Microinjection of mouse phospholipase C zeta complementary RNA into mare oocytes induces long-lasting intracellular calcium oscillations and embryonic development.
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.
Bedford-Guaus
Sylvia J
SJ
Department of Clinical Sciences, Cornell University, College of Veterinary Medicine, Ithaca, NY 14853, USA. sjb55@cornell.edu
Yoon
Sook-Young
SY
Fissore
Rafael A
RA
Choi
Young-Ho
YH
Hinrichs
Katrin
K
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*
D018075:RNA, Complementary; D054801:Phosphoinositide Phospholipase C; C499234:Plcz1 protein, mouse