REPRODUCTIONRESEARCH Effect of genetic background and activating stimulus on the timing of meiotic cell cycle progression in parthenogenetically activated mouse oocytes Elena Iba´n˜ez1, David F Albertini2 and Eric W Overstro¨m1,2 1Department of Biomedical Sciences, Tufts University School of Veterinary Medicine, North Grafton, Massachusetts 01536, USA and 2Department of Anatomy and Cellular Biology, Tufts University School of Medicine, Boston, Massachusetts 02111, USA Correspondence should be addressed to E W Overstro¨m who is now at Department of Biology and Biotechnology, Worcester Polytechnic Institute, 100 Institute Road, Worcester, Massachusetts 01609-2280, USA; Email: [email protected] (E Iba´n˜ez is now at Departament de Biologia Cellular, Fisiologia i Immunologia, Facultat de Cie`ncies, Universitat Auto`noma de Barcelona, 08193 Bellaterra, Spain) (D F Albertini is now at Department of Molecular and Integrative Physiology, Kansas University Medical Center, Kansas City, Kansas 66160–7401, USA) Abstract With the aim of investigating the effects of oocyte genotype and activating stimulus on the timing of nuclear events after acti- vation, oocytes collected from hybrid B6D2F1, inbred C57BL/6 and outbred CF-1 and immunodeficient nude (NU/1) females were activated using ethanol or strontium and fixed at various time-points. Meiotic status, spindle rotation and second polar body (PB2) extrusion were monitored by fluorescence microscopy using DNA-, microtubule- and microfilament-selective probes. Although activation efficiency was similar in all groups of oocytes, a significant percentage of CF-1 and NU/1 oocytes treated with ethanol and of C57BL/6 oocytes treated either with ethanol or strontium failed to complete activation and became arrested at a new metaphase stage (MIII) after PB2 extrusion. C57BL/6 oocytes also showed slower release from MII arrest but faster progression to telophase (TII) after ethanol exposure, and they exhibited the most rapid exit from TII under both activation treatments. Strontium caused delayed meiotic resumption, spindle rotation and PB2 extrusion, but rapid TII exit, in B6D2F1, CF-1 and NU/1 oocytes when compared with ethanol. Compared with all other strains, NU/1 oocytes were significantly slower in completing spindle rotation and PB2 extrusion, irrespective of the activating stimulus, and a significant decrease in activation rates and pace of meiotic progression was observed after strontium exposure. Thus, our findings demon- strated that the kinetics of meiosis resumption and completion, spindle rotation and PB2 extrusion following parthenogenetic activation depends on both genotype-specific factors and on the activation treatment applied. Reproduction (2005) 129 27–38 Introduction oscillations at fertilization also initiate a series of subcellu- lar and biochemical changes in oocytes, including cortical In most mammalian species, ovulated oocytes are arrested granule exocytosis, modifications of zona pellucida glyco- at metaphase of the second meiotic division (MII). This proteins, recruitment of maternal mRNAs, resumption of arrest is characterized by a high maturation-promoting meiosis, second polar body (PB2) extrusion, pronuclear factor (MPF) activity (Nurse 1990), which is maintained by formation, DNA synthesis and first mitotic cleavage a cytostatic factor (CSF) (Masui 1991). Under natural con- (Schultz & Kopf 1995). Collectively, these events are ditions, exit from the MII arrest is accomplished by fertili- referred to as oocyte activation. Meiotic resumption after zation, which triggers a series of transient rises in activation is characterized by chromatid segregation and intracellular Ca2þ concentration that last for several hours spindle elongation during the transition from MII to ana- (Cuthbertson & Cobbold 1985, Swann 1990). These cal- phase (AII) and telophase II (TII). At the same time, in cium transients cause inactivation of CSF, possibly through mouse oocytes, the spindle rotates from a parallel to a per- activation of Ca2þ-calmodulin-dependent protein kinase II pendicular orientation relative to the plasma membrane, (CaMKII), and subsequent inactivation of MPF (Collas et al. and the PB2 is extruded (Maro et al. 1984, Liu et al. 2000, 1995, Zernicka-Goetz et al. 1995). Intracellular Ca2þ Iba´n˜ez et al. 2003). Finally, chromatin is decondensed, q 2005 Society for Reproduction and Fertility DOI: 10.1530/rep.1.00452 ISSN 1470–1626 (paper) 1741–7899 (online) Online version via www.reproduction-online.org 28 E Iba´n˜ez and others pronuclear envelopes form, spindle microtubules depoly- activating stimulus influence nuclear events following arti- merize and the interphase microtubular network is ficial activation. In particular, the effects of these two fac- assembled. MPF inactivation is essential for meiotic tors on the kinetics of meiotic cell cycle resumption and resumption and progression to interphase, whereas completion, spindle rotation and PB2 extrusion were eval- the inactivation of MAP kinase (MAPK), a component of uated. Mouse strains chosen as oocyte donors were CSF, is associated with chromatin decondensation, pro- B6D2F1, C57BL/6 and CF-1, as common representatives of nuclear formation, initiation of DNA synthesis and hybrid, inbred and outbred strains respectively. Oocytes changes in microtubule dynamics (Moos et al. 1995, collected from immunodeficient heterozygous nude Carroll et al. 2000). females (NU/þ), of an outbred background, were also Although sperm supply the natural stimulus responsible included in the study, as delayed PB2 extrusion after acti- for oocyte activation, oocytes may also be activated vation had been observed in these oocytes in previous parthenogenetically, without the contribution of sperm, by studies by our group (E Iba´n˜ez, DF Albertini and a variety of physical and chemical stimuli (reviewed by EW Overstro¨m, unpublished results). Macha´ty & Prather 1998, Alberio et al. 2001). Most artifi- cial stimuli activate oocytes by a mechanism similar to that used by the sperm, i.e. inducing an increase in the Materials and Methods 2þ intracellular Ca levels. In mouse oocytes, ethanol has Collection of MII oocytes been frequently employed as an activating agent (Cuthbertson et al. 1981, Cuthbertson 1983, Winston & Hybrid B6D2F1 (C57BL/6 £ DBA/2), inbred C57BL/6 and Maro 1995). Ethanol activates oocytes by promoting the outbred CF-1 and immunodeficient nude (NU/þ) female formation of inositol 1,4,5-trisphosphate (IP3) at the mice (Charles River Laboratories, Wilmington, MA, USA) plasma membrane and the influx of extracellular Ca2þ were used as oocyte donors. Animal care and procedures (Ilyin & Parker 1992), causing a large, single rise in intra- were conducted according to protocols approved by cellular Ca2þ concentration (Cuthbertson et al. 1981). the Tufts University Institutional Animal Care and Use Strontium is another popular activating agent, currently Committee. Females (8–10 weeks old) were induced to used in mouse nuclear transfer protocols (Wakayama et al. superovulate by intraperitoneal injection of 5 IU equine 1998). It induces repetitive Ca2þ transients, which last for chorionic gonadotropin (eCG; Calbiochem, San Diego, several hours, probably by displacing bound Ca2þ in the CA, USA) followed 48 h later by 5 IU human chorionic oocyte, but also by inducing Ca2þ release from intracellu- gonadotropin (hCG) (Calbiochem). In vivo ovulated MII lar stores (Kline & Kline 1992, Bos-Mikich et al. 1995). oocytes were recovered from oviducts 16 h post-hCG Parthenogenetic activation can be used as a model to injection in Hepes-buffered potassium simplex optimized study biochemical and morphological changes occurring medium (H-KSOM; Speciality Media, Phillipsburg, NJ, in the oocyte during fertilization and early embryonic USA) and cumulus cells were dispersed by incubation in development, and it is a critical component of the cloning 150 U bovine testicular hyaluronidase/ml (Sigma, St Louis, procedure. Because of this, several studies have been per- MO, USA) in H-KSOM at 37 8C for 5 min. Cumulus-free formed to date on the activation of mouse oocytes using oocytes were then washed three times in fresh H-KSOM various artificial stimuli. The majority of these studies and immediately activated. have focused on the comparison of activation rates, usually measured by the presence of a pronucleus several Oocyte activation and culture hours after activation, or on the comparison of partheno- genetic development up to the blastocyst stage, of oocytes Oocytes were parthenogenetically activated using two from the same strain activated with different treatments different chemical treatments. For ethanol activation, (Cuthbertson 1983, O’Neill et al. 1991, Bos-Mikich et al. oocytes were treated for 5 min at 37 8C in H-KSOM con- 1995, 1997, Kishikawa et al. 1999). According to these taining 7% (v/v) ethanol. Oocytes were then washed twice criteria, differences in oocyte activation efficiencies have in H-KSOM and cultured for up to 6 h at 37 8C under 5% been observed depending on the activating stimulus used. CO2 in air in KSOM medium containing both non-essen- Nevertheless, only a few studies have been reported on tial and essential amino acids (Specialty Media) and sup- the activation efficiencies induced by artificial stimuli on plemented with 1 mg bovine serum albumin (BSA)/ml. oocytes from different strains of mice, and the results of When strontium was used as the activating agent, oocytes these studies indicate that parthenogenetic activation and were cultured for up to 6