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

Mirjan Thys1, Hans Nauwynck2, Leen Vandaele1, Jo Bijttebier1, Dominiek Maes1, Herman Favoreel2 and Ann Van Soom1 *1Dept. of Reproduction, Obstetrics and Herd Health, 2Dept. of Virology, Immunology and Parasitology, Faculty of Veterinary Medicine,Ghent University, Belgium* 

#### **1. Introduction**

The complex series of molecular interactions between male and female gametes required for successful fertilization has captured the interest of several research groups for many years (Benoff 1997). Even though substantial insights into these interactions have been elucidated in particular in human and mouse (Fusi *et al.* 1992; 1996*a*; 1996*b*; Almeida *et al.* 1995; Evans *et al.* 1995; Bronson & Fusi 1996), a clear identification of the underlying mechanisms and molecules implicated in bovine fertilization is still required. The involvement of numerous carbohydrates and glycoproteins in adhesion and binding events during several reproductive processes has been described in ruminants, ranging from roles in spermoviduct adhesion (Revah *et al.* 2000; Talevi & Gualtieri 2001; Sostaric *et al.* 2005; Gwathmey *et al.* 2006; Ignotz *et al.* 2007), sperm-oocyte interactions (Gougoulidis *et al.* 1999; Amari *et al.* 2001; Tanghe *et al.* 2004*a*; 2004*b*) to embryo implantation (Spencer *et al.* 2004).

A convenient way to study receptor-ligand interactions is to incubate sperm and/or oocytes with possible ligands in order to inhibit fertilization. Using this approach, Tanghe *et al.* (2004*b*) demonstrated that vitronectin – among other glycoproteins and carbohydrates – when present during bovine *in vitro* fertilization (IVF) inhibited sperm penetration.

Vitronectin (Vn) is a multifunctional 75 kDa glycoprotein - rather exclusively secreted by the liver into the plasma in a monomeric form - and abundantly stored in an essentially multimeric form in diverse extracellular matrices (Stockmann *et al.*1993; Gechtman *et al.* 1997; Francois *et al.* 1999). Like other adhesive proteins (e.g. fibronectin), Vn possesses a heparin binding site and interacts via its Arg-Gly-Asp (RGD) amino acid sequence with integrin receptors (mainly the αvβ3 integrin) at the cell surface (Bronson *et al.* 2000). Interaction of this glycoprotein with a wide range of macromolecules has been described allowing it to participate in several physiological processes, among which complementmediated cell lysis, cell surface proteolysis, cell adhesion, coagulation and fibrinolysis (Gibson *et al.* 1999; Bronson *et al.* 2000).

Vitronectin and Its Receptor (Integrin αvβ3) During Bovine Fertilization *In Vitro* 505

To reconfirm whether the Vn concentration (500 nM) applied in the study of Tanghe *et al.* (2004*b*) was the most appropriate, a preliminary experiment was conducted to assess the dose-response effect of Vn on sperm penetration after bovine IVF. *In vitro* matured COCs were randomly assigned to 6 different fertilization media (Tanghe *et al.* 2004*a*): fertilization medium supplemented with 0 nM, 10 nM, 100 nM, 250 nM, 500 nM and 1 µM Vn. The oocytes were co-incubated with sperm at a final concentration of 106 spermatozoa mL-1 for 20 h (39°C; 5% CO2). Prior to overnight fixation (2% paraformaldehyde – 2% glutaraldehyde in PBS) and staining with 10 µg mL-1 Hoechst 33342 for 10 min, the presumed zygotes were vortexed for 3 min to remove excess spermatozoa and/or cumulus cells. Zygotes were mounted in glycerol with 25 mg mL-1 DABCO and evaluated for sperm penetration using a

*In vitro* matured COCs were randomly assigned to 4 groups (3 replicates). Half of the oocytes (2 groups) were denuded by vortexing (cumulus denuded or CD) and the other half (2 groups) were kept cumulus-enclosed (CE). Both CD and CE oocytes were fertilized under control conditions (in standard fertilization medium) and in the presence of 500 nM Vn (Tanghe *et al.* 2004*b*). The oocytes were co-incubated with sperm at a concentration of 106 sp mL-1 for 20 h (39°C; 5% CO2). Each group contained 76 to 100 oocytes. Prior to fixation the presumed zygotes were vortexed to remove excess spermatozoa. After staining, the oocytes

*In vitro* matured COCs were denuded by vortexing and randomly assigned to two groups (4 replicates). The first group was fertilized under control conditions (in standard fertilization medium), the second group in the presence of 500 nM Vn. The oocytes were co-incubated with sperm at a concentration of 105 sp mL-1. This sperm concentration was previously determined in order to allow unambiguous counting of the number of spermatozoa bound to the ZP. Each group consisted of 77 to 100 oocytes. After 20 h of co-incubation, the oocytes were washed 3 times to remove loosely attached spermatozoa, and subsequently fixed and stained with Hoechst 33342. Per presumed zygote the number of spermatozoa bound to the

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

*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

lysophosphatidyl choline (LPC; L5004, Sigma-Aldrich, Bornem, Belgium).

**2.4 Dose-response effect of Vn on sperm penetration after bovine IVF** 

**2.5 Effect of Vn on sperm penetration of the cumulus oophorus** 

Leica DMR fluorescence microscope.

were evaluated for fertilization and polyspermy.

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

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

ZP was determined.

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 integrity and sperm motility was assessed.
