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Patterns of Intracellular Calcium Oscillations in Horse Oocytes Fertilized by Intracytoplasmic Sperm Injection: Possible Explanations for the Low Success of This Assisted Reproduction Technique in the Horse¹

Department of Veterinary and Animal Sciences,³ University of Massachusetts, Amherst, Massachusetts 01003 Department of Physiology and Pharmacology,⁴ College of Veterinary Medicine, Texas A&M University, College Station, Texas 77843

In all species studied, fertilization induces intracellular Ca²⁺ ([Ca²⁺]_i) oscillations required for oocyte activation and embryonic development. This species-specific pattern has not been studied in the equine, partly due to the difficulties linked to in vitro fertilization in this species. Therefore, the objective of this study was to use intracytoplasmic sperm injection (ICSI) to investigate fertilization-induced [Ca²⁺]_i signaling and, possibly, ascertain problems linked to the success of this technology in the horse. In vivo- and in vitro-matured mare oocytes were injected with a single motile stallion sperm. Few oocytes displayed [Ca²⁺]_i responses regardless of oocyte source and we hypothesized that this may result from insufficient release of the sperm-borne active molecule (sperm factor) into the oocyte. However, permeabilization of sperm membranes with Triton-X or by sonication did not alleviate the deficient [Ca²⁺]_i responses in mare oocytes. Thus, we hypothesized that a step downstream of release, possibly required for sperm factor function, is not appropriately accomplished in horse oocytes. To test this, ICSI-fertilized horse oocytes were fused to unfertilized mouse oocytes, which are known to respond with [Ca²⁺]_i oscillations to injection of stallion sperm, and [Ca²⁺]_i monitoring was performed. Such pairs consistently displayed [Ca²⁺]_i responses demonstrating that the sperm factor is appropriately released into the ooplasm of horse oocytes, but that these are unable to activate and/or provide the appropriate substrate that is required for the sperm factor delivered by ICSI to initiate oscillations. These findings may have implications to improve the success of ICSI in the equine and other livestock species.

¹ This work was funded by a Healey Endowment Grant at the University of Massachusetts to S.J.B. and R.A.F and was also supported by funds provided by a USDA/Hatch grant to R.A.F, a USDA grant (02-2078) to R.A.F., a Lalor foundation fellowship to M.K., and the Link Equine Research Endowment (Texas A&M).

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

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