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Reproduction (2006) 132 45-57
DOI: 10.1530/rep.1.01059
Copyright © 2006 Society for Reproduction and Fertility
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RESEARCH

The absence of a Ca2+ signal during mouse egg activation can affect parthenogenetic preimplantation development, gene expression patterns, and blastocyst quality

N T Rogers, G Halet1, Y Piao2, J Carroll1, M S H Ko2 and K Swann3

Department of Anatomy and Developmental Biology, University College London, Gower Street, London WC1E 6BT, UK, 1 Department of Physiology, University College London, Gower Street, London WC1E 6BT, UK, 2 Developmental Genomics and Aging Section, Laboratory of Genetics, National Institute on Aging, National Institute of Health, 333 Cassell Drive, Suite 3000, Baltimore, MD 21224, USA and 3 Department of Obstetrics and Gynaecology, School of Medicine, Heath Park, Cardiff University, Cardiff CF14 4XN, UK

Correspondence should be addressed to K Swann; Email: swannk1{at}cf.ac.uk


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
A series of Ca2+ oscillations during mammalian fertilization is necessary and sufficient to stimulate meiotic resumption and pronuclear formation. It is not known how effectively development continues in the absence of the initial Ca2+ signal. We have triggered parthenogenetic egg activation with cycloheximide that causes no Ca2+ increase, with ethanol that causes a single large Ca2+ increase, or with Sr2+ that causes Ca2+ oscillations. Eggs were co-treated with cytochalasin D to make them diploid and they formed pronuclei and two-cell embryos at high rates with each activation treatment. However, far fewer of the embryos that were activated by cycloheximide reached the blastocyst stagecompared tothose activated by Sr2+ orethanol. Any cycloheximide-activated embryos that reached the blastocyst stage had a smaller inner cell mass number and a greater rate of apoptosis than Sr2+-activated embryos. The poor development of cycloheximide-activated embryos was due to the lack of Ca2+ increase because they developed to blastocyst stages at high rates when co-treated with Sr2+ or ethanol. Embryos activated by either Sr2+ or cycloheximide showed similar signs of initial embryonic genome activation (EGA) when measured using a reporter gene. However, microarray analysis of gene expression at the eight-cell stage showed that activation by Sr2+ leads to a distinct pattern of gene expression from that seen with embryos activated by cycloheximide. These data suggest that activation of mouse eggs in the absence of a Ca2+ signal does not affect initial parthenogenetic events, but can influence later gene expression and development.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
During fertilisation in mammals, the sperm triggers a series of repetitive Ca2+ oscillations that last for several hours and eventually stop around the time of pronuclear formation (Cuthbertson et al. 1981, Kline & Kline 1992, Jones et al. 1995, Marangos et al. 2003). These sperm-induced Ca2+ oscillations release the egg from meiotic arrest by targeting cyclin B for destruction by the proteosome (Hyslop et al. 2004). Similar to meiotic resumption, the Ca2+ increase at fertilisation is responsible for triggering cortical granule exocytosis and pronuclear formation. These events are part of the initial egg activation. A Ca2+ increase during fertilization is known to be essential for stimulating egg activation because inhibition of Ca2+ oscillations with the Ca2+ chelator BAPTA-AM completely prevents these early events of development (Kline & Kline 1992). The role of Ca2+ in egg activation is also underlined by the fact that most parthenogenetic stimuli cause a Ca2+ increase in the egg. Chemicals treatments such as 7% ethanol, ionomycin, or injection of Ca2+ cause a single prolonged rise in Ca2+ (Cuthbertson et al. 1981, Colonna et al. 1989, Swann & Ozil 1994). Incubation of eggs in Sr2+-containing media causes a series of Ca2+ oscillations (Kline & Kline 1992, Swann & Ozil 1994, Liu et al. 2002). Whilst the single rise in Ca2+ induced by agents such as ethanol is an effective stimulus for aged mammalian eggs, triggering Ca2+ oscillations appears to be more efficient in stimulating egg activation (Ozil 1998, Ducibella et al. 2002). Consequently, the series of Ca2+ oscillations at fertilization seems to have a role in guaranteeing that both the early (e.g. cortical granule exocytosis) and the later events of activation (pronuclear formation) are stimulated in a timely and effective sequence (Ducibella et al. 2002).

There is evidence that the Ca2+ oscillations in mammalian eggs may affect development beyond the first cell cycle. In rabbit eggs, the amplitude and duration of Ca2+ transients can be manipulated by means of electrical field pulses (Ozil 1990). Such methods have been used to demonstrate that the pattern of Ca2+ transients during parthenogenetic activation, influences the numbers of embryos reaching compacted morula and blastocyst stages (Ozil 1990, Ozil & Huneau 2001). Applying extra Ca2+ transients to rabbit eggs after fertilization has also been shown to influence the number of embryos that successfully implant (Ozil 1998). In the mouse egg, it has been shown that imposing different patterns of Ca2+ transients during activation and the first cell cycle can affect the number of cells in the inner cell mass of the resulting blastocysts (Bos-Mikich et al. 1997), however, it is not clear how the Ca2+ transients during the first cell cycle of development could affect the later events. Since Ca2+ oscillations of different frequencies can exert specific effects on gene expression in somatic cells (Dolmetsch et al. 1998), it is possible that some of the long-term effects of egg activation are mediated through the EGA. However, as yet this idea is untested. Furthermore, there have been no data to show whether the pattern of Ca2+ increase during egg activation can affect the pattern of gene expression in mammalian embryos.

One of the difficulties in trying to study an effect of Ca2+ transients upon embryo development is the fact that a Ca2+ increase is normally associated with, and necessary for, both fertilization and parthenogenetic activation. Consequently, it is difficult to demonstrate whether an effect of Ca2+ changes on the later embryo development are independent of the actions of Ca2+ on starting development. One potential method to separate the effect of Ca2+ on activation from an effect on later development is to use a method of starting development that does not rely upon Ca2+ increase. Protein synthesis inhibitors such as cycloheximide and puromycin can activate mouse eggs after prolonged periods of incubation (Siracusa et al. 1978, Moses & Kline 1995). Eggs activated with cycloheximide, form pronuclei, have been reported to undergo cleavage divisions. Cycloheximide is also reported to be an effective activator without inducing any increase in Ca2+ (Moses & Kline 1995). Protein synthesis inhibitors probably activate mammalian eggs by blocking the continuous synthesis of cyclin B that is required to stimulate the cell cycle protein kinase CDK1. Inhibitors of protein kinases such as roscovitine also activate mammalian eggs (Phillips et al. 2002). Although both cycloheximide and roscovitine-activated eggs undergo cleavage division, the later development of these embryos has not been documented systematically.

In this study, we have used cycloheximide to activate mouse eggs in the absence of a Ca2+ increase. The development of these eggs was compared to eggs that were exposed to Sr2+ or ethanol in addition to cycloheximide. Our data suggest that a Ca2+ increase during egg activation plays a role in the completion of cell divisions that lead to blastocyst formation. The effects we see do not appear to be due to an alteration in the timing or the amount of global gene expression during EGA. However, we use microarray analysis to show that the lack of a Ca2+ increase during activation appears to lead to differences in the pattern of genes expressed in embryos at the mid preimplantation stages.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Acknowledgements
 References
 
Egg and embryo handling
Female MF1 strain mice of 21–24 days were super-ovulated by i.p. injection of pregnant mare serum gonadotrophin and human chorionic gonadotrophin (hCG) (both from Intervet, Milton Keynes, UK) as described previously (Lawrence et al. 1997). Eggs were collected from oviducts 14–16 h posthCG injection and maintained in HEPES-buffered potassium simplex optimised medium (HKSOM) buffer (Summers et al. 2000). Parthenogenetic activation in a Ca2+-dependent manner was carried out by exposing eggs to 7% ethanol for 7 min, or by incubation for 2 h in media with 10 mM Sr2+. Parthenogenetic activation in a Ca2+-independent manner took place by incubating eggs in 20 µg/ml cycloheximide for 4 h. When eggs were activated using a combined treatment, they were first incubated in cycloheximide for 2 h, embryos were then placed in HKSOM medium containing cycloheximide and Sr2+ for the next 2 h. Alternatively, eggs were incubated in cycloheximide for 2 h, removed to a medium containing cycloheximide and 7% ethanol for 7 min at room temperature, and then returned to cycloheximide media for 1 h 53 minutes. All eggs were cultured in media containing 2 µg/ml cytochalasin D during 2 hours of their activation protocol in order to produce diploid parthenotes. Cytochalasin D itself does not cause any alteration in the intracellular Ca2+ in eggs (McAvey et al. 2002, K Swann, unpublished observations). Activated eggs were cultured at 37 °C in KSOM/AA in 5% CO2 (Summers et al. 2000).

Ca2+ measurements
The eggs were loaded with 2 µM Fura-2 AM (Sigma) for 15 min. Zona pellucidae were removed with acidified Tyrode’s solution and eggs transferred to a heated chamber on to the stage of a Nikon Diaphot microscope. Fluorescence ratios from eggs were recorded as described previously (Lawrence et al. 1997, Halet et al. 2004).

Monitoring genome activation with a luciferase reporter gene
EGA was assayed by monitoring luminescence from embryos injected with the luciferase reporter gene pGL3 (Promega) as described previously (Miranda et al. 1993, Ram & Schultz 1993). Pronuclei of in vivo fertilised or in vitro-activated parthenotes were injected with 50 ng/µl of pGL3-control vector DNA in the buffer containing 120 mM KCl and 20 mM Hepes. After injection, the pronucleate embryos were incubated in HKSOM media containing luciferin (100 µM). The luminescence from groups of embryos was monitored continuously for 25 h using a 10 x 0.5 NA (numerical aperture) objective and an imaging photon detector system supplied by Science Wares (www.sciencewares.com). This system uses an inverted microscope with an Imaging Photon Detector (Photek Ltd, St Leonards on Sea, UK).

Differential staining of inner cell mass and trophectoderm
Zona pellucidae were removed from blastocysts (103 h after activation) with acidified Tyrode’s solution and blastocysts were then exposed to 100 µg/ml fluorescein isothiocyanate (FITC)-labelled wheat germ lectin (WGA, Sigma) for 20 min at 37 °C followed by fixation in 4% paraformaldeyde in PBS at room temperature for 30 min. Blastocysts were then exposed to 0.05% Triton-X in PBS for 1 h followed by incubation in 5 µg/ml propidium iodide for 1 h. After permeabilisation and staining, blastocysts were washed in PBS before being viewed on a confocal microscope (model LSM510, Carl Zeiss, UK, Welvyn Garden City, UK). Fluorescein was excited with a 488-nm line of an Argon laser with emission at 450–490 nm, and propidium iodide was excited with at 543 nm with a He–Ne laser with emission at 585–615 nm.

Terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) labelling to assay apoptosis
Mouse blastocysts were washed through drops of PBS containing 1 mg/ml poly vinyl propylene (PVP) and then fixed in 4% paraformaldehyde in PBS for 20 min at room temperature. Following fixation blastocysts were per-meabilised in PBS containing 0.5% Triton-X (Sigma) for 1 h. Positive controls were incubated in 5 U/ml RQ1 DNase for 20 min at 37 °C. Blastocysts were then washed in PBS/PVP and incubated in fluorescein-conjugated dUTP and terminal deoxynucleotidyl transferase (TUNEL reagents, Roche) for 1 h in the dark at 37 °C. In order to count the number of cells in the blastocyst, the nuclei were stained by, incubation in 5 µg/ml propidium iodide and 50 µg/ml RNase A for 1 h in the dark at room temperature. Blastocysts were then washed in PBS/PVP and mounted on a glass slide in 10% glycerol. Slides were viewed on a confocal microscope as explained previously for fluorescein and propidium iodide. FITC-labelled cells were counted as apoptotic cells and propidium iodide-stained nuclei were counted as total number of live cells. The apoptotic index was calculated by the total number of TUNEL-stained cells divided by the total number of cells in each blastocyst.

Embryo collection for microarray work
Eggs were activated parthenogenetically using Sr2+ or cycloheximide as described previously. After 60 h of culture, only eight-cell embryos exhibiting good morphology were selected. Batches of 34 eight-cell embryos were washed four times through drops of HKSOM media and rapidly frozen in 4 µl of the same media. Eight-cell embryos were stored at –80 °C until they were used for RNA extraction.

RNA extraction labelling and hybridisation on the NIA 22K 60-Mer Oligo microarray
Two batches of 34 embryos were collected for each group of cycloheximide and Sr2+-activated eight-cell embryos. Total mRNA was extracted from each group of embryos using a Quickprep micro poly-A RNA Extraction Kit (Amersham) and linear acrylamide as a carrier (Ambion, Austin, TX, USA). Total RNA samples from each extraction were labelled with Cy3-dye by two-round linear amplification labelling reaction for cRNA targets using a Fluorescent Linear Amplification Kit (Agilent Technologies, Palo Alto, CA, USA). Quantification of target was determined using a microscale spectrophotometer (NanoDrop, Wilmington, DE, USA). Universal mouse reference RNA (Stratagene, La Jolla, CA, USA) was labelled with Cy5-dye by a one-round amplification-labelling reaction and used as a control for all hybridization reactions, allowing cross-comparisons between all data sets. The cRNA targets from both the sets of parthenogenetically activated embryos and universal mouse reference RNA were hybridised on the NIA 22k 60-mer oligo microarray (Carter et al. 2003). The data presented are from two biological and two technical replicates.

Analysis of microarray data
The intensity of 21 045 gene features per array was extracted from scanned microarray images using Feature Extraction 5.1.1 software (Agilent Technologies, Palo Alto, CA, USA). Statistically significant genes were determined quantitatively using the ANOVA-false discovery rate (ANOVA-FDR) = 10% (Sharov et al. 2005). Assignment of gene function was carried out using gene ontology (GO) terms (Ashburner et al. 2000), which characterized genes into basic groups such as cell cycle and cell adhesion. GO terms were used in conjunction with GenMapp/MAPP-Finder (Doniger et al. 2003) to identify the genes and functional groups associated with embryos parthenogenetically activated with Sr2+ or cycloheximide. The microarray data are available from the public databases (GEO and ArrayExpress) and the website: http://lgsun.grc.nia.nih.gov/microarray/data.html.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Acknowledgements
 References
 
Development of embryos stimulated with- or without- a Ca2+ increase
To stimulate parthenogenetic activation of mouse eggs, we used cycloheximide and Sr2+ or ethanol. Consistent with previous studies (Siracusa et al. 1978, Moses & Kline 1995, Moos et al. 1996), we found that eggs treated with cycloheximide formed pronuclei, but they failed to show any Ca2+ increase during a 4-h incubation (Fig. 1aGo). Other agents such as the protein kinase inhibitor roscovitine have also been reported to activate mouse eggs in the absence of a Ca2+ increase (Phillips et al. 2002). However, we found that they were not as effective, on their own, in activating eggs as cycloheximide, so they were not used in further studies (data not shown). In contrast to cycloheximide treatment, Sr2+ treatment caused a series of Ca2+ oscillations and 7% ethanol caused a singular Ca2+ increase (Fig. 1b and cGo). Both of these treatments also lead to pronuclear formation. These data confirm previous findings and show that, under our conditions, we can stimulate activation with agents that cause no Ca2+ increase, a single large Ca2+ increase, or a series of Ca2+ oscillations.


Figure 1
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  		Figure 1 Ca2+ measurements in mouse eggs during parthenogenetic activation. Ca2+ was monitored in eggs by changes in the Fura-2 fluorescence ratio (see Materials and Methods). (a) An example of an egg treated with cycloheximide from the start of the recording (typical of 14/14 eggs); (b) an example of egg undergoing oscillations after addition of media containing 10 mM Sr2+ from the start of the trace (typical of 13/14 eggs); (c) an egg was exposed to 7% ethanol for 7 min starting at times indicated by the bar (similar to 5/5 eggs); (d) An egg had been exposed to cycloheximide for 2 h before being placed in media containing both Sr2+ and cycloheximide at the start of the recording (typical of 16/16 eggs).

 
The overall degree of egg activation, as judged by pronuclear formation, was found to be similar with these three different treatments (Fig. 2Go). At 4 h after starting the activation treatments, a higher number of Sr2+-activated embryos had two fully formed pronuclei (58%) when compared to cycloheximide or ethanol-activated embryos (26%). However, by 6 h postactivation, a similar proportion (>75%) of embryos from all three treatment groups had two fully formed pronuclei (Fig. 2Go). At 6 h after activation, the majority of one-cell embryos (86%) produced by cycloheximide-induced activation underwent a temporary phenotypic stage in which they formed irregular shapes and furrows. This phenotype persisted for 1–2 h before the embryos returned to a well-defined spherical shape. Eggs activated with Sr2+ or ethanol alone did not produce embryos with this phenotype.


Figure 2
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  		Figure 2 Egg activation and development with cycoheximide, Sr2+ and ethanol. The timing of pronuclear formation during pathenogenetic activation is shown for mouse eggs treated with Sr2+, cycloheximide (CX) or ethanol, or combinations of thereof. The data are taken from 10 replicates with a total of 505 eggs for Sr2+ treatment, 575 eggs for cycloheximide treatment, 321 eggs for treatment with Sr2+ plus cycloheximide, 290 eggs for ethanol treatment and 270 eggs for treatment with ethanol plus cycloheximide.

 
We found that egg activation by means of Sr2+, cycloheximide, or ethanol was sufficient to produce high numbers of two-cell embryos. With cycloheximide and Sr2+ 90%, and with ethanol 89% of the pronucleate, embryos reached the two-cell stage. Most embryos in each group also cleaved to the four-cell stage. These data are shown in detail in Table 1Go and represents the continuation of development of the eggs presented in Fig. 2Go. However, despite rates of the initial development, a major difference was seen with the different parthenogenetic agents when embryos were cultured beyond the four-cell stage. The number of embryos reaching the eight-cell stage dropped dramatically when the embryos activated by means of cycloheximide were compared with those activated by Sr2+ or ethanol. The effect on activation was clearly related to events after the first cell cycle because we found that approximately 70% of the two-cell embryos reached the blastocyst stage when they had been activated by Sr2+ and ethanol (Table 1Go). In contrast, only 24% of two-cell embryos reached the blastocyst stage when they have been activated by cycloheximide (Table 1Go).


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  		Table 1 Preimplantation development after parthenogenetic activation.
 
The poor development of embryos activated by cycloheximide is rescued by a Ca2+ increase
The relatively poor development of embryos after activation with cycloheximide could be due to their lack of Ca2+ signal (as shown in Fig. 1aGo). To test this hypothesis, we treated eggs with combinations of cycloheximide and agents that can cause a Ca2+ increase in eggs, either Sr2+ or ethanol. Sr2+ media is reported to be less effective in causing Ca2+ oscillations as embryos progress through the first cell cycle and we found that when eggs were incubated for 4 h in cycloheximide, many of them failed to respond to generate Ca2+ oscillations in the Sr2+ media (data not shown). However, Fig. 1dGo shows that treating eggs with Sr2+ media was still effective in causing Ca2+ changes after they had been incubated for 2 h in cycloheximide and hence this protocol was adopted for subsequent activation studies. We found that the combined treatments of cycloheximide with either Sr2+ or ethanol were associated with similar times for pronuclear formation as that seen with each activating agent on its own (Fig. 2Go). When eggs were treated with cycloheximide first and then with Sr2+ plus cycloheximide for 2 h, the numbers of embryos developing to the eight-cell stage (77%) were also comparable to embryos activated by Sr2+ alone (Table 1Go). We also found similar results when eggs were treated with cycloheximide and ethanol compared to activation with ethanol alone (Table 1Go). Hence, these data show that Ca2+-elevating agents reversed the detrimental effect of cycloheximide activation upon embryo development. This shows that the poor development of embryos activated by cycloheximide is unlikely due to its effect upon protein synthesis per se, but can be explained by the lack of a Ca2+ increase during the activation phase.

Cell proliferation and apoptosis in parthenogenetic embryos
Although preimplantation development is compromised by activation with cycloheximide, about 20% of embryos reached the blastocyst stage. Previous studies of mouse embryos have assessed the quality of blastocysts by the number of cells and the degree of apoptosis (Hardy 1997). We first examined our blastocyst-stage embryos by differential staining to count the number of cells in the inner cell mass and trophectoderm. An example of a blastocyst stained with propidium iodide and FITC lectin is shown in Fig. 3Go. Figure 4Go shows the results of analysis of embryos that had been activated by either Sr2+ or cycloheximide. The data show that there was a significant reduction in the overall cell numbers in blastocysts that had been activated by cycloheximide compared with those activated by Sr2+ (Fig. 4aGo). The difference in the number of trophectoderm cells was not significantly different between the two groups of embryos (Fig. 4bGo). However, a significant difference was seen in the number of cells in the inner cell mass where cycloheximide-activated embryos had, on an average, 8 compared with 14.3 cells in Sr2+-activated embryos (Fig. 4cGo). This led to a significant difference in the ratio of the inner cell mass number to the trophectoderm cell number in the two types of embryos (Fig. 4dGo). These data suggest that even those cycloheximide-activated embryos that develop to the blastocyst stages are compromised in the number of cells in the inner mass.


Figure 3
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  		Figure 3 Differential staining of cells in parthenogenetic blastocysts. Confocal images are shown of sample mouse blastocyst activated by cycloheximide. The image shows the trophectoderm cells alone stained using WGA-FITC (a), the nuclei of all cells stained with propidium iodide (b), and the merged section is shown in (c). The images in (d)–(f) are sections through another blastocyst activated by Sr2+ illustrating the top of the blastocyst with all the trophectoderm cells stained green and red (d), a layer further down showing the red inner cell mass nuclei in the middle (e), and a section directly afterwards showing the large cavity with trophectoderm cells only (f).

 

Figure 4
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  		Figure 4 Composition of blastocysts derived from cycloheximide or Sr2+-induced egg activation. Bar charts with error bars showing differences in mean inner cell mass (A), mean trophectoderm (B), mean total cell number (C) and ratio of ICM/TE (D) in blastocysts generated from either Sr2+ or cycloheximide-induced parthenogenetic activation. Each asterisk indicates that Sr2+ and cyclohexmide groups are significantly different (Student’s t-test, P < 0.01). The data represent the mean numbers and S.E.M. from 4 replicate experiments. A total of 22 and 21 blastocysts derived from Sr2+ and CX-induced parthenogenetic egg activation were used for staining respectively.

 
One possible explanation of a reduced inner cell mass in a blastocyst could be that more cells have undergone apoptosis in these embryos. We used TUNEL staining to assess apoptosis in our embryos (Brison & Schultz 1997). Figure 5aGo shows examples of parthenogenetic embryos stained with TUNEL reagents and propidium iodide. Figure 5bGo shows the apoptotic index for embryos activated by Sr2+ or cycloheximide, where the index is a measure of the proportion of cells undergoing programmed cell death. The data indicate that there is an increase in apoptosis in embryo that were activated by cycloheximide compared with those activated by Sr2+. These results are consistent with those obtained for the cell counts and suggests that any blastocysts that are generated without having undergone a Ca2+ increase during egg activation are not of the same quality as those that have been exposed to Ca2+ increases.


Figure 5
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  		Figure 5 Apoptosis in embryos activated by cycloheximide or Sr2+. The staining for apoptosis is illustrated in (a) which shows blastocysts derived from a cycloheximide- (i) or Sr2+ (ii)-activated embryo. The red colour shows propidium iodide staining the nuclei of all cells and the green shows the fluorescein conjugated TUNEL reagent used as an indicator of apoptosis; (b) the bar chart shows the mean and S.E.M. for the apoptotic index of blastocyst parthenotes. A significantly larger apoptotic index occurs in blastocysts derived from cycloheximide activation (n = 26) compared to Sr2+ activation (n = 19) (P < 0.0001). Blastocysts were from three biological replicates.

 
Parthenogenetic activation and the onset of EGA
EGA is essential for development and can be first detected at the one-cell stage. We investigated if EGA was relatively deficient in cycloheximide-activated embryos compared to those activated by Sr2+. We monitored total gene expression during EGA by imaging embryos injected with firefly luciferase-encoding DNA into pronuclei at the one-cell stage as described previously (Miranda et al. 1993, Ram & Schultz 1993). We found that gene expression during EGA in all embryos occurs in a bell-shaped pattern with an initial sharp rise to the peak followed by a slower and more staggered decrease in expression. The start of EGA occurred 15–20 h postactivation and this phase of gene expression continued for approximately 20 h. Figure 6a and bGo shows some examples of the pattern of luciferase luminescence from Sr2+ and cycloheximide-activated embryos respectively. Figure 6cGo shows a luminescence image of embryos expressing luciferase. The patterns of increased luciferase luminescence we observed in these embryos were similar to fertilized zygotes (data not shown). For the parthenogenetic embryos, we took the area under the curve of the luminescence as a quantitative way of measuring the total relative amount of gene expression. We measured a total sum of 1.7±0.7x105 photons (S.E.M., n = 19) from Sr2+-activated embryos and 1.5±0.8x105 photons (S.E.M., n = 18) from cycloheximide-activated embryos. We also took the time to reach half the maximum peak of luminescence as a measure of the rate of EGA onset. We found the half-time for the onset of EGA was 9.6±0.8 h (S.E.M., n = 19) for Sr2+-activated embryos and 10.9±0.9 h (S.E.M., n = 18) for cycloheximide-activated embryos. There was a considerable variation in the degree of EGA from one embryo to another, so we cannot exclude small differences in EGA in our embryos. However, these data do suggest that there is no major difference in the timing or overall degree of EGA in embryos activated with or without a Ca2+ increase.


Figure 6
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  		Figure 6 EGA in parthenogenetic embryos using a luciferase reporter gene. The expression of luciferase is measured in one-cell embryos by the luminescence (photons/5 min). (a) A representative profile of luciferase expression from a Sr2+-activated embryo and (b) a similar representative recording of expression from a cycloheximide-activated embryo. The time is measured after the start of the activation treatments; (c) luminescence and bright field image of some Sr2+-activated embryos. The luminescence image is made from light integrated over 5 min at about 25 h after activation.

 
Microarray analysis of gene expression in parthenogenetically activated embryos
It is possible that changes in gene expression underlie the poor development and the relatively poor quality of blastocysts of the cycloheximide-activated embryos. To gain insight into the effect of Ca2+ signals on later gene expression, we used eight-cell embryos activated with either Sr2+ or cycloheximide for expression profiling. A universal mouse reference RNA (Strategene) was used and a pair-wise comparison of gene expression using microarray data was carried out to highlight differentially expressed genes in Sr2+ or cycloheximide-activated groups. Quantitative statistical analysis using a FDR≤10% identified 807 genes that were differentially expressed between the two groups of embryos. There were 239 genes that were identified as being expressed at higher levels in the Sr2+-activated group compared to 568 found to be expressed at higher levels in the cycloheximide-activated group. MAPPFinder (Doniger et al. 2003) was used to assign functional categories to differentially expressed genes by identifying the major GO-terms associated with them. As it is impossible to be conclusive about a function when very few genes are differentially expressed in a functional category, Tables 2Go and 3Go are compiled by only selecting those categories containing more than three differentially expressed genes in either group of embryos. The eight-cell embryos resulting from Sr2+-activation had many genes associated with the GO-terms ‘cell proliferation, cell adhesion and ion transport’ (Table 3Go), whereas eight-cell embryos resulting from cycloheximide activation were characterized by greater expression of genes associated with the GO-terms ‘cell cycle, apoptosis and cell differentiation’ (Table 2Go). It is important to note that a gene can be common to more than one biological process, for example RAD21 is associated with the ‘cell cycle’ and ‘apoptosis’ gene-expression pathways. The data in Tables 2Go and 3Go suggest that the presence or absence of a Ca2+ signal during egg activation does have a distinctive influence upon the pattern of gene expression during preimplantation development. Although there were differences in expression pattern for many GO groups, it was notable that cycloheximide-activated embryos show greater expression of genes associated with apoptosis. This is consistent with our other observations on cell numbers and TUNEL staining in intact blastocysts.


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  		Table 2 Genes that were more highly expressed in cycloheximide-activated embryos than in Sr2+-activated embryos.
 

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  		Table 3 Genes that were more highly expressed in Sr2+-activated embryos than in cycloheximide-activated embryos.
 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Acknowledgements
 References
 
A number of previous studies in the mouse and rabbit have found that imposing different type of Ca2+ oscillations during egg activation can influence post-implantation embryo development (Ozil 1990, Ozil & Huneau 2001). This is the first study that compares the development of preimplantation embryos that have been activated using both Ca2+-dependent and-independent means. This provides a simple and reproducible protocol for studying the specific influence of a Ca2+ signal on development that can be separated from the proximal effect of Ca2+ upon egg activation. Our developmental data showed that a larger number of embryos that underwent Ca2+ oscillations or a single Ca2+ increase, developed to the eight-cell and blastocyst stages compared to those from the cycloheximide-activated group, which experienced no Ca2+ increase. Blastocysts that were generated after cycloheximide activation also had a reduced inner cell mass number compared to those seen after Sr2+ activation. Furthermore, TUNEL staining of blastocysts demonstrated that a higher incidence of apoptosis occurred in blastocysts developed after cycloheximide activation. An increase in apoptosis has been demonstrated in blastocyst-stage embryos from mouse and a number of other mammalian species after either fertilization or parthenogenetic activation. Apoptosis is particularly prevalent in human blastocysts where it has been suggested that it is related to events occurring during the first cell cycle (Hardy et al. 2001). It is noteworthy that we found that either brief application of 7% ethanol or longer incubations in Sr2+ media were able to rescue the poor development seen after cycloheximide activation. These are very different chemical agents applied for very different times, and yet they both share in common the ability to increase Ca2+ in mouse eggs. Consequently, our data suggest that the presence of a Ca2+ increase during egg activation is the relevant early event that has later effects upon cell cycle progression in embryos and development to the blastocysts stage.

The method we used to activate eggs in the absence of a Ca2+ increase is incubation in cycloheximide. Although cycloheximide has been known to activate mammalian eggs for many years, there have been no systematic studies on the developmental capacity of cycloheximide-activated embryos. Some of the early studies on the cycloheximide activation would have involved haploid embryos, which itself is known to lead to impaired preimplantation development (Liu et al. 2002). In our study, we used incubation in cytochalasin D for all parthenogenetic activation treatments and, since our activated embryos formed two pronuclei, they were diploid. Consequently, it is clear that there is a systematic impairment of development after cycloheximide activation. It has been reported that incubation of mouse eggs in cycloheximide can delay the onset of EGA (Wang & Latham 1997, Aoki et al. 2003). This might be seen as an explanation for why cycloheximide-treated embryos showed poor development up to the blastocyst stage. However, the studies where cycloheximide was shown to delay EGA-used treatments of 6–14 h, whereas in this study we incubated eggs only in cycloheximide for 4 h. More significantly, we showed that treating eggs with either Sr2+ or ethanol could reverse the poor development seen with cycloheximide activation, and it seems unlikely that the ability to inhibit protein synthesis is reversed by ethanol or Sr2+. In addition, when we monitored EGA using a luciferase reporter we found that there was no significant difference in the onset, timing or degree of luciferase expression in parthenogenetic embryos that had been activated with Sr2+ or cycloheximide. Consequently, our data suggest that the poor development of embryos activated by cycloheximide alone is due to a lack of a Ca2+ increase. This implies that a Ca2+ increase during activation has later consequences for preimplantation development.

There are several lines of evidence in somatic cells that cytosolic Ca2+ increases can induce gene expression (Dolmetsch et al. 1998). The ability of Ca2+ to exert an influence upon later embryo development could potentially be due to some effect upon EGA. The onset of EGA in mouse is set by a clock that is initiated during fertilization (Schultz 1993). Since activation during fertilization involves robust Ca2+ oscillations, it is possible that the Ca2+ signal is the trigger for the start of a ‘zygotic clock’. However, our experiments to monitor EGA with a reporter gene found no significant difference in embryos activated either with or without a Ca2+ increase. To monitor EGA, we used a luciferase-based reporter gene that contains an SV40 promoter that is activated by Sp1. The Sp1 transcription factor is thought to play a major role in stimulating EGA in mouse embryos (Worrad et al. 1994). Therefore, our data suggest that any Sp1-dependent EGA in mouse embryo has no strict requirement for a Ca2+ increase during egg activation. It remains possible that any effect of Ca2+ upon later development is mediated by other transcription factors. Alternatively, the initial phase of genome activation in mammals could be independent of Ca2+ signalling and may instead reflect a genome-wide release from the inhibition of transcription (Schultz 1993, Zeng et al. 2004).

Although we could not detect any large-scale differences in gene expression, using an exogenous probe in one-cell embryos, we did find differences in the pattern of gene expression in later stages of development using microarray analysis. We chose to gain snapshot of the differences in our two main groups of embryos at the eight-cell stage to maximize the chances of detecting changes in genes. The eight-cell stage also represents the mid-preimplantation gene activation that is proximal to the point when poor development is evident in cycloheximide-activated embryos (Hamatani et al. 2004). This stage is also the prelude to the blastocyst stage where differences in gene expression will be relevant to the differences in blastocyst quality that we observed between the two groups of embryos.

Our microarray analysis of parthenogenetic embryos showed that a number of genes associated with the cell cycle were expressed at higher levels in eight-cell embryos from the cycloheximide-activated compared with Sr2+-activated embryos (Tables 2Go and 3Go). Overexpression of some these cell cycle-related genes such as Ccnb3, Cdk7 and Cdkn2d can have detrimental consequences for the cell cycle (Okuda et al. 1995, Nishiwaki et al. 2000, Nguyen et al. 2002). Consistent with our TUNEL staining, we found that cycloheximide-activated embryos also had higher expression of a number apoptosis-related genes. Gadd45 g in particular has been shown to cause cell cycle arrest and apoptosis in vitro (Mak & Kultz 2004), and overexpression of RAD21 (Pati et al. 2002), DAPK2 (Kawaii et al. 1999), plk4 (Mundt et al. 1997) and Cdk9 (Foskett et al. 2001) render cells sensitive to apoptosis. The overexpression of these apoptotic related-genes could contribute to the increased incidence of apoptosis, we observed in blastocysts derived from cycloheximide activation.

The greater expression of genes such as Sp3, Icsbp1, Cepbp, Socs2, Jak2 and Xdh, which are associated with ‘cell differentiation’, were also found to be associated with the cycloheximide-activated group of embryos (Table 3Go). Overexpression of Sp3 can cause down-regulation of N-cadherin (Le Mee et al. 2005), which is important for the embryos during the process of compaction. However, expression of some other genes associated with cell adhesion, cell proliferation and ion transport pathways were also characteristic of Sr2+-induced activation. Embryos activated by Sr2+ also showed a relative increase in levels of genes for ‘ion transport’such as Trpc7, Slc12a7, Scn8a and Kcnj11. The high levels of Trpc7 expression were notable because it codes for a Ca2+ permeable cation channel, which is activated by diacylglycerol and Ca2+ store depletion (Hoffman et al. 1999). Some genes related to the cell adhesion processes, such as Pcdh7 and Pcdha, or cell proliferation, such as Pdgfa, were also identified as being expressed at higher levels in Sr2+-activated embryos. These genes could also play some role in promoting embryo development of Sr2+-activated embryos over cycloheximide activated ones.

It is not clear how the presence or absence of a Ca2+ increase during activation could exert such an influence upon the pattern of gene expression in embryos at much later stages of preimplantation development. The Ca2+ increase during egg activation is known to stimulate a number of protein kinases such as calmodulin-dependent protein kinase II and PKC (Halet et al. 2004, Markoulaki et al. 2004, Madgwick et al. 2005). These protein kinases are involved in causing the immediate events of egg activation such as meiotic resumption and cortical granule exocytosis. However, these or other Ca2+-dependent protein kinases may have other substrates that exert a long-term influence. This might involve protein phosphorylation leading to changes in protein synthesis that could have a selective effect upon particular genes during EGA. Since each wave of gene expression has an influence upon the next phase (Hamatani et al. 2004, Wang et al. 2004), any difference in the pattern of gene expression at these earlier stages could have a knock-on effect upon the pattern of expression during the mid-preimplantation stage of development. The exact form of the Ca2+ increase during activation may not be critical because either ethanol or Sr2+ rescue poor development seen with cycloheximide activation, and these agents cause a single large Ca2+ increase or a series of Ca2+ spikes respectively. This is consistent with other parthenogenetic studies that suggest that any stimulus that causes a sufficient amount of Ca2+ release during activation is effective in stimulating early development (Toth et al. 2006).

One of the implications of our current findings is that the way the egg is activated can influence the pattern of gene expression and the way the embryo develops up to the blastocyst stage. This has some practical implications since mammalian embryos are prone to developmental arrest and apoptosis during preimplantation stages (Hardy 1997). So far, attention has been focussed upon the use of different types of culture media to optimise preimplantation development (Rinaudo & Schultz 2004). Our data raise the possibility that failure of embryos to cleave, or the poor quality blastocysts, after in vitro culture, may also be a consequence of a deficient Ca2+ signal during egg activation.


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
References
 
This work was supported by a Wellcome Trust Grant awarded to K S, by a Bogue Fellowship and an MRC Studentship awarded to NTR, and also in part by the Intramural Research Program of the National Institute on Aging, NIH. We thank Mark Larman, Kazuhiro Aiba, and Alexei A Sharov for technical advice.


   Footnotes
 
Received 3 November 2005
First decision 31 January 2006
Revised manuscript received 3 March 2006
Accepted 20 April 2006


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 

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