Characterization of calcium oscillation patterns in caprine oocytes induced by IVF or an activation technique used in nuclear transfer.
Routine activation of nuclear transfer (NT) eggs involves the application of a single intracellular calcium [Ca2+]i rise, stimulated by an electrical pulse, as opposed to [Ca2+]i oscillations, which is the natural mode of sperm-induced activation at fertilization in all mammalian species tested to date. It has yet to be shown that caprine oocytes exhibit an increase in calcium at fertilization in a manner similar to other mammals. The objective of the present study was to evaluate and characterize the ([Ca2+]i) oscillation patterns of caprine metaphase II (MII) oocytes during IVF and during an activation techniques used in nuclear transfer. Additionally, the effect of cytochalasin B (cyto B) in the NT process was evaluated for its impact on [Ca2+]i oscillations and subsequent embryo development. Mature in vitro and in vivo derived caprine oocytes were activated by 5 microM ionomycin, an electrical pulse(s), or IVF. The intracellular Ca2+ response was determined using the [Ca2+]i indicator Fura-2 dextran (Fura-2D). Ova treated with ionomycin or stimulated by an electrical pulse exhibited a single [Ca2+]i rise, whereas IVF-derived oocytes showed oscillations. IVF [Ca2+]i showed some variation, with 62% of in vitro matured oocytes exhibiting oscillations, whereas 8% of in vivo matured oocytes exhibited oscillations demonstrating a correlation between [Ca2+]i responses and maturation technique. Knowing the [Ca2+]i profile of activated eggs, one may be able to optimize the activation methodology used in a production nuclear transfer setting which could potentially improve development to term for NT embryos.
Jellerette
Teru
T
GTC Biotherapeutics Inc., 175 Crossing Boulevard, Framingham, MA 01702, USA.
Melican
David
D
Butler
Robin
R
Nims
Scott
S
Ziomek
Carol
C
Fissore
Rafael
R
Gavin
William
W
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D003571:Cytochalasin B / Q000494:pharmacology*; D004558:Electric Stimulation; D005260:Female; D005307:Fertilization in Vitro / Q000662:veterinary*; D006041:Goats / Q000502:physiology*; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D008677:Metaphase / Q000502:physiology; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000378:metabolism*; D013084:Sperm-Ovum Interactions
D007476:Ionophores; D003571:Cytochalasin B; D015759:Ionomycin; D002118:Calcium