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Research Article |
Division of Animal Sciences,4
Comparative Medicine Center,5
and Departments of Biology6
and Microbiology and Immunology,7 University of Missouri-Columbia, Columbia, Missouri 65211
Monsanto Company,8 St. Louis, Missouri 63167
ABSTRACT
This study was designed to determine the role of osteopontin (SPP1) in in vitro fertilization (IVF) in swine. The initial objective was to evaluate the effect of various concentrations of SPP1 (0, 0.001, 0.01, 0.1 and 1 µg/ml) on spermatozoa and oocytes during IVF. The results demonstrate that SPP1 reduced the rate of polyspermy in a dose-dependent manner (P < 0.05). SPP1 also reduced both the number of sperm in oocytes as compared to the control and the number of spermatozoa bound to the zona pellucida (ZP) (P < 0.05). High doses of SPP1 (1 µg/ml) reduced penetration and male pronucleus formation as compared to the control (P < 0.05). Interestingly, compared to the control group, medium doses of SPP1 increased fertilization efficiency (42.6% and 44.6% vs. 31.6%; P < 0.05), representing a 41% improvement for 0.1 µg/ml SPP1). The ZP of 0.1 µg/ml SPP1-treated oocytes was more difficult to digest than control oocytes (P < 0.05). The percentage of acrosome-reacted spermato zoa bound to the ZP during IVF increased after 4 h of 1.0 µg/ml SPP1 treatment compared to 0 or 0.1 µg/ml SPP1. SPP1 did not have an effect on sperm motility, progressive motility, and sperm viability. To confirm that the reduction of polyspermy was specific to SPP1, a mixture of pregnancy-associated glycoproteins was included in the IVF protocol and shown to have no effect on polyspermy. Furthermore, Western blotting demonstrated that a 50-kDa SPP1 form was present in the oviducts on Days 0, 3, and 5 in pregnant and nonpregnant gilts, and the concentration of SPP1 on Day 0 was higher than on Days 3 and 5. The current study represents the first report to demonstrate that SPP1 plays an important role in the regulation of pig polyspermic fertilization; it decreases polyspermy and increases fertilization efficiency during IVF.
fertilization, in vitro fertilization, oocyte, osteopontin, oviduct, pig, polyspermy, sperm, spermatozoa
2 Correspondence: Randall S. Prather, Division of Animal Sciences, University of Missouri-Columbia, E125D ASRC, 920 East Campus Drive, Columbia, MO 65211. FAX: 573 884 7827; PratherR{at}Missouri.edu
3 Visiting scientist from Life Science and Biotechnology Research Center, Northeastern Agricultural University, Harbin, People's Republic of China.
1 Supported by the Monsanto Company and Food for the 21st Century.
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