**2.3 Fixation and staining of oocytes**

After fertilization, the presumed zygotes were fixed overnight (2% paraformaldehyde – 2% glutaraldehyde in PBS) and stained with 10 µg mL-1 Hoechst 33342 (Molecular Probes, Leiden, The Netherlands) for 10 min. Zygotes were mounted in a droplet of glycerol with (25 mg mL-1) 1,4-diazabicyclo (2.2.2) octane (DABCO; Acros, Ghent, Belgium) and evaluated using a Leica DMR fluorescence microscope (Leica Microsystems, Groot-Bijgaarden, Belgium). The presence of two pronuclei was indicative for a successful fertilization of the oocyte. Penetration percentage was defined as the sum of the fertilization and polyspermy (more than two pronuclei) percentage per experimental group.

RGD sequences, present in Vn as well as in other extracellular matrix proteins (Fusi *et al.* 1992), are believed to take part in various integrin-mediated recognition systems involved in cell-to-cell and cell-to-matrix adhesion (Ruoslahti & Pierschbacher 1986). Since - in human integrins have been detected on both male and female gametes and spermatozoa express Vn on their surface following capacitation (Bronson & Fusi 1996), Vn may be involved in spermegg interaction. The present study was conducted to determine whether the inhibitory effect of exogenously supplemented Vn on bovine IVF appeared during a) the sperm penetration of the cumulus oophorus, b) the sperm-zona binding, c) the sperm-oolemma binding or d) the sperm-oocyte fusion. Subsequently, the expression of endogenous Vn and integrin subunit αv (subunit of the Vn receptor) on bovine oocytes and sperm cells was evaluated using indirect immunofluorescence, and the effect of exogenous Vn on sperm membrane

Oocytes were derived from bovine ovaries randomly collected at a local abattoir and prepared following the protocol of Tanghe *et al.* (2004*a*). Immature cumulus-oocyte complexes (COC) were aspirated from follicles with a diameter ranging from 2 to 8 mm. Only COCs displaying a multilayered compact cumulus and a homogeneous ooplasm were selected. Frozen-thawed bull semen from the same ejaculate was used for all inhibition experiments. Straws were thawed in a water bath (37°C) for 60 s. Subsequently, the semen was centrifuged on a discontinuous Percoll gradient (90% and 45%; Pharmacia, Uppsala,

Media and chemicals were analogous to those used by Tanghe *et al.* (2004*a*). Vitronectin from bovine plasma (V9881) used in all experiments was purchased from Sigma-Aldrich

The cumulus oophorus of the matured COCs was removed mechanically by vortexing (8 min). Subsequently, the cumulus-denuded oocytes were incubated in 0.1% protease (P5147, Sigma-Aldrich, Bornem, Belgium) in phosphate-buffered saline (PBS) for 5 to 15 min at 37°C to dissolve their zona pellucida (ZP). Afterwards the oocytes were washed and transferred to the incubator to allow recovery of the oolemma for at least 30 min (Tanghe *et al.* 2004*a*).

After fertilization, the presumed zygotes were fixed overnight (2% paraformaldehyde – 2% glutaraldehyde in PBS) and stained with 10 µg mL-1 Hoechst 33342 (Molecular Probes, Leiden, The Netherlands) for 10 min. Zygotes were mounted in a droplet of glycerol with (25 mg mL-1) 1,4-diazabicyclo (2.2.2) octane (DABCO; Acros, Ghent, Belgium) and evaluated using a Leica DMR fluorescence microscope (Leica Microsystems, Groot-Bijgaarden, Belgium). The presence of two pronuclei was indicative for a successful fertilization of the oocyte. Penetration percentage was defined as the sum of the fertilization and polyspermy

integrity and sperm motility was assessed.

Sweden) as described by Thys *et al.* (2009*a*).

**2.2 Removal of the zona pellucida** 

**2.3 Fixation and staining of oocytes** 

(more than two pronuclei) percentage per experimental group.

**2. Materials and methods** 

(Bornem, Belgium).

**2.1 Oocyte and semen preparation** 
