Microinjection of mouse phospholipase Cζ complementary RNA into mare oocytes induces long-lasting intracellular calcium oscillations and embryonic development
Sylvia J. Bedford-Guaus A D , Sook-Young Yoon B , Rafael A. Fissore B , Young-Ho Choi C and Katrin Hinrichs C
+ Author Affiliations
- Author Affiliations
A Department of Clinical Sciences, Cornell University, College of Veterinary Medicine, Ithaca, NY 14853, USA.
B Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, MA 01003, USA.
C Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine, Texas A&M University, College Station, TX 77843-4466, USA.
D Corresponding author. Email: sjb55@cornell.edu
Reproduction, Fertility and Development 20(8) 875-883 https://doi.org/10.1071/RD08115
Submitted: 21 May 2008 Accepted: 27 July 2008 Published: 10 October 2008
Abstract
Methods presently used to activate mare oocytes for assisted reproduction technologies provide low rates of advanced embryonic development. Because phospholipase Cζ (PLCζ) is the postulated sperm-borne factor responsible for oocyte activation at fertilisation, the aim of the present study was to investigate the pattern of [Ca2+]i oscillations and developmental rates achieved by microinjection of three concentrations of mouse PLCζ complementary (c) RNA (1, 0.5 or 0.25 μg μL–1) into mare oocytes. The frequency of [Ca2+]i oscillations was no different (P > 0.05) after injection of 1, 0.5 or 0.25 μg μL–1 PLCζ cRNA (41.1 ± 5.3, 47 ± 4.0 and 55.4 ± 9.0, respectively). However, [Ca2+]i oscillations persisted longest (P < 0.05) for oocytes injected with 0.5 μg μL–1 PLCζ cRNA (570.7 ± 64.2 min). There was no significant difference in cleavage rates after injection of the three concentrations of PLCζ (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 μg μL–1 PLCζ cRNA (3%) than for those injected with 1 μg μL–1 PLCζ cRNA (15%). Based on these results, microinjection of PLCζ 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 [Ca2+]i signalling in this species.
Additional keywords: assisted reproduction, oocyte activation.
Acknowledgements
This study was funded by faculty start-up funds at Cornell University, College of Veterinary Medicine, and by the Link Equine Research Endowment Fund, Texas A&M University. The authors thank Changli He (University of Massachusetts) for assistance with the preparation of the mouse PLCζ cRNA construct and Lori McPartlin (Cornell University) and Linda Love (Texas A&M University) for assistance with the preparation of mare oocytes for experiments.
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