Influence of Osteopontin in Bovine Uterine Tube Fluid on Sperm Binding and Fertilization in RCA-1 Lectin-treated Oocytes

This study was conducted to determine the effect of pre-exposure of oocytes to Ricinus communis (RCA-1) lectin and osteopontin (OPN) in uterine tube fluid (UTF) on in vitro sperm-egg binding and fertilization. In vitro-matured bovine oocytes were incubated (39°C, 5% CO₂ in air) for 2 h in the following treatments: (i) 500 μl of fertilization medium (FM); (ii) 250 μl of FM with 0.25 ml of non-luteal ampullary uterine tube fluid (NLAUTF); (iii) 250 μl of FM with 250 μl of NLAUTF and 4 μl of RCA-1 lectin; (iv) 250 μl of FM with 250 μl of NLAUTF, a rabbit polyclonal antibody (1:200) against purified bovine milk OPN, and RCA-1 lectin; (v) 500 μl of FM and RCA-1 lectin. Following incubation, oocytes were washed, placed in FM with 2 μg heparin, and incubated with 1 x 10⁵ frozen-thawed spermatozoa per 10 oocytes. Oocytes used to assess sperm binding were stained with Hoescht 33342, and the number of sperm bound per zona pellucida counted. The remaining oocytes were fixed in acid alcohol, stained with 1% acetate-orcein and observed to determine the presence of pronuclei. More sperm bound to the zona pellucida (mean ± SEM) when oocytes were incubated in treatment 3 (59.0 ± 5.5) than in treatments 2 (46.4 ± 5.6), 4 (18.1 ± 5.4), 5 (33.4 ± 5.6) or 1 (32.5 ± 5.6). More oocytes were fertilized when incubated in treatment 3 (91% ± 3.0) than in 2 (84% ± 3.0), 4 (40% ± 3.0), 5 (77% ± 3.0) or 1 (76% ± 3.0). As in previous studies, this study suggests that RCA-1 lectin enhances binding of UTF-derived OPN to bovine oocytes, resulting in increased sperm-egg binding and fertilization in vitro and a possible role in fertilization.