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Fertilization and Inositol 1,4,5-Trisphosphate (IP₃)-Induced Calcium Release in Type-1 Inositol 1,4,5-Trisphosphate Receptor Down-Regulated Bovine Eggs¹

Department of Veterinary and Animal Sciences,⁵ University of Massachusetts, Amherst, Massachusetts 01003 Laboratorium voor Fysiologie,⁶ Katholieke Universiteit Leuven, B-3000 Leuven, Belgium Hematech, Inc.,⁷ Sioux Falls, South Dakota 57106

It is widely believed that stimulation of the phosphoinositide pathway and production of 1,4,5-inositol trisphosphate (IP₃) underlies the oscillatory changes in the concentration of intracellular free calcium ions ([Ca²⁺]_i) seen during mammalian fertilization. IP₃ promotes Ca²⁺ release in eggs by binding to its receptor, the type-1 IP₃ receptor (IP₃R-1, also known as ITPR1), a ligand-gated Ca²⁺ channel located in the membrane of the endoplasmic reticulum, the main Ca²⁺ store of the cell. While IP₃R-1 has been shown to mediate all Ca²⁺ release during mouse fertilization, whether or not it plays such an essential role in fertilization-induced Ca²⁺ release in large domestic species such as bovine and porcine is presently not known. Accordingly, we have generated metaphase II bovine eggs with a 70%–80% reduction in the number of intact IP₃R-1 by inducing receptor down-regulation during oocyte maturation. We did so by injecting the nonhydrolyzable IP₃ analogue, adenophostin A. Functional Ca²⁺ release analysis revealed that IP₃R-1 is the predominant Ca²⁺ release channel in bovine eggs, requiring as little as 20% of total intact receptor to mount persistent [Ca²⁺]_i oscillations in response to fertilization, expression of PLC (also known as PLCZ1), and adenophostin A. However, lower concentrations of IP₃ and near-physiological concentrations of porcine sperm extract were unable to trigger [Ca²⁺]_i oscillations in this reduced IP₃R-1 model. Furthermore, we present evidence that the sensitivity of bovine IP₃R-1 is impaired at the first embryonic interphase. Together, these results demonstrate the essential role of IP₃R-1-mediated Ca²⁺ release during fertilization in bovine eggs, and identify cell cycle regulatory mechanisms of [Ca²⁺]_i oscillations at the level of IP₃R-1.

calcium, fertilization, gamete biology, in vitro fertilization

² Correspondence: Rafael A. Fissore, Department of Veterinary and Animal Sciences, Paige Laboratory, University of Massachusetts, Amherst, MA 01003. FAX: 413 545 6326; rfissore{at}vasci.umass.edu

³ These authors contributed equally to this work

⁴ Current address: Department of Biology, The University of Pennsylvania, Philadelphia, PA 19104

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				S.-Y. Yoon and R. A Fissore Release of phospholipase C {zeta}and [Ca2+]i oscillation-inducing activity during mammalian fertilization Reproduction, November 1, 2007; 134(5): 695 - 704. [Abstract] [Full Text] [PDF]