In vitro association of six 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 bovine oviductal fluid proteins that associate with the bovine zona pellucida. Oviductal fluid was obtained daily from two dairy cows with normal oestrous cycles via indwelling oviductal cannulae. Fluid was collected from the ampullary and isthmic regions of the same oviduct. Oviductal fluid samples were pooled by oviduct region and according to stage of the oestrous cycle as determined by the concentration of serum progesterone. Samples collected when serum progesterone concentrations were > 1.5 ng ml(- 1) were combined into luteal pools. Non-luteal pools consisted of oviductal fluid samples collected on days when serum progesterone concentrations were less than or equal to 1.5 ng ml(- 1). Each oviductal fluid sample was assayed for protein concentration, and an aliquot equivalent to 10 mg ml(- 1) was biotinylated using biotinamidocaproate N-hydroxysuccinimide ester at a concentration of 1 mg ml(- 1). Cumulus-free, non-viable bovine oocytes were incubated in the biotinylated oviductal fluid samples for 3.5 h. Oocytes were washed, the zonae mechanically ruptured, solubilized, and subjected to one-dimensional SDS-PAGE. Separated proteins were transferred to nitrocellulose and probed with avidin-horseradish peroxidase. Five biotinylated oviductal fluid proteins were found to associate with the zona pellucida in all 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 fluid of both regions, but not from oocytes incubated in luteal fluid. This protein was shown to be bovine oestrus-associated protein by using a monospecific polyclonal antibody.