REPRODUCTIONRESEARCH C 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,MSHKo2 and K Swann3 Department of Anatomy and Developmental Biology, University College London, Gower Street, London WC1E 6BT, UK, 1Department of Physiology, University College London, Gower Street, London WC1E 6BT, UK, 2Developmental 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 3Department of Obstetrics and Gynaecology, School of Medicine, Heath Park, Cardiff University, Cardiff CF14 4XN, UK Correspondence should be addressed to K Swann; Email: [email protected] Abstract C AseriesofCa2 oscillations during mammalian fertilization is necessary and sufficient to stimulate meiotic resumption and C pronuclear formation. It is not known how effectively development continues in the absence of the initial Ca2 signal. We have C triggered parthenogenetic egg activation with cycloheximide that causes no Ca2 increase, with ethanol that causes a single large C C C 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 C activated by cycloheximide reached the blastocyst stage compared tothose activated by Sr2 orethanol. Any cycloheximide-activated C embryos that reached the blastocyst stage had a smaller inner cell mass number and a greater rate of apoptosis than Sr2 -activated C embryos. The poor development of cycloheximide-activated embryos was due to the lack of Ca2 increase because they developed to C C 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 C gene expression at the eight-cell stage showed that activation by Sr2 leads to a distinct pattern of gene expression from that seen with C 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. Reproduction (2006) 132 45–57 C of Ca2 in egg activation is also underlined by the fact Introduction C that most parthenogenetic stimuli cause a Ca2 increase During fertilisation in mammals, the sperm triggers a C in the egg. Chemicals treatments such as 7% ethanol, 2 C series of repetitive Ca oscillations that last for several ionomycin, or injection of Ca2 causeasingle C hours and eventually stop around the time of pronuclear prolonged rise in Ca2 (Cuthbertson et al. 1981, formation (Cuthbertson et al. 1981, Kline & Kline 1992, Colonna et al. 1989, Swann & Ozil 1994). Incubation Jones et al. 1995, Marangos et al. 2003). These sperm- 2C 2C 2C of eggs in Sr -containing media causes a series of Ca induced Ca oscillations release the egg from meiotic oscillations (Kline & Kline 1992, Swann & Ozil 1994, Liu C arrest by targeting cyclin B for destruction by the et al. 2002). Whilst the single rise in Ca2 induced by proteosome (Hyslop et al. 2004). Similar to meiotic agents such as ethanol is an effective stimulus for aged 2C C resumption, the Ca increase at fertilisation is respon- mammalian eggs, triggering Ca2 oscillations appears to sible for triggering cortical granule exocytosis and be more efficient in stimulating egg activation (Ozil pronuclear formation. These events are part of the initial 1998, Ducibella et al. 2002). Consequently, the series of C C egg activation. A Ca2 increase during fertilization is Ca2 oscillations at fertilization seems to have a role in known to be essential for stimulating egg activation guaranteeing that both the early (e.g. cortical granule C C because inhibition of Ca2 oscillations with the Ca2 exocytosis) and the later events of activation (pronuclear chelator BAPTA-AM completely prevents these early formation) are stimulated in a timely and effective events of development (Kline & Kline 1992). The role sequence (Ducibella et al. 2002). q 2006 Society for Reproduction and Fertility DOI: 10.1530/rep.1.01059 ISSN 1470–1626 (paper) 1741–7899 (online) Online version via www.reproduction-online.org Downloaded from Bioscientifica.com at 09/25/2021 06:58:24AM via free access 46 N T Rogers and others C C There is evidence that the Ca2 oscillations in that were exposed to Sr2 or ethanol in addition to C mammalian eggs may affect development beyond the cycloheximide. Our data suggest that a Ca2 increase first cell cycle. In rabbit eggs, the amplitude and during egg activation plays a role in the completion of C duration of Ca2 transients can be manipulated by cell divisions that lead to blastocyst formation. The means of electrical field pulses (Ozil 1990). Such effects we see do not appear to be due to an alteration in the methods have been used to demonstrate that the timing or the amount of global gene expression during 2C pattern of Ca transients during parthenogenetic EGA. However, we use microarray analysis to show that C activation, influences the numbers of embryos reaching the lack of a Ca2 increase during activation appears to compacted morula and blastocyst stages (Ozil 1990, lead to differences in the pattern of genes expressed in 2C Ozil & Huneau 2001). Applying extra Ca transients embryos at the mid preimplantation stages. 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 2C shown that imposing different patterns of Ca Materials and Methods transients during activation and the first cell cycle can affect the number of cells in the inner cell mass of the Egg and embryo handling resulting blastocysts (Bos-Mikich et al. 1997), however, 2C Female MF1 strain mice of 21–24 days were super- it is not clear how the Ca transients during the first ovulated by i.p. injection of pregnant mare serum cell cycle of development could affect the later events. 2C gonadotrophin and human chorionic gonadotrophin Since Ca oscillations of different frequencies can exert (hCG) (both from Intervet, Milton Keynes, UK) as specific effects on gene expression in somatic cells described previously (Lawrence et al. 1997). Eggs were (Dolmetsch et al. 1998), it is possible that some of the collected from oviducts 14–16 h posthCG injection and long-term effects of egg activation are mediated through maintained in HEPES-buffered potassium simplex the EGA. However, as yet this idea is untested. Further- optimised medium (HKSOM) buffer (Summers et al. more, there have been no data to show whether the pattern 2C 2C 2000). Parthenogenetic activation in a Ca -dependent of Ca increase during egg activation can affect the pattern of gene expression in mammalian embryos. manner was carried out by exposing eggs to 7% ethanol for 7 min, or by incubation for 2 h in media with 10 mM One of the difficulties in trying to study an effect of C C C 2 2 Ca2 transients upon embryo development is the fact Sr . Parthenogenetic activation in a Ca -independent C that a Ca2 increase is normally associated with, and manner took place by incubating eggs in 20 mg/ml necessary for, both fertilization and parthenogenetic cycloheximide for 4 h. When eggs were activated using a activation. Consequently, it is difficult to demonstrate combined treatment, they were first incubated in C whether an effect of Ca2 changes on the later embryo cycloheximide for 2 h, embryos were then placed in C 2C development are independent of the actions of Ca2 on HKSOM medium containing cycloheximide and Sr starting development. One potential method to separate for the next 2 h. Alternatively, eggs were incubated in C the effect of Ca2 on activation from an effect on later cycloheximide for 2 h, removed to a medium containing development is to use a method of starting development cycloheximide and 7% ethanol for 7 min at room C that does not rely upon Ca2 increase. Protein synthesis temperature, and then returned to cycloheximide inhibitors such as cycloheximide and puromycin can media for 1 h 53 minutes. All eggs were cultured in activate mouse eggs after prolonged periods of incu- media containing 2 mg/ml cytochalasin D during 2 hours bation (Siracusa et al. 1978, Moses & Kline 1995). Eggs of their activation protocol in order to produce diploid activated with cycloheximide, form pronuclei, have parthenotes. Cytochalasin D itself does not cause any C been reported to undergo cleavage divisions. Cyclohex- alteration in the intracellular Ca2 in eggs (McAvey et al. imide is also reported to be an effective activator without 2002, K Swann, unpublished observations). Activated 2C inducing any increase in Ca (Moses & Kline 1995). eggs were cultured at 37 8C in KSOM/AA in 5% CO2 Protein synthesis inhibitors probably activate mamma- (Summers et al. 2000). lian eggs by blocking the continuous synthesis of cyclin B that is required to stimulate the cell cycle protein C kinase CDK1. Inhibitors of protein kinases such as Ca2 measurements roscovitine also activate mammalian eggs (Phillips et al. 2002). Although both cycloheximide and roscov- The eggs were loaded with 2 mM Fura-2 AM (Sigma) for itine-activated eggs undergo cleavage division, the later 15 min. Zona pellucidae were removed with acidified development of these embryos has not been documen- Tyrode’s solution and eggs transferred to a heated ted systematically. chamber on to the stage of a Nikon Diaphot microscope. In this study, we have used cycloheximide to activate Fluorescence ratios from eggs were recorded as C mouse eggs in the absence of a Ca2 increase.