Ionic calcium levels in oviduct explant-conditioned media from an Australian marsupial, the brushtail possum (Trichosurus vulpecula) and its relevance to in vitro fertilization
Gametes from the brushtail possum (Trichosurus vulpecula), an Australian marsupial, require exposure to oviductal cells and/or their secretions before sperm binding and penetration of the zona pellucida can occur. Sperm-egg fusion, the next critical step in fertilization has not previously been reported in vitro. Here we describe the refinement of an oviduct epithelial cell (OEC) explant culture system using two different media to obtain in vitro sperm-egg fusion in the brushtail possum for the first time. Conditioned media from OEC explant cultures were supplemented with either 1% fetal calf serum (FCS) or 1mg/ml polyvinyl alcohol and used for co-culture of epididymal sperm and superovulated eggs. Under these conditions zona penetration rates varied from 0 to 46% and sperm-egg fusion from 0 to 20%. Analysis of explant conditioned media indicated that qualitative and quantitative differences between batches could account, at least partially, for the large variability in zona penetration rates. Conditioned media that contained approximately 1mM of ionic calcium were most effective for achieving sperm capacitation, zona binding, and penetration and sperm-egg fusion. The reorientation of the sperm head to T-shape, an indicator of capacitation in the brushtail possum, was closely linked with the concentration of calcium present in vitro.(Received March 3 2003)
(Accepted May 25 2003)
Key Words: Sperm capacitation; oviductal condition media; ionic calcium; zona penetration rate; in vitro fertilization.
c1 All correspondence to: Dr K.S.Sidhu, Senior Scientist, Marsupial CRC, Department of Biological Sciences, Macquarie University, NSW 2109 Australia. e-mail: email@example.com Fax: +61 2 9850 9254
p1 Department of Biological Sciences, Macquarie University, Australia 2109
p2 Landcare Research PO Box 69, New Zealand 8152
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