al., 2002Go; Swann et al., 2006Go). Injection of cRNA encoding human PLC{zeta} (hPLC{zeta}) into mouse or human oocytes that had failed to fertilize, triggers a long lasting series of Ca2+ oscillations (Cox et al., 2002Go; Rogers et al., 2004Go), and can lead to oocyte activation and embryo development to the blastocyst stage (Rogers et al., 2004Go). The successful application of this novel, sperm-specific hPLC{zeta} therefore represents an alternative parthenogenetic activation protocol. However, although the demonstrably effective way of introducing hPLC into oocytes currently relies on expression of its cognate cRNA, the previous studies have not quantified the relationship between the amount of PLC{zeta} protein expressed in the unfertilized oocyte, and subsequent embryo development. To overcome this deficiency, in this study we have injected cRNA for hPLC{zeta} tagged with firefly luciferase, and we have quantified the level of hPLC{zeta}-luc protein expression by monitoring luciferase luminescence, whilst simultaneously monitoring Ca2+ oscillations and embryo development. We have used mouse oocytes as a test bed for analysing the effects of hPLC{zeta} since they exhibit a robust response to the human PLC{zeta} and provide a uniform gamete population in regard to post-ovulatory oocyte and genetic background. Mouse oocytes have also been successfully used to predict the ability of human sperm to activate human oocytes (Araki et al., 2004Go; Heindryckx et al., 2005Go). Our studies indicate that there is a precise range of concentrations in which PLC{zeta} can effectively trigger both oocyte activation and embryo development and this phenomenon may have implications for our understanding of early development of human embryos.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Funding
 References
 
Handling and microinjection of oocytes
MF1 female mice ~4–6 weeks were superovulated as described previously (Saunders et al., 2002Go; Larman et al., 2004Go). Oocytes were released from the oviduct ampulae into M2 medium (Sigma–Aldrich, Poole, Dorset, UK) with a needle 13–15 h post-HCG followed by a removal of the cumulus mass by 0.3 mg/ml hyaluronidase. cRNA encoding the 608-residue human PLC, tagged via the C-terminus with firefly luciferase (hPLC{zeta}-luc), was prepared as described previously (Nomikos et al., 2005Go). Microinjection procedures were carried out as previously described (Saunders et al., 2002Go). Oocytes were collected and microinjected within 13–16 h after injecting female mice with HCG. Oocytes were microinjected to 3–5% oocyte volume with a solution containing different concentrations of hPLC{zeta}-luc cRNA (0.05–0.5 µg/µl) and 1 mM Oregon Green BAPTA dextran (Invitrogen Ltd, Paisley, UK). In the control experiment, 0.5 µg/µl luciferase RNA was injected into oocytes before they were activated by SrCl2 or hPLC{zeta}-luc cRNA injection.

Measurement of intracellular ca2+ and luciferase expression
Some of the injected oocytes were placed in a chamber with M2 medium (Sigma–Aldrich, Poole, Dorset, UK) containing 1 mM luciferin, on the temperature-controlled stage of an inverted microscope. Ca2+ oscillations were monitored by measuring the fluorescence of Oregon Green BAPTA dextran and luciferase expression was monitored by the luminescence. These measurements were both carried out on the same sets of oocytes using a Zeiss Axiovert S100 microscope with light from the stage directed towards a cooled intensified CCD camera (ICCD) with a bialkali-type photocathode-based intensifier cooled to 10°C. The microscope and ICCD camera were placed inside a custom-made dark box. This photon counting camera, dark box and associated software was supplied by Photek Ltd (St Leonards on Sea, East Sussex, UK). In most experiments, the fluorescence was recorded first by exposing oocytes to excitation light (450–490 nm) and reducing the sensitivity of the ICCD camera to 10%, and then the luminescence was recorded by removing the excitation light and switching the ICCD camera to maximum sensitivity. The luminescence values in experiments represent the absolute number of measured photon counts per second (cps), whereas the intensity of fluorescence is displayed in arbitrary units of intensity. The levels of luciferase protein corresponding to a level of luminescence were estimated by injecting oocytes with known amounts of recombinant luciferase protein (Sigma–Aldrich, Poole, Dorset, UK) and then measuring the luminescence of these oocytes under the same conditions as those injected with hPLC{zeta}-luc cRNA.

Culture and analysis of embryos
On each experimental day, some of the hPLC{zeta}-luc-injected oocytes were imaged and some from the same batch were put into potassium simplex optimized media (KSOM) media (synthetic oviductal medium enriched with potassium) (Summers et al., 2000Go), containing 5 µg/ml cytochalasin B for 6 h. A separate batch of oocytes that were not injected with hPLC{zeta}-luc was activated by 10 mM SrCl2 in Ca2+-free KSOM medium for 4 h. After the pronuclei formation was checked, both types of activated oocytes were cultured in KSOM medium at 37°C in a 5% CO2 incubator for 96 h. All the resulting blastocysts were incubated in 0.5% pronase to remove the zona pellucida. After washing in M2 medium, blastocysts were incubated in 10% rabbit anti-mouse whole serum for 30 min, washed again with M2 medium, and then incubated in M2 medium containing 20% guinea pig complement, 30 µg/ml propidium iodide and 10 µg/ml Hoechst 33 342 for 15 min. The embryos were rinsed quickly and mounted in glycerol onto a glass slide. The data were expressed as mean ± SE. To evaluate the statistical significance of differences between groups, we applied the Student's t-test to test for mean comparisons. A P level of ≤0.05 was considered statistically significant. All chemicals not otherwise specified were obtained from Sigma–Aldrich (Poole, Dorset, UK).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Funding
 References
 
Expression of hPLC{zeta}-luciferase and generation of Ca2+ oscillations
When mouse oocytes were microinjected with hPLC{zeta}-luc cRNA, the luminescence level (an indicator of luciferase protein concentration), as measured in photon cps, started to increase within the first hour and detection of luminescence continued for over a 20 h period (Fig. 1). Fig. 1b shows that the luminescence level gradually accumulated until ~3 h after cRNA injection when a plateau level of ~0.4 cps was achieved for ~6 h, after which there was a gradual decline over ~3 h to a luminescence level of ~0.1 cps. Since Ca2+ signalling and oocyte activation generally occurs within 6 h of injection, in subsequent experiments we measured Ca2+ changes in the first 6 h period post-injection followed by determination of luminescence from the same oocytes for 30 min in the presence of luciferin to obtain the level of PLC{zeta}-luc protein.


Figure 1
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  		Figure 1: Luminescence and time course of expression of hPLC{zeta} in mouse oocytes

(a) Luminescence image (integrated photon counts accumulated over 20 h) of a typical group of mouse oocytes that had been injected with luciferase-tagged hPLC{zeta} (hPLC{zeta}-luc) cRNA and placed in luciferin containing media. (b) An example of the luminescence trace (in photon cps) from one of the mouse oocytes in (a).

 
To assess the effects of different amounts of PLC{zeta}-luc protein on activation, we injected various pipette concentrations of 0.05–0.5 µg/µl of hPLC{zeta}-luc cRNA into groups of mouse oocytes. A total of 233 oocytes were microinjected with cRNA and subsequently monitored for Ca2+ oscillations and luciferase expression. Fig. 2 shows some typical examples of the distinct patterns of Ca2+ oscillations occurring in oocytes due to different levels of hPLC{zeta}-luc expression. Increasing expression levels from 0.01 to 0.3 cps resulted in an enhanced frequency of spikes that was maintained for over 5 h. However, higher expression levels of >0.3 cps caused a cessation of the spikes after 1–3 h, preceded by a gradual decrease in spike amplitude. The scatter plots in Fig. 3 (left column) illustrate the relationship between the pattern of Ca2+ oscillations, as indicated by the number, duration and interval of responses, and the level of luciferase expression. Fig. 3 also presents a histogram analysis (right column) where patterns of oscillations (number, duration and interval) are grouped according to the increasing level (I–IV) of luciferase expression and to changes in Ca2+ oscillations pattern. Luminescence levels of 0.2, 1.0 and 2.5 cps were considered to be the transition points and hence the levels of hPLC{zeta}-luc expression were classified into four different ranges; I (0–0.2 cps), II (0.2–1.0 cps), III (1.0–2.5 cps) and IV (2.5–6 cps). Fig. 3a identifies expression levels I and II as optimal for spike number, whereas the higher levels III and IV cause a reduction. The duration of the train of the spikes also decreases with increasing expression levels, with maximal duration occurring with I (Fig. 3b). From Fig. 3c, it can be seen that interspike interval also is reduced with increasing hPLC{zeta}-luc expression, and hence there is an increase in the frequency of Ca2+ oscillations. Since the duration of the whole train of Ca2+ oscillations decreases as the frequency increases there is only a small increase in the number of Ca2+ spikes in oocytes with high hPLC{zeta}-luc concentrations. Moreover, with high concentrations of hPLC{zeta}-luc, it is evident that the cessation of Ca2+ oscillations at high expression levels (e.g. after 1–2 h in the 1.0–7.0 cps traces in Fig. 2) occurs well before the peak of protein expression has occurred (after 3–4 h in Fig. 1b). These data support previous studies suggesting that the pattern of Ca2+ oscillations is affected by the amount of PLC{zeta} cRNA injected into each oocyte (Cox et al., 2002Go; Saunders et al., 2002Go), but it is now evident that the variation in the pattern of Ca2+ oscillations is seen over a relatively small range of PLC{zeta} protein concentrations.


Figure 2
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  		Figure 2: The pattern of Ca2+ oscillations caused by different amounts of hPLC{zeta}-luc expression in individual mouse oocytes

Fluorescence of the Ca2+ sensitive dye Oregan Green BAPTA is shown on the y-axis in arbitrary units (a.u.). The 12 traces represent 12 different oocytes, each with a different level of hPLC{zeta}-luc expression. Luminescence expression (cps) is shown for each fluorescence trace, indicating Ca2+ levels.

 

Figure 3
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  		Figure 3: The relationship of Ca2+ oscillations and hPLC{zeta}-luc expression

A total of 233 mouse oocytes were imaged, analysed and plotted as a scatter of dots which show that there were distinct trends in the number, duration and intervals of Ca2+ increases that correlated with the hPLC{zeta}-luc expression levels in individual oocytes. According to the changes of Ca2+ oscillation pattern (left), cps of 0.2, 1.0 and 2.5 (arrows) were thought to be transition points and levels of hPLC{zeta}-luc expression were classified into four different ranges; I (0–0.2 cps), II (0.2–1.0 cps), III (1.0–2.5cps) and IV (2.5–6 cps). The bar charts on the right show that there are statistically significant differences in pattern of Ca2+ oscillations (number of spikes, duration of train, or the intervals between spikes) as the expression levels of hPLC{zeta}-luc increase (*indicates a significant difference versus any other group in the histogram, P < 0.05).

 
We estimated the protein expression level in these hPLC{zeta}-luc-injected oocytes by comparing the amount of light emitted from embryos on the imaging system with that from oocytes injected with known amounts of luciferase protein. We found that 1 cps of luminescence corresponded to ~250 fg of luciferase protein. Since Ca2+ oscillations were triggered with expression levels as low as 0.01 cps, we can estimate that as little as 2.5 fg of hPLC{zeta}-luc protein at 6 h is associated with Ca2+ release. However, since we also know that Ca2+ oscillations in these cases started ~1–2 h after injection (Fig. 2) we can estimate that levels of around 1 fg of hPLC{zeta}-luc are sufficient to trigger Ca2+ release in mouse oocytes.

Developmental potential of embryos activated by different levels of HPLC{zeta}-luc
The imaging experiments described above were carried out on a small group of oocytes taken from a larger cohort. For the remaining oocytes, we assessed their developmental potential by placing them in KSOM media with cytochalasin B for 6 h, followed by sustained culture in normal KSOM media. To evaluate the potential of hPLC{zeta}-luc to activate mouse eggs and trigger subsequent development, we monitored both pronuclei and blastocyst formation at 6 and 96 h after injection, respectively. We also compared the results from hPLC{zeta}-luc-injected oocytes to the developmental potential of a group of Sr2+-activated oocytes that had been collected from the same set of mice on the same day.

Fig. 4a shows that at a very low level of PLC{zeta}-luc expression (0.05 cps) mouse oocytes can be effectively activated to form pronuclei. Furthermore, over a large range of expression levels (0.05–6 cps) nearly all oocytes formed pronuclei at 6 h after hPLC{zeta}-luc injection (95.9%, n = 850). This is comparable to the pronuclei formation efficiency observed for SrCl2 activation (85.7 ± 2.4%, n = 1013), and is also consistent with previous observations showing mouse oocyte activation upon microinjection of various cRNA concentrations of the untagged hPLC{zeta} (Cox et al., 2002Go). However, we found that the further development of hPLC{zeta}-luc-injected oocytes beyond pronuclei formation at 6 h was markedly dependent upon the precise level of PLC{zeta}-luc expression. Fig. 4b shows the rate of development to the blastocyst stage for PLC{zeta}-luc-injected oocytes was >50% (n = 563) (i.e. similar to Sr2+-activated oocytes) only when the level of hPLC{zeta}-luc expression was between narrowly defined limits of 0.12–2.7 cps. Most notably, when the expression of hPLC{zeta}-luc was >2.7 cps, the embryos all failed to develop into blastocysts. Most of these high hPLC{zeta}-luc expression embryos (>2.7 cps) arrested at the two-cell stage with only 38% (n = 235) reaching the four-cell stage. These data show that there is a wide range of concentrations where hPLC{zeta}-luc can fully activate oocytes (i.e. induce pronuclei formation), but that ability to activate in itself does not guarantee that embryos will develop into blastocysts. These observations suggest that only a specific, narrow window of hPLC{zeta} levels is consistent with successful pre-implantation development.


Figure 4
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  		Figure 4: The effects of hPLC{zeta}-luc cRNA injection on mouse oocyte activation and development

In (a) the rate of oocyte activation is scored as pronuclear formation rate divided by the number of injected oocytes. The luminescence level is plotted for each group of oocytes injected with hPLC{zeta}-luc (closed circles). The open circles represent the activation rates for groups of Sr2+ treated oocytes that were not luminescent but that were treated in parallel on the same day. In (b) the development rate (number of blastocysts formed divided by the number of activated oocytes) is plotted against range of luminescence values. The right hand panels for (a) and (b) show examples of hPLC{zeta}-luc activated oocytes and blastocysts, respectively.

 
Assessments of blastocysts obtained after hPLC{zeta}-luc injection
We evaluated the blastocyst embryos obtained from hPLC{zeta}-luc injection by analysis of the total cell number, and the cell number ratio between inner cell mass (ICM) and trophoblast cells within the blastocysts. This was done by differential staining (Fig. 5), which has been widely used to evaluate the quality of blastocysts (Van Soom et al., 2001Go). We examined blastocysts derived from ten separate experiments in which the expression levels of hPLC{zeta}-luc ranged between 0.1 and 2.7 cps. Data shown in Fig. 5 demonstrate that all blastocysts obtained after PLC{zeta}-luc injection have a similar ratio of cells allocated between the ICM and the trophectoderm, and this ratio is similar to that seen after Sr2+-induced oocyte activation (Fig. 5b). However, the total cell number in the blastocysts does show some dependency upon hPLC{zeta}-luc level. With lower levels of expression (0.1–0.4 cps), the total cell number is significantly lower than that in blastocysts activated by SrCl2 (39.8 ± 3.6, n = 17 versus 63.6 ± 2.5, n = 30 for 0.1 cps and 40.4 ± 7.4, n = 13 versus 68.4 ± 2.6, n = 13 for 0.4 cps; Fig. 5a). In contrast, blastocysts induced with higher level of hPLC{zeta}-luc expression (0.6–2.7 cps) have the same cell number as that activated with SrCl2 (Fig. 5a). These data suggest that a specific level of PLC{zeta}-luc expression may be required to achieve an optimal number of cells in a blastocyst.


Figure 5
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  		Figure 5: The distribution of cells in mouse blastocysts analysed by differential staining

The plots in (a) show the total cell number and (b) the ratio of ICM cells to trophoblastic cells, in blastocysts activated by hPLC{zeta}-luc. Each bar represents a group of embryos that had been activated by a different expression level of hPLC{zeta}-luc, as plotted on the x-axis. The number and distribution of cells for each group of Sr2+-activated embryos is shown alongside the parallel hPLC{zeta} group. In each experiment, at least 10 blastocysts were analysed, and * indicates a significant difference versus Sr2+-activated oocytes, P < 0.05. In (c) a typical image is shown for an hPLC{zeta}-luc activated blastocyst with the ICM and trophoblastic cells stained in blue and pink, respectively.

 
The effects of luciferase cRNA injection
From the experiments described above, it is clear that injection of hPLC{zeta}-luc cRNA can readily activate oocytes but hPLC{zeta}-luc expression can also have a detrimental effect on further development at higher concentrations. In order to establish that these effects are not due to non-specific effects associated with the over-expression of luciferase, or with the injection of exogenous cRNA, we injected mouse oocytes with a control cRNA encoding luciferase protein alone. Mouse oocytes were injected with luciferase cRNA at the same maximal concentration that we used for hPLC{zeta}-luciferase (0.5 µg/µl). These luc-injected oocytes were then subsequently activated either by incubation with SrCl2 or by hPLC{zeta}-luc injection (0.1 µg/µl) and cultured in KSOM medium. Images are shown in Fig. 6a where the average luminescence from these luc-injected oocytes was 19 ± 5.2 cps, which is much greater than the maximal signals we obtained with hPLC{zeta}-luc injection alone (Fig. 1). Despite the high level of luciferase expression, we found that the blastocyst formation rate of luc-injected oocytes was not different from the control oocytes activated by either SrCl2, or by a single injection of 0.1 µg/µl hPLC{zeta}-luc (Fig. 6b). These results indicate that neither luciferase, nor exogenous RNA injection, can account for any attenuation of development potential of hPLC{zeta}-luc-activated oocytes.


Figure 6
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  		Figure 6: The development of mouse embryos injected with control luciferase cRNA

Possible non-specific effects associated with over-expression of luciferase or the injection of exogenous cRNA were investigated. Panel (a) shows an image of oocytes injected with either control luciferase RNA (i) giving 23 cps, or with a typical amount (0.1 µg/ml) of hPLC{zeta}-luc cRNA (ii), which showed 0.83 cps. The image is from integrated counts over 20 min. Development rates of embryos are shown in (b). Mouse oocytes were injected with excess luciferase cRNA and activated either by incubation with SrCl2 or by hPLC{zeta}-luc injection. The blastocyst formation rate of luc-injected oocytes (solid bars) was not different from the control oocytes (open bars), activated by either SrCl2, or by a single injection of hPLC{zeta}-luc. The numbers above each box indicate the number of oocytes in each experimental group.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Funding
 References
 
In this study, we have injected cRNA encoding the human PLC{zeta} fused with luciferase into mouse oocytes to investigate the most effective range over which the PLC{zeta} can stimulate development up to the blastocyst stage. We previously reported that human PLC{zeta} cRNA injection stimulates mouse and human oocyte activation and can lead to embryo development to the blastocyst stage (Cox et al., 2002Go; Rogers et al., 2004Go). However, these studies did not measure the amount of protein expression associated with the cRNA injection method and so the precise relationship between PLC{zeta}, Ca2+ oscillations and embryo development could not be quantitatively assessed. We have enabled direct measurement of PLC{zeta} protein in the current study by monitoring the luminescence of luciferase that has been fused to the C-terminus of the hPLC{zeta}. The suitability of this method for studying development is vindicated since control experiments showed that neither the oocyte microinjection procedure nor expression of luciferase protein impairs Sr2+- or PLC{zeta}-induced embryo development to the blastocyst stage in mouse (Fig. 6). Consequently, we can conclude that the observed effects on Ca2+ oscillations and embryo development are due to different levels of PLC{zeta} protein expressed in the oocyte, rather than our method of monitoring expression. With this direct quantification, we were able to estimate that ~1 fg of human PLC{zeta} protein can effectively initiate Ca2+ signals in mouse oocytes. This 1 fg threshold is noticeably less than the estimated 20–50 fg of mouse PLC{zeta} required to trigger Ca2+ oscillations (Saunders et al., 2002Go) and is consistent with the suggestion that the human form of PLC{zeta} may be more potent than mouse PLC{zeta} (Cox et al., 2002Go).

Consistent with several previous studies using untagged, or mouse PLC{zeta}, we found that hPLC{zeta}-luc cRNA injection reliably triggered Ca2+ oscillations in mouse oocytes. By quantifying hPLC{zeta} level in oocytes, we showed that the higher the level of hPLC{zeta} protein expression the earlier the first Ca2+ increase occurs after injection (Fig. 2). Increased expression of PLC{zeta} protein also correlates with a higher frequency of Ca2+ transients, but a reduction in the duration for the overall Ca2+ oscillation response (Figs 2 and 3). The finding that more rapid PLC{zeta} synthesis correlates with a quicker onset and higher frequency of Ca2+ changes is entirely consistent with models of Ca2+ oscillations. The enzymatic product of PLC{zeta} is inositol triphosphate (IP3), and the level of IP3 in a cell determines both the latency and frequency of oscillations (Dupont and Goldbeter, 1993Go; Keizer et al., 1995Go). A surprising result is the finding that Ca2+ oscillations terminate more quickly in oocytes expressing high levels of PLC{zeta}-luc protein (Fig. 2). The termination of Ca2+ oscillations in these cases occurs during the phase of increasing synthesis of PLC{zeta}, suggesting that there is a feedback mechanism regulating the activity of PLC{zeta}, or a desensitization of the oocyte to its effects. Previous work has shown that mouse PLC{zeta} undergoes nuclear localization upon pronuclear formation, and that this nuclear localization may be responsible for the termination of oscillations (Larman et al., 2004Go). However, this is unlikely to explain the cessation of Ca2+ oscillations in the current study since Ca2+ increase often stopped well before (1–2 h) the observation of pronuclearformation (6 h). Nuclear sequestration of PLC{zeta} may also be limited in capacity and it is unlikely that all the hPLC{zeta} is localized to the nuclei at high expression levels. There may be other mechanisms for termination of Ca2+ oscillations involving other aspects of desensitization such as IP3 receptor down-regulation or Ca2+ store depletion (Jellerette et al., 2000Go). These and other mechanisms for hPLC{zeta}-dependent termination of oscillations are currently being investigated. Regardless of the specific mechanism(s), our data suggest that mouse oocytes possess an innate ability to limit the duration and intensity of Ca2+ signals initiated by PLC{zeta}.

The Ca2+ oscillations triggered by hPLC{zeta}-luc lead to high rates of oocyte activation as judged by pronuclear formation. This is consistent with previous data using untagged or Venus-tagged PLC{zeta} (Cox et al., 2002Go; Rogers et al., 2004Go; Kuroda et al., 2006Go), and confirms that PLC{zeta} is a very effective activation stimulus for mouse oocytes. The present data demonstrate that the activation of oocytes occurs over a wide range of PLC{zeta} levels and that the activation process is not very sensitive to the pattern of Ca2+ oscillations, apparently requiring only that a sufficient amount of Ca2+ release is achieved during the activation phase (Toth et al., 2006Go). In direct contrast to oocyte activation, we found that subsequent development of mouse embryos is markedly affected by the level of PLC{zeta}-luc expression. First, we found that low levels of hPLC{zeta}-luc expression (0.05 cps) induced a reasonable number of Ca2+ spikes resulting in high rates of oocyte activation (95%, Fig. 4), however, this did not lead to any development to the blastocyst stage. Moreover, oocytes expressing high levels of PLC{zeta}-luc also resulted in Ca2+ oscillations and oocyte activation, but these similarly failed to develop further to the blastocyst stage. These data strongly imply that, in contrast to oocyte activation, there is a relatively narrow window of PLC{zeta} expression required to trigger successful development to the blastocyst stage in mouse.

This developmental requirement for a precise window of PLC{zeta} expression could be caused by a number of factors. The poor development with low levels of hPLC{zeta}-luc may result from inadequacy with the pattern of Ca2+ oscillations in these oocytes, such as low frequency or reduced spike number. Similarly, the low efficiency of development demonstrated with high levels of PLC{zeta} expression could also be related to the nature of the Ca2+ signals during activation, such as elevated frequency or early termination. These explanations would be broadly consistent with studies in rabbit and human oocytes that used high-voltage electric field pulses to propagate different patterns of Ca2+ oscillations. Such studies suggest that the type of Ca2+ changes specifically occurring during oocyte activation can have dramatic effects on later embryo development processes (Ozil, 1998Go; Ozil and Huneau, 2001Go). However, in our PLC{zeta}-luc studies, there are some cases where the pattern of Ca2+ oscillations that lead to good activation but poor development, is not substantially different from the pattern of Ca2+ oscillations that gave good development (e.g. comparing embryos in Group I with Group II, or Group III with Group IV). If the distinctive pattern of Ca2+ oscillations is the critical factor, then even very subtle differences in the pattern are presumably translated into a major effect upon later embryo development to the blastocyst stage. An alternative possibility is that Ca2+ signalling may not be the only factor responsible for further development. One of the consequences of PLC{zeta}s enzymatic action is that it will generate diacylglycerol that can activate protein kinase C, and this could also have effects on the early embryos that become manifest as development proceeds. The stimulation of some other pathways by PLC{zeta} may also be involved in explaining why the pattern of Ca2+ oscillations could change (e.g. oocytes in Group II compared to Group III) and yet development to the blastocyst stage was similar. As yet, however, the effects of diacylglycerol-dependent signaling events associated with PLC{zeta} remain to be investigated.

It is notable that a previous study has demonstrated that Ca2+ oscillations affect cell composition and number in resulting blastocysts (Mikich-Bos et al., 1997Go). In our experiments, we found that the ratio of ICM cell number to the total cell number in blastocysts induced by hPLC{zeta} is not significantly different from that for SrCl2 stimulation. However, a small effect was present in conditions where low levels of hPLC{zeta}-luc affected the total cell number in hPLC{zeta}-derived blastocysts compared to Sr2+-activated controls. This result may be a reflection of small differences in the cell cycle rate in the two conditions, although in comparison with our other data on PLC{zeta} concentration-dependent changes, this is a relatively minor effect and suggests that the quality of PLC{zeta}-induced blastocysts is not strictly controlled by the amount of PLC{zeta} protein present during activation.

Mouse oocytes have previously been used to investigate the ability of human sperm to cause oocyte activation (Rybouchkin et al., 1995Go; Lee et al., 1996Go; Araki et al., 2004Go). ICSI with human sperm injected into mouse oocytes is effective in causing both Ca2+ oscillations and oocyte activation. This suggests that the sperm factors involved in oocyte activation in humans and mice are conserved (Yanagida et al., 2000Go). Consequently, we anticipate that our general conclusions for hPLC{zeta}-induced development in mouse oocytes will reasonably extrapolate to studies of human oocytes. If we assume that our work on mouse will extrapolate to human oocytes then our results may have implications for our understanding of male factor infertility. If the fertilization process proceeds with an otherwise normal human sperm but which contains little or no PLC{zeta}, we would expect that successful oocyte activation would not occur. It is possible that such extreme cases of PLC{zeta} deficiency could account for previously documented cases of failed oocyte activation after ICSI (Battaglia et al., 1997Go; Heindryckx et al., 2005Go; Moaz et al., 2006Go). It is in these potential cases of PLC{zeta} deficiency where fertilization may be rescued by the application of stimuli, including PLC{zeta}, that can generate a Ca2+ increase (Eldar-Geva et al., 2003Go; Murase et al., 2004Go; Moaz et al., 2006Go). However, our current study raises the possibility of more subtle developmental effects relevant to putative therapeutic applications of PLC{zeta} for oocyte activation. The effective level of PLC{zeta} expression consistent with good preimplantation development occurs within a narrow window spanning only about a 4-fold concentration range. If the level of functional PLC{zeta} in sperm is abnormally low or high, and hence outside of the required physiological window, it is possible that the fertilizing sperm may cause oocyte activation, but later embryo development does not proceed. These cases would not be noticed as overt activation failure in ICSI or IVF treatment and in most clinical scenarios they may only be evident as failures to establish pregnancy. Successful delivery of appropriately calibrated levels of functional PLC{zeta} that triggers both oocyte activation and development to blastocyst may be an effective therapy in these situations.


   Funding
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Funding
References
 
This work was supported by the BBSRC and by start up funds for KS from Cardiff University School of Medicine.


   Acknowledgments
 
We thank Nazar Amso for advice and discussions.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Funding
 References
 
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Submitted on June 14, 2007; resubmitted on July 24, 2007; accepted on July 28, 2007.


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