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Phospholipase Cζ Causes Ca2+ Oscillations and Parthenogenetic Activation of Human Oocytes

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Phospholipase Cζ Causes Ca2+ Oscillations and Parthenogenetic Activation of Human Oocytes REPRODUCTIONRESEARCH Phospholipase Cz causes Ca21 oscillations and parthenogenetic activation of human oocytes N T Rogers1, E Hobson2, S Pickering2, F A Lai3, P Braude2 and K Swann4 1Department of Anatomy and Developmental Biology, University College London, Gower Street, London WC1E 6BT, 2Assisted Conception Unit and GKT Department of Women’s Health, Guy’s and St Thomas’s NHS Trust, Guy’s Hospital, London SE1 9RT, 3Wales Heart Research Institute, Wales College of Medicine, Heath Park, Cardiff University, Cardiff CF14 4XN and 4Department of Obstetrics and Gynaecology, Wales College of Medicine, Heath Park, Cardiff University, Cardiff CF14 4XN, UK Correspondence should be addressed to K Swann; Email: [email protected] Abstract At fertilization in mammals the sperm activates development of the oocyte by inducing a prolonged series of oscillations in the cytosolic free Ca21 concentration. One theory of signal transduction at fertilization suggests that the sperm cause the Ca21 oscillations by introducing a protein factor into the oocyte after gamete membrane fusion. We recently identified this sperm-specific protein as phospholipase Cz (PLCz), and we showed that PLCz triggers Ca21 oscillations in unfertilized mouse oocytes. Here we report that microinjection of the complementary RNA for human PLCz causes prolonged Ca21 oscillations in aged human oocytes that had failed to fertilize during in vitro fertilization or intracytoplasmic sperm injection. The fre- quency of Ca21 oscillations was related to the concentration of complementary RNA injected. At low concentrations, PLCz stimulated parthenogenetic activation of oocytes. These embryos underwent cleavage divisions and some formed blastocysts. These data show that PLCz is a novel parthenogenetic stimulus for human oocytes and that it is unique in its ability to mimic the repetitive nature of the Ca21 stimulus provided by the sperm during human fertilization. Reproduction (2004) 128 697–702 Introduction the finding that most of the treatments that cause the parthenogenetic activation of development in mammals Mammalian oocytes are ovulated in a state of arrest at are effective because they cause a marked rise in the cyto- metaphase of the second meiotic division. During fertili- solic Ca2þ concentration (Whittingham 1980, Swann & zation the sperm releases the oocyte from metaphase Ozil 1994). In mouse and domestic animal oocytes arrest and initiates development by inducing a series of parthenogenetic activating agents such as ethanol, Ca2þ repeated rises in the cytosolic free Ca2þ concentration ionophores and electrical-field stimulation cause a single (Whittingham 1980, Cuthbertson & Cobbold 1985, and prolonged rise in Ca2þ (Swann & Ozil 1994). In Stricker 1999). Such repeated rises in Ca2þ,orCa2þ oscil- mouse, and some other species, parthenogenetic activa- lations, occur at intervals of several minutes for the first ting agents such as Sr2þ ions or thimerosal have been few hours after sperm–oocyte interaction. These Ca2þ shown to cause Ca2þ oscillations that are similar, but not oscillations have been observed during in vitro fertiliza- identical, to those seen at fertilization (Kline & Kline tion (IVF) in a range of mammalian species including 1992, Cheek et al. 1993). oocytes from humans (Taylor et al. 1993). In addition, in The mechanism by which the sperm stimulates the human and mouse oocytes prolonged Ca2þ oscillations Ca2þ oscillations at fertilization is not fully resolved have also been demonstrated following intracytoplasmic (Stricker 1999). However, one theory is that the sperm sperm injection (ICSI; Tesarik & Sousa 1994, Nakano et al. introduces a protein factor into the egg cytosol after 1997). The Ca2þ oscillations seen in mammals appear to gamete fusion, and that this sperm factor protein initiates be both necessary and sufficient for the activation of Ca2þ release in the oocyte via the generation of inositol development. In the mouse, for example, it has been 1,4,5-trisphosphate (InsP3; Swann et al. 2004). This theory shown that the Ca2þ oscillations are necessary for second- is supported by the finding that injecting cytosolic extracts polar-body emission and pronuclear formation (Kline & from sperm can cause Ca2þ oscillations in a range of Kline 1992). The importance of Ca2þ is also underlined by different mammalian oocytes, including those from q 2004 Society for Reproduction and Fertility DOI: 10.1530/rep.1.00484 ISSN 1470–1626 (paper) 1741–7899 (online) Online version via www.reproduction-online.org Downloaded from Bioscientifica.com at 09/28/2021 11:54:41AM via free access 698 N T Rogers and others humans (Swann 1990, Homa & Swann 1994, Wu et al. Materials and Methods 1997). We recently demonstrated that mouse sperm pos- sess a novel and specific isoform of phospholipase C Obtaining and handling of human oocytes (PLC) referred to as PLCz (Saunders et al. 2002). PLCz was Human oocytes were obtained from patients whose shown to be the protein present in the sperm extracts that gametes had failed to fertilize following conventional IVF 2þ is responsible for generating Ca release and InsP3 pro- or ICSI. Ethical approval for the project was obtained from duction (Saunders et al. 2002). Most critically it was St Thomas’s Hospital Local Research Ethics Committee demonstrated that PLCz is an effective mimic of the sperm and from the Human Fertilization and Embryology Auth- because microinjection of complementary RNA (cRNA) ority who issued a licence for the work (R0147). Consent 2þ encoding for PLCz into mouse oocytes causes Ca oscil- for the use of unfertilized oocytes was obtained from lations identical to those seen during fertilization. Injec- patients before starting their treatment. For treatment the tion of cRNA for PLCz also leads to egg activation and patients underwent pituitary downregulation and con- development to the blastocyst stage in the mouse (Saun- trolled ovarian hyperstimulation using gonadotrophins. ders et al. 2002). As well as mice, both humans and mon- Ovarian stimulation was achieved using a daily dose of keys have been shown to possess a sperm-specific PLCz 150–450 IU of recombinant follicle-stimulating hormone (Cox et al. 2002). Previous work suggested that the sperm (Gonal F; Serono Laboratories, Welwyn Garden City, factor protein was not species specific and, consistent Herts, UK; or Puregon; Organon, Cambridge, UK). with this, we found that injection of cRNA for the human Human chorionic gonadotrophin, 10 000 IU (Profasi from z 2þ or monkey isoforms of PLC is able to cause Ca oscil- Serono Laboratories or Pregnyl from Organon), was lations in mouse oocytes and stimulate embryo develop- administered when at least three follicles had reached a ment up to the blastocyst stage at similar rates to those mean diameter of 18 mm or more. Transvaginal follicular seen after in vivo fertilization (Cox et al. 2002). However, aspiration was performed 34–36 h after human chorionic the effect of PLCz in human oocytes has not previously gonadotrophin injection and 3–6 h later oocytes were been reported. prepared for IVF or ICSI dependent upon earlier semen Like other mammals human oocytes can also be parthe- analysis. Following IVF insemination or ICSI oocytes were nogenetically activated by stimuli that elevate Ca2þ levels. cultured overnight and checked for signs of fertilization Calcium ionophores such A23187 have been shown to 19–20 h later. Only those oocytes that appeared to be at cause both fresh and aged oocytes to undergo pronuclear metaphase II or I and that showed no signs of fertilization formation and begin early cleavage divisions (Winston þ were used for the project. Such unfertilized oocytes were et al. 1991). However, some studies have found that Ca2 transferred from the Assisted Conception Unit at Guy’s ionophore alone does not reliably activate human oocytes Hospital to laboratories at University College London and (Balakier & Casper 1993, Rinaudo et al. 1997). So the most common current activation protocols combine Ca2þ microinjected within the next 1–2 h. Unless otherwise ionophore with protein synthesis or protein kinase inhibi- stated, all manipulations in the laboratory were carried tors such as 6-dimethyl aminopurine (6-DMAP) or puro- out on ooyctes in Hepes/KSOM (HKSOM) medium (Saunders et al. 2002). mycin (Cibelli et al. 2001, Nakagawa et al. 2001, Lin et al. 2þ 2003). These combination protocols have proved effective In experiments where Ca was monitored oocytes were in stimulating human oocytes to form pronuclei, but the injected with solutions containing various concentrations success rates of subsequent preimplantation development of PLCz cRNA and 1 mM Oregon Green BAPTA dextran are still poor compared with embryo development after (Molecular Probes, Eugene, OR, USA) in a buffered salt fertilization. In order to try and improve activation and solution (120 mM KCl/20 mM Hepes, pH 7.4) that had development after parthenogenesis, attempts have been been treated with Chelex 100 beads (Sigma) to remove made to use stimuli that mimic IVF in causing repetitive divalent cations. In developmental experiments where 2þ Ca2þ increases. Data in mouse and rabbit oocytes that Ca was not monitored the Oregon Green BAPTA dextran were exposed to repeated electrical-field pulses have was omitted from the injection buffer. cRNA encoding the suggested that repeated rises in Ca2þ can improve acti- 608-amino-acid sequence of the human form of PLCz was vation rates, and subsequent development, compared with prepared as described previously (Saunders et al. 2002). stimuli that cause a single Ca2þ increase (Ozil 1990, Ozil The cRNA was stored in aliquots at 280 8C until being & Huneau 2001). Limited application of this technology thawed immediately prior to injection. For injection the has suggested that the repetitive stimuli may also be useful oocytes were placed on a Nikon Diaphot stage and micro- in activating human oocytes (Zhang et al. 1999). In this injected by application of brief pressure pulses as study we demonstrate that microinjection of cRNA enco- described previously (Swann 1990). The injection was þ ding human PLCz protein can cause a prolonged series between 3 and 5% of the oocyte’s total volume.
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