Analysis by two-dimensional electrophoresis of proteins secreted by sheep ovarian follicles: effects of the FecB gene, follicle size and atresia

in Reproduction

Proteins secreted by sheep ovarian follicles at different stages of maturation (small healthy, large healthy or large atretic) and originating from ewes with different ovulation rates (homozygous carriers or noncarriers of the FecB gene) were analysed by two-dimensional electrophoresis (PAGE) followed by computerized image analysis. Follicles were incubated intact for 1 h to assess steroidogenesis, and then incubated for 24 h in the presence of [35S]methionine. Secreted proteins were then resolved by iso-electric focusing followed by migration on 10% acrylamide slab gels and fluorography. Incorporation of label into proteins was similar irrespective of genetic type or health status of the follicles (4–6%). Small follicles incorporated significantly less (P < 0.05) label. Over 100 spots were detected on the fluorographs. The presence of the FecB gene induced qualitative and quantitative changes in the follicular protein patterns. Frequency of detection of spots 116 and 129 was significantly higher (P < 0.01) in Fec+Fec+ compared with FecBFecB follicles (80% versus 8%). In addition, amounts of proteins present in spots 2 and 12 were increased in FecBFecB follicles, while those in spots 9 and 21 were decreased. Size and atresia affected protein patterns only quantitatively. Increased amounts of proteins in spots 1, 10, 38, 44 and 113 were associated with atresia (P < 0.05). Follicle enlargement was associated with increased (P < 0.05) amounts of proteins in spots 5 and 6 and decreased amounts of proteins in spot 16. Amounts of three proteins (33, 40 and 58) were related positively to oestradiol production in vitro before labelling.

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