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BOR - Papers in Press, published online ahead of print February 19, 2003.
Biol Reprod 2003, 10.1095/biolreprod.102.014696
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BIOLOGY OF REPRODUCTION 69, 106–116 (2003)
DOI: 10.1095/biolreprod.102.014696
© 2003 by the Society for the Study of Reproduction, Inc.


Embryo

Ligand-Activated Signal Transduction in the 2-Cell Embryo1

David P. Lu3, Yan Li3, Roslyn Bathgate3, Margot Day4, and Christopher O'Neill2,3

Human Reproduction Unit, Royal North Shore Hospital,3 Developmental Physiology Laboratory,4 Department of Physiology, University of Sydney, Sydney, New South Wales, 2065 Australia

Platelet-activating factor (PAF) is an autocrine trophic/survival factor for the preimplantation embryo. PAF induced an increase in intracellular calcium concentration ([Ca2+]i) in the 2-cell embryo that had an absolute requirement for external calcium. L-type calcium channel blockers (diltiazem, verapamil, and nimodipine) significantly inhibited PAF-induced Ca2+ transients, but inhibitors of P/Q type ({omega}-agatoxin; {omega}-conotoxin MVIIC), N-type ({omega}-conotoxin GVIA), T-type (pimozide), and store-operated channels (SKF 96365 and econazole) did not block the transient. mRNA and protein for the {alpha}1-C subunit of L-type channels was expressed in the 2-cell embryo. The L-type calcium channel agonist (±) BAY K 8644 induced [Ca2+]i transients and, PAF and BAY K 8644 each caused mutual heterologous desensitization of each other's responses. Depolarization of the embryo (75 mM KCl) induced a [Ca2+]i transient that was inhibited by diltiazem and verapamil. Whole-cell patch-clamp measurements detected a voltage-gated channel (blocked by diltiazem, verapamil, and nifedipine) that was desensitized by prior responses of embryos to exogenous or embryo-derived PAF. Replacement of media Ca2+ with Mn2+ allowed Mn2+ influx to be observed directly; activation of a diltiazem-sensitive influx channel was an early response to PAF. The activation of a voltage-gated L-type calcium channel in the 2-cell embryo is required for normal signal transduction to an embryonic trophic factor.

1 This work was supported by a grant from the Australian National Health and Medical Research Council. D.P.L. and Y.L. contributed equally to this paper.

2 Correspondence. FAX: 61 2 9926 6343; chriso{at}med.usyd.edu.au




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