title: Microinjection of mouse phospholipase C zeta complementary RNA into mare oocytes induces long-lasting intracellular calcium oscillations and embryonic development.
abstract: Methods presently used to activate mare oocytes for assisted reproduction technologies provide low rates of advanced embryonic development. Because phospholipase Czeta (PLCzeta) is the postulated sperm-borne factor responsible for oocyte activation at fertilisation, the aim of the present study was to investigate the pattern of [Ca(2+)](i) oscillations and developmental rates achieved by microinjection of three concentrations of mouse PLCzeta complementary (c) RNA (1, 0.5 or 0.25 microg microL(-1)) into mare oocytes. The frequency of [Ca(2+)](i) oscillations was no different (P > 0.05) after injection of 1, 0.5 or 0.25 microg microL(-1) PLCzeta cRNA (41.1 +/- 5.3, 47 +/- 4.0 and 55.4 +/- 9.0, respectively). However, [Ca(2+)](i) oscillations persisted longest (P < 0.05) for oocytes injected with 0.5 microg microL(-1) PLCzeta cRNA (570.7 +/- 64.2 min). There was no significant difference in cleavage rates after injection of the three concentrations of PLCzeta (P > 0.05; range 97-100%), but the proportion of oocytes reaching advanced stages of embryonic development (>64 nuclei) was significantly lower for oocytes injected with 0.25 microg microL(-1) PLCzeta cRNA (3%) than for those injected with 1 microg microL(-1) PLCzeta cRNA (15%). Based on these results, microinjection of PLCzeta may prove an effective and consistent method for the parthenogenetic activation of mare oocytes for nuclear transfer and provides a physiologically relevant tool with which to study fertilisation-dependent [Ca(2+)](i) signalling in this species.
authors.affiliation: Department of Clinical Sciences, Cornell University, College of Veterinary Medicine, Ithaca, NY 14853, USA. sjb55@cornell.edu
authors.forename: Sylvia J
authors.initials: SJ
authors.lastname: Bedford-Guaus
authors.forename: Sook-Young
authors.initials: SY
authors.lastname: Yoon
authors.forename: Rafael A
authors.initials: RA
authors.lastname: Fissore
authors.forename: Young-Ho
authors.initials: YH
authors.lastname: Choi
authors.forename: Katrin
authors.initials: K
authors.lastname: Hinrichs
mesh_terms: D000818:Animals; D020013:Calcium Signaling / Q000187:drug effects* / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D047108:Embryonic Development / Q000187:drug effects* / Q000502:physiology; D005260:Female; D006736:Horses / Q000502:physiology*; D051379:Mice; D008845:Microinjections; D009865:Oocytes / Q000187:drug effects* / Q000502:physiology; D054801:Phosphoinositide Phospholipase C / Q000235:genetics* / Q000494:pharmacology; D018075:RNA, Complementary / Q000008:administration & dosage / Q000494:pharmacology*; D027724:Reproductive Techniques, Assisted / Q000662:veterinary*
chemical_list: D018075:RNA, Complementary; D054801:Phosphoinositide Phospholipase C; C499234:Plcz1 protein, mouse