Association of oviductal fluid (ODF) proteins with the bovine zona pellucida.

The objective of this study was to determine using confocal microscopy whether bovine serum albumin (BSA), osteopontin (OPN) and lipocalin-type prostaglandin D synthase (L-PGDS) were associated with the bovine zona pellucida (ZP) of immature bovine oocytes and after in vitro matured oocytes were incubated in ODF. Ampullary (A) and isthmic (I) non luteal (NL) ODFs from two Holstein cows were used. To investigate if BSA, OPN, L-PGDS bind to the ZP alone or to cumulus cells and plasma membrane, mature cumulus oocyte complexes, denuded and ZP-free mature oocytes were incubated in 1) TALP medium (without BSA, with 0.1% PVA) (control), 2) TALP medium and 50% ANL-ODF, 3) TALP medium and 50% INL-ODF. After 2.5 h the eggs were washed and incubated for 1 h with primary antibodies against one of the three proteins, washed again and then incubated for 30 min with goat anti-rabbit FITC labeled secondary antibody. Immature denuded oocytes were incubated for 1 h in 1) TCM (control), 2) TCM and primary antibody to one of the three proteins and then for 30 min with FITC-labeled secondary antibody. In all cases, the ZP of mature denuded eggs probed with primary and secondary FITC labeled antibodies fluoresced when eggs were pre-exposed to either TALP medium alone or TALP medium and ANL/INL-ODF. In no case did the cumulus cells or the plasma membrane have an affinity for any of the three proteins. However, L-PGDS distribution differed from that of BSA and OPN in that it was detected in the perivitelline space. These studies suggest the presence and in vivo binding of these proteins to the eggs before they reach the oviduct. Because the ZP of immature denuded oocytes incubated in TCM and probed with primary and secondary FITC labeled antibodies also fluoresced like the mature eggs, we suggest that BSA, OPN and L-PGDS are first acquired by the ZP from follicular fluid during follicular development. (USDA grant 2004-34437-15106).