IN-VITRO ASSOCIATION OF 6 OVIDUCTAL FLUID PROTEINS WITH THE BOVINE ZONA-PELLUCIDA

Oviductal fluid proteins have been shown to associate with gametes in several species. The objective of the present study was to identify bo vine oviductal fluid proteins that associate with the bovine zona pell ucida. Oviductal fluid was obtained daily from two dairy cows with nor mal oestrous cycles via indwelling oviductal cannulae. Fluid was colle cted from the ampullary and isthmic regions of the same oviduct. Ovidu ctal fluid samples were pooled by oviduct region and according to stag e of the oestrous cycle as determined by the concentration of serum pr ogesterone. Samples collected when serum progesterone concentrations w ere > 1.5 ng ml(- 1) were combined into luteal pools. Non-luteal pools consisted of oviductal fluid samples collected on days when serum pro gesterone concentrations were less than or equal to 1.5 ng ml(- 1). Ea ch oviductal fluid sample was assayed for protein concentration, and a n aliquot equivalent to 10 mg ml(- 1) was biotinylated using biotinami docaproate N-hydroxysuccinimide ester at a concentration of 1 mg ml(- 1). Cumulus-free, non-viable bovine oocytes were incubated in the biot inylated oviductal fluid samples for 3.5 h. Oocytes were washed, the z onae mechanically ruptured, solubilized, and subjected to one-dimensio nal SDS-PAGE. Separated proteins were transferred to nitrocellulose an d probed with avidin-horseradish peroxidase. Five biotinylated oviduct al fluid proteins were found to associate with the zona pellucida in a ll treatments. These proteins had apparent molecular masses of 80, 74, 60, 45, and 30 kDa. An additional protein, of molecular mass 95 kDa, was found associated with zonae from oocytes incubated in non-luteal f luid of both regions, but not from oocytes incubated in luteal fluid. This protein was shown to be bovine oestrus-associated protein by usin g a monospecific polyclonal antibody.