**2.7 Effect of Vn on sperm-oolemma binding**

The sperm pellet – obtained after Percoll centrifugation – was diluted with fertilization medium to a concentration of 5 x 105 sp mL-1 and incubated for 30 min (39°C; 5% CO2) to allow capacitation of the sperm cells. Subsequently, the acrosome reaction was induced by incubation of the sperm suspension for 15 min (39°C; 5% CO2) in 100 µg mL-1 lysophosphatidyl choline (LPC; L5004, Sigma-Aldrich, Bornem, Belgium).

*In vitro* matured ZP-free oocytes (3 replicates) were randomly assigned to two different media: standard fertilization medium or fertilization medium supplemented with 500 nM Vn. The female gametes were co-incubated with sperm at a final concentration of 2.5 x 105 sp

Vitronectin and Its Receptor (Integrin αvβ3) During Bovine Fertilization *In Vitro* 507

PBS for 30 min (at RT). Subsequently, they were incubated with 10% goat serum in PVP for 30 min (37°C), with mouse monoclonal antibody A18 to Vn (1/300) for 1 to 2 h (37°C) and with goat-anti-mouse FITC antibody (1/100) for 1 h (37°C). To stain the nuclei, all sperm fractions were treated with 10 µg mL-1 Hoechst 33342 for 10 min (RT). Between each treatment the sperm fractions were centrifuged (10 min, 200g) and re-suspended in PVP. They were mounted in glycerol with 25 mg mL-1 DABCO and evaluated for the presence of Vn using fluorescence microscopy (Olympus IX81 inverted fluorescence microscope and a Hamamatsu Orca B/W camera using Olympus Cell\*R software, Aartselaar, Belgium) and flow cytometry (FacsCanto II, BD, Belgium). Additionally, frozen-thawed semen originating from the same ejaculate was stained to evaluate Vn-expression (as described above) without

previous fixation and permeabilization. The latter samples were processed on ice.

mouse IgG1 antibody prior to the FITC-labeled secondary antibody treatment.

**gametes** 

indirect immunofluorescence.

with ice-cold methanol during 15 min.

secondary antibody was included as negative control.

The mouse monoclonal antibody A18 is claimed to be highly specific for Vn, since there is no evidence for cross reactivity with other connective tissue proteins (fibronectin, elastin, collagen, laminin). Nevertheless, two negative controls were additionally included: 1) a sample processed without primary antibody prior to the incubation with the secondary FITC-labeled goat-anti-mouse antibody, and 2) a sample incubated with an isotype-matched

**2.10 Localization of αv (subunit of the Vn integrin receptor) on female and male bovine** 

With respect to the female bovine gamete, *in vitro* matured CD oocytes were sampled (as described above) and fixed with 2% paraformaldehyde in PBS for 30 min (4°C) prior to

To assess the presence of αv on the male gamete, frozen-thawed semen originating from the same ejaculate was centrifuged on a discontinuous Percoll gradient, and the sperm pellet was diluted to a concentration of 10x106 sp mL-1. Subsequently, the sample was split into 3 fractions. Each fraction was processed as described before, resulting in a non-treated (NT), capacitated (CAP) and acrosome reacted (AR) sperm fraction. All sperm samples were fixed

This time, the primary antibody used was rabbit polyclonal antibody to integrin subunit α<sup>v</sup> (AB1930; Chemicon – Millipore, Belgium) (1/100), which was fluorescently labeled with goat anti-rabbit FITC antibody (Molecular Probes, Leiden, The Netherlands) (1/100). The primary antibody is guaranteed to have no-cross reactivity with α1, α2, α3, α4 or α6 integrin subunits. To evaluate the specificity of the rabbit polyclonal antibody to integrin subunit αv, a sample processed without primary antibody prior to the incubation with the FITC-labeled

**2.11 Effect of sperm incubation with Vn on membrane integrity and sperm motility** 

Frozen-thawed bull semen originating from the same ejaculate (3 replicates) was centrifuged on a discontinuous Percoll gradient and diluted to a concentration of 60x106 spermatozoa mL-1 (with medium containing Tyrode balanced salt solution supplemented with 25 mM NaHCO3, 10 mM sodium lactate, 0.2 mM sodium pyruvate, 10 µg mL-1 gentamycin sulphate, 6 mg mL-1 fatty acid-free BSA, and 20 µg mL-1 heparin). Subsequently, the sperm suspension was split into three fractions, which were diluted (1:1) respectively with the modified

mL-1 in 50 µl droplets of medium (10 oocytes per droplet) covered with paraffin oil (Tanghe *et al.* 2004*a*). The number of oocytes per experimental group ranged from 28 to 44. One hour after insemination the oocytes were washed 3 times to remove loosely attached spermatozoa, fixed and stained. Of each presumed zygote the number of spermatozoa bound to the oolemma was evaluated.

#### **2.8 Effect of Vn on sperm-oocyte fusion**

The experimental setup was identical to the one described in the previous experiment, except that the ZP-free oocytes were fixed 20 h after insemination. All presumed zygotes were evaluated for sperm-oolemma fusion (defined as the presence of two or more pronuclei).
