Oviductal fluid proteins associated with the bovine zona pellucida and the effect on in vitro sperm-egg binding, fertilization and embryo development.

Studies have demonstrated that oviductal fluid (ODF) proteins associate with eggs of numerous species including the bovine. In this study, the association of three ODF proteins, the bovine oestrus-associated protein, osteopontin (OPN), lipocalin-type prostaglandin D synthase (L-PGDS), with the bovine zona pellucida (ZP) was demonstrated by immunohistochemistry and western blot. The biological function of ODF derived egg-associated OPN and L-PGDS in sperm binding, fertilization and embryonic development was also explored. In vitro matured bovine oocytes were pre-incubated with ODF collected by cannula from cows in oestrus, or ODF with antibodies to OPN, L-PGDS and bovine serum albumin (BSA). Following incubation, oocytes were inseminated with 1 x 10(5) frozen-thawed spermatozoa, and they were evaluated for sperm binding, fertilization and embryonic development in vitro. Pre-treatment of ODF with antibodies to all of proteins reduced sperm binding to the ZP and fertilization in vitro. Cleavage rates were not significantly different among incubations, but rates of embryo development were significantly decreased. We conclude that antibodies to OPN, L-PGDS and BSA react with oocytes incubated with ODF and inhibit sperm binding, fertilization and embryonic development in vitro, suggesting a potential role of these proteins in these events.

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 < or = 1.5 ng ml-1. Each oviductal fluid sample was assayed fpr 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.