**2.9 Localization of endogenous Vn on female and male bovine gametes**

With respect to the female bovine gamete, immature COCs, *in vitro* matured COCs, CD oocytes as well as ZP-free oocytes were sampled following the protocol of Tanghe *et al.* (2004*a*). Cumulus cells were removed mechanically by vortexing (8 min), and the ZP was dissolved by incubation of the CD oocytes in 0.1% protease in PBS for 5 to 15 min (at 37°C). Since Vn is an extracellular matrix protein, expression of Vn was also analyzed in cumulus monolayers that were grown *in vitro* for a week (as described by Vandaele et al. 2007). All female gamete samples were fixed with 4% paraformaldehyde (P6118, Sigma-Aldrich, Bornem, Belgium) in PBS for 1 h (4°C) and permeabilized with 0.5% Triton X-100 (Sigma-Aldrich, Bornem, Belgium) in PBS for 30 min at room temperature (RT). Subsequently, they were incubated with 10% goat serum (16210-064, Invitrogen, Merelbeke, Belgium) in polyvinyl pyrrolidone (PVP, 0.1% in PBS) solution for 30 min (37°C), with mouse monoclonal antibody A18 to Vn (Abcam, Cambridge, UK) (1/300) for 1 to 2 h (37°C) and with goat-anti-mouse FITC antibody (Molecular Probes, Leiden, The Netherlands) (1/100) for 1 h (37°C). To stain the nuclei, all oocyte types were treated with 2% Propidium Iodide (Molecular Probes, Leiden, The Netherlands) in PBS for 30 min. Between each treatment the samples were washed in PVP. They were mounted in a droplet of glycerol with 25 mg mL-1 DABCO and evaluated for the presence of Vn using a Leica DM/RBE laser scanning confocal fluorescence microscope (Leica Microsystems, Groot-Bijgaarden, Belgium).

To evaluate the expression of Vn on the male gamete, frozen-thawed semen was thawed in water of 37°C for 60 s and centrifuged on a discontinuous Percoll gradient. Next, the sample was split into 3 fractions. Fraction 1 was diluted to a concentration of 10x106 sp mL-1 (with medium consisting of a HEPES-buffered Tyrode balanced salt solution supplemented with 25 mM NaHCO3, 10 mM sodium lactate, 0.2 mM sodium pyruvate, 10 µg mL-1 gentamycin sulphate) prior to indirect immunofluorescence, and represented non-treated (NT) sperm. Fraction 2 and 3 were diluted to a concentration of 5x106 sp 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) and subsequently incubated for 30 min (39°C; 5% CO2) to induce capacitation. Then, fraction 2 (representing capacitated – CAP – sperm) was processed in the same way as fraction 1. Fraction 3 was supplemented with 100 µg mL-1 LPC and incubated for 15 min (39°C; 5% CO2) in order to induce the acrosome reaction (acrosome reacted – AR – sperm). All three sperm fractions were fixed with 1% paraformaldehyde (in PBS) for 30 min (at 4°C) and permeabilized with 0.5% Triton X-100 in

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

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).

With respect to the female bovine gamete, immature COCs, *in vitro* matured COCs, CD oocytes as well as ZP-free oocytes were sampled following the protocol of Tanghe *et al.* (2004*a*). Cumulus cells were removed mechanically by vortexing (8 min), and the ZP was dissolved by incubation of the CD oocytes in 0.1% protease in PBS for 5 to 15 min (at 37°C). Since Vn is an extracellular matrix protein, expression of Vn was also analyzed in cumulus monolayers that were grown *in vitro* for a week (as described by Vandaele et al. 2007). All female gamete samples were fixed with 4% paraformaldehyde (P6118, Sigma-Aldrich, Bornem, Belgium) in PBS for 1 h (4°C) and permeabilized with 0.5% Triton X-100 (Sigma-Aldrich, Bornem, Belgium) in PBS for 30 min at room temperature (RT). Subsequently, they were incubated with 10% goat serum (16210-064, Invitrogen, Merelbeke, Belgium) in polyvinyl pyrrolidone (PVP, 0.1% in PBS) solution for 30 min (37°C), with mouse monoclonal antibody A18 to Vn (Abcam, Cambridge, UK) (1/300) for 1 to 2 h (37°C) and with goat-anti-mouse FITC antibody (Molecular Probes, Leiden, The Netherlands) (1/100) for 1 h (37°C). To stain the nuclei, all oocyte types were treated with 2% Propidium Iodide (Molecular Probes, Leiden, The Netherlands) in PBS for 30 min. Between each treatment the samples were washed in PVP. They were mounted in a droplet of glycerol with 25 mg mL-1 DABCO and evaluated for the presence of Vn using a Leica DM/RBE laser scanning

**2.9 Localization of endogenous Vn on female and male bovine gametes** 

confocal fluorescence microscope (Leica Microsystems, Groot-Bijgaarden, Belgium).

To evaluate the expression of Vn on the male gamete, frozen-thawed semen was thawed in water of 37°C for 60 s and centrifuged on a discontinuous Percoll gradient. Next, the sample was split into 3 fractions. Fraction 1 was diluted to a concentration of 10x106 sp mL-1 (with medium consisting of a HEPES-buffered Tyrode balanced salt solution supplemented with 25 mM NaHCO3, 10 mM sodium lactate, 0.2 mM sodium pyruvate, 10 µg mL-1 gentamycin sulphate) prior to indirect immunofluorescence, and represented non-treated (NT) sperm. Fraction 2 and 3 were diluted to a concentration of 5x106 sp 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) and subsequently incubated for 30 min (39°C; 5% CO2) to induce capacitation. Then, fraction 2 (representing capacitated – CAP – sperm) was processed in the same way as fraction 1. Fraction 3 was supplemented with 100 µg mL-1 LPC and incubated for 15 min (39°C; 5% CO2) in order to induce the acrosome reaction (acrosome reacted – AR – sperm). All three sperm fractions were fixed with 1% paraformaldehyde (in PBS) for 30 min (at 4°C) and permeabilized with 0.5% Triton X-100 in

bound to the oolemma was evaluated.

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

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.

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

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

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 indirect immunofluorescence.

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 with ice-cold methanol during 15 min.

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 secondary antibody was included as negative control.

#### **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

Vitronectin and Its Receptor (Integrin αvβ3) During Bovine Fertilization *In Vitro* 509

penetration was not statistically significant when comparing the CE and the CD groups (P=0.106). Nevertheless, considering the small sample size (n=6), the mean difference of 30.2% in inhibition of penetration between cumulus-enclosed and cumulus-denuded groups

Fig. 1. Dose-response effect of vitronectin (Vn) on sperm penetration after bovine IVF. Data represent mean ± SEM. \*Values significantly different from control with 0 nM Vn (P < 0.05).

CD 0 249 15.0a ± 2.06 0.4a ± 0.43 15.4a ± 1.86 - 500 258 1.6b ± 1.60 0.4a ± 0.40 2.0b ± 1.44 87.0 CE 0 296 62.9a ± 7.23 7.7a ± 1.04 70.6a ± 7.71 - 500 273 26.8b ± 12.16 3.7b ± 0.87 30.5b ± 12.42 56.8 Table 1. Fertilization, polyspermy and penetration percentages of cumulus-denuded (CD) and cumulus-enclosed (CE) oocytes inseminated in standard fertilization medium and in fertilization medium supplemented with 500 nM of vitronectin (Vn). a,b Values with a different superscript in the same column within the CD and the CE groups differ

The number of spermatozoa bound to the ZP in the Vn supplemented group was slightly but significantly higher than that in the control group (50.1 ± 2.1 versus 42.9 ± 1.9

Vitronectin supplementation did not significantly influence the sperm-oolemma binding. However, a slight numerical decrease in sperm adherence (from 27.4 ± 1.9 to 23.0 ± 2.8

spermatozoa per oocyte) was observed in the presence of Vn (P>0.05).

Polyspermy (%)

Penetration (%)

Inhibition of penetration (%)

suggests a relevant effect of cumulus denudation.

(nM) No. Fertilization

(%)

Oocytes Vn

significantly (P<0.05).

**3.3 Effect of Vn on sperm-zona binding** 

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

spermatozoa per oocyte; P<0.05).

Tyrode balanced salt solution (control), modified Tyrode balanced salt solution supplemented with 200 nM Vn (100 nM Vn) and modified Tyrode balanced salt solution supplemented with 1 µM Vn (500 nM Vn). Three aliquots from each sperm fraction were incubated (39°C; 5% CO2), and at three different time points of incubation (1 h, 3 h and 6 h, respectively) one aliquot per fraction was evaluated for membrane integrity and total versus progressive sperm motility.

Membrane integrity was evaluated using a fluorescent SYBR14-Propidium Iodide (PI) staining technique (L7011; Molecular Probes, Leiden, The Netherlands). A stock solution of 1 mmol L-1 SYBR14 reagent was diluted (1:50) in HEPES-TALP, stored frozen at –20°C and thawed just before use. From each sperm aliquot, 100 l was used and 1 L SYBR14 was added. After 5 min of incubation (at 37°C), 1 L PI was added prior to another 5 min incubation (at 37°C). Per aliquot 200 spermatozoa were examined using a Leica DMR fluorescence microscope. Three populations of sperm cells were identified: living (membrane intact; stained green), dead (membrane damaged; stained red), and moribund (double stained; green-orange) spermatozoa. The moribund sperm cells were considered to be part of the dead sperm population.

Total and progressive motility were determined by means of computer-assisted sperm analysis (Hamilton-Thorne CEROS 12.3) (Tanghe *et al.* 2004*a*).

### **2.12 Statistical analyses**

Differences in fertilization and penetration percentages, and differences in number of Vnpositive cells were analyzed by means of binary logistic regression (including the effect of replicate). To evaluate the differences in mean number of spermatozoa bound to the ZP, the non parametric Kruskal Wallis test was applied, since the concerning variable was not normally distributed. Differences in mean number of sperm cells binding the oolemma were analyzed using ANOVA. Differences in membrane integrity and (total and progressive) sperm motility were evaluated using repeated measures analysis of variance. Hypothesis testing was performed using a significance level of 5% (2-sided test) and results were cited as mean ± S.E.M. (SPSS 15.0).
