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

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).

Vitronectin and Its Receptor (Integrin αvβ3) During Bovine Fertilization *In Vitro* 511

Fig. 2. Fluorescent images of frozen-thawed bovine semen labeled with mouse monoclonal to vitronectin and goat-anti-mouse FITC after fixation and permeabilization (*a*, *c* and *e*) and without prior fixation and permeabilization (*b*, *d* and *f*) (Original Magnification x600; Bar = 10 µm) (*a*-*b*) non-treated sperm; (*c*-*d*) capacitated sperm; (*e*-*f*) acrosome reacted sperm.

Fig. 3. Indirect immunofluorescent staining with mouse monoclonal to vitronectin and goat-

permeabilization (*a*) cumulus cell monolayer (Original Magnification x600; Bar = 25 µm), (*b*)

anti-mouse FITC combined with Propidium Iodide staining after fixation and

*in vitro* matured COC (Original Magnification x400; Bar = 50 µm).

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

A significant decrease in fertilization percentage (from 25.4% to 14.2%) and sperm penetration percentage (from 28.0% to 16.0%) was found when 500 nM Vn was supplemented during IVF (P<0.05). Compared to the ZP-free control group, the sperm penetration was inhibited with 42.9%.

#### **3.6 Localization of endogenous Vn on female and male bovine gametes**

After fixation and permeabilization, the percentage of Vn positive sperm cells was very high and consistent in all three sperm fractions (≥ 99.4% positive cells for NT, CAP and AR sperm). However, the intensity of fluorescence was 3 times higher in the AR group compared to the NT and CAP group (Table 2) and the predominant fluorescent pattern observed in the AR fraction (Fig.2*e* – fluorescence at the acrosomal region and midpiece) also differed from the one mainly observed in the NT and CAP groups (Fig.2*a* and 2*c* – fluorescence at the postacrosomal region and midpiece). When using fluorescence microscopy for the evaluation of unfixed spermatozoa, fluorescence was only observed in the AR fraction, displaying a green signal at the apical sperm head region (Fig.2*f)*. No fluorescence could be visualized in the NT and CAP sperm fractions (Fig.2*b* and *d*). Subsequently, flow cytometry (a far more sensitive technique) was applied, resulting in detection of fluorescent spermatozoa in all three sperm fractions: 5.8% in NT sperm, 14.4% in CAP sperm and 49.5% in AR sperm respectively. The intensity of fluorescence was much lower compared to the equivalent fixed and permeabilized sperm fractions (Table 2).


Table 2. Percentage of vitronectin positive cells, mean and median of relative fluorescence intensity per sperm fraction after flow cytometric evaluation of frozen-thawed bovine sperm cells labeled with mouse monoclonal to vitronectin and goat-anti-mouse FITC. NT: nontreated spermatozoa; CAP: capacitated spermatozoa; AR: acrosome-reacted spermatozoa.

With respect to the cumulus monolayer, the cytoplasm of approximately 100% of the cumulus cells stained positively for Vn, and fluorescent mesh forming structures were observed in the extracellular matrix (Fig.3*a*). In immature COCs a relatively small number of cumulus cells expressed Vn in their cytoplasm. After *in vitro* maturation, the number of Vn positive cumulus cells was considerably increased in the COCs (Fig.3*b*). Cumulus-denuded oocytes appeared to express Vn on the surface of the ZP, including fluorescent spurs penetrating the ZP (Fig.4). Protease treated (ZP-free) oocytes did not show membrane expression.

A significant decrease in fertilization percentage (from 25.4% to 14.2%) and sperm penetration percentage (from 28.0% to 16.0%) was found when 500 nM Vn was supplemented during IVF (P<0.05). Compared to the ZP-free control group, the sperm

After fixation and permeabilization, the percentage of Vn positive sperm cells was very high and consistent in all three sperm fractions (≥ 99.4% positive cells for NT, CAP and AR sperm). However, the intensity of fluorescence was 3 times higher in the AR group compared to the NT and CAP group (Table 2) and the predominant fluorescent pattern observed in the AR fraction (Fig.2*e* – fluorescence at the acrosomal region and midpiece) also differed from the one mainly observed in the NT and CAP groups (Fig.2*a* and 2*c* – fluorescence at the postacrosomal region and midpiece). When using fluorescence microscopy for the evaluation of unfixed spermatozoa, fluorescence was only observed in the AR fraction, displaying a green signal at the apical sperm head region (Fig.2*f)*. No fluorescence could be visualized in the NT and CAP sperm fractions (Fig.2*b* and *d*). Subsequently, flow cytometry (a far more sensitive technique) was applied, resulting in detection of fluorescent spermatozoa in all three sperm fractions: 5.8% in NT sperm, 14.4% in CAP sperm and 49.5% in AR sperm respectively. The intensity of fluorescence was much

lower compared to the equivalent fixed and permeabilized sperm fractions (Table 2).

Table 2. Percentage of vitronectin positive cells, mean and median of relative fluorescence intensity per sperm fraction after flow cytometric evaluation of frozen-thawed bovine sperm cells labeled with mouse monoclonal to vitronectin and goat-anti-mouse FITC. NT: nontreated spermatozoa; CAP: capacitated spermatozoa; AR: acrosome-reacted spermatozoa.

With respect to the cumulus monolayer, the cytoplasm of approximately 100% of the cumulus cells stained positively for Vn, and fluorescent mesh forming structures were observed in the extracellular matrix (Fig.3*a*). In immature COCs a relatively small number of cumulus cells expressed Vn in their cytoplasm. After *in vitro* maturation, the number of Vn positive cumulus cells was considerably increased in the COCs (Fig.3*b*). Cumulus-denuded oocytes appeared to express Vn on the surface of the ZP, including fluorescent spurs penetrating the ZP (Fig.4). Protease treated (ZP-free) oocytes did not show membrane

fraction No. Positive cells (%) Mean Median

NT 10 000 99.4 1586 1452 CAP 10 000 99.7 1458 1226 AR 10 000 99.8 4635 4728

NT 10 000 5.8 134 116 CAP 10 000 14.4 172 139 AR 10 000 49.5 284 214

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

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

penetration was inhibited with 42.9%.

Treatment Sperm

+ fixation + permeabilization


expression.

Fig. 2. Fluorescent images of frozen-thawed bovine semen labeled with mouse monoclonal to vitronectin and goat-anti-mouse FITC after fixation and permeabilization (*a*, *c* and *e*) and without prior fixation and permeabilization (*b*, *d* and *f*) (Original Magnification x600; Bar = 10 µm) (*a*-*b*) non-treated sperm; (*c*-*d*) capacitated sperm; (*e*-*f*) acrosome reacted sperm.

Fig. 3. Indirect immunofluorescent staining with mouse monoclonal to vitronectin and goatanti-mouse FITC combined with Propidium Iodide staining after fixation and permeabilization (*a*) cumulus cell monolayer (Original Magnification x600; Bar = 25 µm), (*b*) *in vitro* matured COC (Original Magnification x400; Bar = 50 µm).

Vitronectin and Its Receptor (Integrin αvβ3) During Bovine Fertilization *In Vitro* 513

Fig. 5. Fluorescent images of frozen-thawed bovine semen labeled with rabbit polyclonal antibody to integrin subunit αv and goat-anti-rabbit FITC combined with Hoechst 33342 staining after fixation (*a*-*c*-*e*) and without prior fixation (*b*-*d*-*f*) (Original Magnification x600; Bar = 10 µm) (*a*-*b*) non-treated sperm; (*c*-*d*) capacitated sperm; (*e*-*f*) acrosome reacted sperm.

However, incubation of the same *fixed* sperm fractions with heat-inactivated rabbit serum instead of the primary rabbit polyclonal antibody - appeared to induce a similar fluorescent

Integrin subunit αv was detected in *in vitro* matured CD bovine oocytes (Fig.6). The fluorescent pattern varied between the sampled cells: some oocytes (12.5%) expressed integrin subunit αv only at their oolemma (Fig.6*a*), whereas the greater part (87.5%) appeared to express the receptor molecule also at (the exterior side of) the ZP, including fluorescent spurs penetrating the ZP (Fig.6*b*). ZP-free bovine oocytes displayed green

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

to the inhibitory effect of Vn supplementation on sperm penetration during IVF.

Sperm membrane integrity was negatively affected in the presence of 500 nM Vn (P < 0.05), but was not altered by sperm incubation with 100 nM Vn (Fig.7). Total and progressive motility differed significantly (P < 0.05) between all sampled groups (control, 100 nM Vn and 500 nM Vn; Fig.8). However, especially the substantial twofold decrease in progressive motility in presence of 500 nM Vn should be considered as an important factor contributing

pattern in NT and CAP sperm cells (data not shown).

fluorescent spots at their surface.

Fig. 4. Confocal fluorescent image of cumulus-denuded ZP intact bovine oocytes labeled with mouse monoclonal to vitronectin and goat-anti-mouse FITC combined with Propidium Iodide staining after fixation and permeabilization (Original Magnification x200; Bar = 100 µm). fluorescent spur penetrating the ZP; ZP

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

After fixation and permeabilization, all sampled sperm cells were positive for integrin subunit αv, irrespective of their functional state (NT, CAP or AR sperm). All spermatozoa displayed the same fluorescent pattern (Fig.5*a*, 5*c* and 5*e*). When staining NT, CAP and AR sperm without prior fixation and permeabilization, no integrin expression was visually observed in the NT group (Fig.5*b*). The CAP sperm fraction showed faint fluorescence (Fig.5*d*), whereas a bright signal was detected in the AR sperm cells (Fig.5*f*). Flow cytometric evaluation of unfixed sperm cells confirmed these subjective observations (Table 3). The number of integrin subunit αv positive cells increased after heparin treatment, whereas the relative fluorescence intensity was substantially increased after artificial induction of the acrosome reaction.


Table 3. Percentage of integrin subunit αv positive cells, mean and median of relative fluorescence intensity per sperm fraction after flow cytometric evaluation of unfixed frozenthawed bovine sperm cells labeled with rabbit polyclonal to integrin subunit αv and goatanti-rabbit FITC. NT: non-treated spermatozoa; CAP: capacitated spermatozoa; AR: acrosome-reacted spermatozoa.

Fig. 4. Confocal fluorescent image of cumulus-denuded ZP intact bovine oocytes labeled with mouse monoclonal to vitronectin and goat-anti-mouse FITC combined with Propidium Iodide staining after fixation and permeabilization (Original Magnification x200; Bar = 100

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

After fixation and permeabilization, all sampled sperm cells were positive for integrin subunit αv, irrespective of their functional state (NT, CAP or AR sperm). All spermatozoa displayed the same fluorescent pattern (Fig.5*a*, 5*c* and 5*e*). When staining NT, CAP and AR sperm without prior fixation and permeabilization, no integrin expression was visually observed in the NT group (Fig.5*b*). The CAP sperm fraction showed faint fluorescence (Fig.5*d*), whereas a bright signal was detected in the AR sperm cells (Fig.5*f*). Flow cytometric evaluation of unfixed sperm cells confirmed these subjective observations (Table 3). The number of integrin subunit αv positive cells increased after heparin treatment, whereas the relative fluorescence intensity was substantially increased after artificial induction of the

Sperm fraction No. Positive cells (%) Mean Median NT 10 000 55.7 5105 4964 CAP 10 000 69.4 6373 6273 AR 10 000 69.9 8018 7808

Table 3. Percentage of integrin subunit αv positive cells, mean and median of relative fluorescence intensity per sperm fraction after flow cytometric evaluation of unfixed frozenthawed bovine sperm cells labeled with rabbit polyclonal to integrin subunit αv and goatanti-rabbit FITC. NT: non-treated spermatozoa; CAP: capacitated spermatozoa; AR:

µm). fluorescent spur penetrating the ZP; ZP

**gametes** 

acrosome reaction.

acrosome-reacted spermatozoa.

Fig. 5. Fluorescent images of frozen-thawed bovine semen labeled with rabbit polyclonal antibody to integrin subunit αv and goat-anti-rabbit FITC combined with Hoechst 33342 staining after fixation (*a*-*c*-*e*) and without prior fixation (*b*-*d*-*f*) (Original Magnification x600; Bar = 10 µm) (*a*-*b*) non-treated sperm; (*c*-*d*) capacitated sperm; (*e*-*f*) acrosome reacted sperm.

However, incubation of the same *fixed* sperm fractions with heat-inactivated rabbit serum instead of the primary rabbit polyclonal antibody - appeared to induce a similar fluorescent pattern in NT and CAP sperm cells (data not shown).

Integrin subunit αv was detected in *in vitro* matured CD bovine oocytes (Fig.6). The fluorescent pattern varied between the sampled cells: some oocytes (12.5%) expressed integrin subunit αv only at their oolemma (Fig.6*a*), whereas the greater part (87.5%) appeared to express the receptor molecule also at (the exterior side of) the ZP, including fluorescent spurs penetrating the ZP (Fig.6*b*). ZP-free bovine oocytes displayed green fluorescent spots at their surface.

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

Sperm membrane integrity was negatively affected in the presence of 500 nM Vn (P < 0.05), but was not altered by sperm incubation with 100 nM Vn (Fig.7). Total and progressive motility differed significantly (P < 0.05) between all sampled groups (control, 100 nM Vn and 500 nM Vn; Fig.8). However, especially the substantial twofold decrease in progressive motility in presence of 500 nM Vn should be considered as an important factor contributing to the inhibitory effect of Vn supplementation on sperm penetration during IVF.

Vitronectin and Its Receptor (Integrin αvβ3) During Bovine Fertilization *In Vitro* 515

Fig. 8. Effect of 100 nM and 500 nM vitronectin (Vn) on total motility (*a*) and progressive motility (*b*) of bovine frozen-thawed spermatozoa during incubation (evaluated by means of CASA). Data represent mean ± SD. \*Values significantly different from control with 0 nM Vn

binding requires a multitude of receptor-ligand interactions (Lyng & Shur 2007). In the present study, we have confirmed that high concentrations of the extracellular matrix glycoprotein Vn had a negative effect on bovine sperm-oocyte interaction (Tanghe *et al.* 2004*b*). Furthermore, the main inhibitory effect of 500 nM exogenous Vn was observed at the level of sperm-oolemma fusion, implicating that Vn might be one of the ligands involved in

Vitronectin strongly reduced sperm penetration in CD oocytes, but did only moderately so in CE oocytes. These results suggest that the cumulus oophorus is able to capture a substantial part of the supplemented Vn allowing still a fair sperm penetration rate. Vitronectin (serving as a cell-to-substrate adhesion molecule) is likewise known to interact with glycosaminoglycans and proteoglycans and is recognized by certain members of the integrin family (http://www.uniprot.org/uniprot/P04004). Integrin expression has been

A slightly augmented number of spermatozoa binding the ZP was noted in the presence of 500 nM Vn. This was an intriguing finding, since despite the increased sperm binding in the presence of Vn, oocyte penetration was substantially reduced. In mice, it has been demonstrated that sperm binding to the ZP is not sufficient to induce acrosomal exocytosis (Baibakov *et al.* 2007). The actual sperm passage through the pores of the ZP is believed to mechanically trigger the acrosome reaction: contact between motile sperm and the small ZPpores would generate sufficient shear force to bring forth a mechanosensory signal and acrosomal exocytosis. Binding to the ZP is suggested to slow down the forward progression of motile sperm and the forceful thrusting of the tail in order to transduce a mechanosensory signal mobilizing acrosomal Ca2+ stores and – consequently – to induce the acrosome reaction (Baibakov *et al.* 2007). The substantial twofold decrease in progressive sperm motility noted in presence of 500 nM Vn may therefore well be responsible for a defective sperm-ZP penetration. Furthermore, considerable head-to-head agglutination was observed when incubating bovine spermatozoa in the presence of Vn, especially at the high concentration of 500 nM (data not shown). Probably, the sperm is able to bind the ZP (assisted by the exogenous Vn connecting the integrin αv at the sperm cell surface to the integrin αv at the exterior side of the ZP), but is not capable of successful penetration of the

observed in bovine cumulus cells before (Sutovsky *et al.* 1995).

(P < 0.05).

sperm-egg recognition in cattle.

Fig. 6. Confocal fluorescent images of cumulus-denuded ZP intact bovine oocytes labeled with rabbit polyclonal antibody to integrin subunit αv and goat-anti-rabbit FITC after fixation (*a*) fluorescent signal confined to the oolemma, (*b*) fluorescent signal at the level of the oolemma, the ZP and exterior side of the ZP (Original Magnification x400; bar = 50 µm).

Fig. 7. Effect of 100 nM and 500 nM Vn on membrane integrity of bovine frozen-thawed spermatozoa during incubation (evaluated by means of SYBR14-PI staining). Data represent mean ± SD. \*Values significantly different from control with 0 nM Vn (P < 0.05).

#### **4. Discussion**

In cattle, cumulus cells are shed from the oocyte in the oviduct within a few hours (Lorton & First 1979) to 10 h after ovulation (Hyttel *et al.* 1988). However, it is not entirely clear whether the cumulus cells or the matrix are necessary for bovine fertilization *in vivo*: sperm cells probably need to penetrate the cumulus matrix first, before they can pass through the ZP and subsequently fuse with the oolemma (Van Soom *et al.* 2002). The molecular basis of each of these processes has not been resolved yet, but it is now accepted that sperm-egg

Fig. 6. Confocal fluorescent images of cumulus-denuded ZP intact bovine oocytes labeled with rabbit polyclonal antibody to integrin subunit αv and goat-anti-rabbit FITC after fixation (*a*) fluorescent signal confined to the oolemma, (*b*) fluorescent signal at the level of the oolemma, the ZP and exterior side of the ZP (Original Magnification x400; bar = 50 µm).

Fig. 7. Effect of 100 nM and 500 nM Vn on membrane integrity of bovine frozen-thawed spermatozoa during incubation (evaluated by means of SYBR14-PI staining). Data represent

In cattle, cumulus cells are shed from the oocyte in the oviduct within a few hours (Lorton & First 1979) to 10 h after ovulation (Hyttel *et al.* 1988). However, it is not entirely clear whether the cumulus cells or the matrix are necessary for bovine fertilization *in vivo*: sperm cells probably need to penetrate the cumulus matrix first, before they can pass through the ZP and subsequently fuse with the oolemma (Van Soom *et al.* 2002). The molecular basis of each of these processes has not been resolved yet, but it is now accepted that sperm-egg

mean ± SD. \*Values significantly different from control with 0 nM Vn (P < 0.05).

**4. Discussion** 

Fig. 8. Effect of 100 nM and 500 nM vitronectin (Vn) on total motility (*a*) and progressive motility (*b*) of bovine frozen-thawed spermatozoa during incubation (evaluated by means of CASA). Data represent mean ± SD. \*Values significantly different from control with 0 nM Vn (P < 0.05).

binding requires a multitude of receptor-ligand interactions (Lyng & Shur 2007). In the present study, we have confirmed that high concentrations of the extracellular matrix glycoprotein Vn had a negative effect on bovine sperm-oocyte interaction (Tanghe *et al.* 2004*b*). Furthermore, the main inhibitory effect of 500 nM exogenous Vn was observed at the level of sperm-oolemma fusion, implicating that Vn might be one of the ligands involved in sperm-egg recognition in cattle.

Vitronectin strongly reduced sperm penetration in CD oocytes, but did only moderately so in CE oocytes. These results suggest that the cumulus oophorus is able to capture a substantial part of the supplemented Vn allowing still a fair sperm penetration rate. Vitronectin (serving as a cell-to-substrate adhesion molecule) is likewise known to interact with glycosaminoglycans and proteoglycans and is recognized by certain members of the integrin family (http://www.uniprot.org/uniprot/P04004). Integrin expression has been observed in bovine cumulus cells before (Sutovsky *et al.* 1995).

A slightly augmented number of spermatozoa binding the ZP was noted in the presence of 500 nM Vn. This was an intriguing finding, since despite the increased sperm binding in the presence of Vn, oocyte penetration was substantially reduced. In mice, it has been demonstrated that sperm binding to the ZP is not sufficient to induce acrosomal exocytosis (Baibakov *et al.* 2007). The actual sperm passage through the pores of the ZP is believed to mechanically trigger the acrosome reaction: contact between motile sperm and the small ZPpores would generate sufficient shear force to bring forth a mechanosensory signal and acrosomal exocytosis. Binding to the ZP is suggested to slow down the forward progression of motile sperm and the forceful thrusting of the tail in order to transduce a mechanosensory signal mobilizing acrosomal Ca2+ stores and – consequently – to induce the acrosome reaction (Baibakov *et al.* 2007). The substantial twofold decrease in progressive sperm motility noted in presence of 500 nM Vn may therefore well be responsible for a defective sperm-ZP penetration. Furthermore, considerable head-to-head agglutination was observed when incubating bovine spermatozoa in the presence of Vn, especially at the high concentration of 500 nM (data not shown). Probably, the sperm is able to bind the ZP (assisted by the exogenous Vn connecting the integrin αv at the sperm cell surface to the integrin αv at the exterior side of the ZP), but is not capable of successful penetration of the

Vitronectin and Its Receptor (Integrin αvβ3) During Bovine Fertilization *In Vitro* 517

sperm penetration, whereas a negative influence was noted in the presence of high concentrations (500 nM – 1 µM). This inhibitory effect may well be – at least partially – due to compromised membrane integrity. Furthermore, the observed substantial twofold decrease in progressive motility in the presence of 500 nM Vn (compared to the control) should most likely be considered as an important factor contributing to the inhibitory effect of Vn supplementation on sperm penetration during IVF. Combining these results with the explicit head-to-head agglutination noted when incubating sperm with 500 nM of Vn, high concentrations of Vn should obviously be regarded as detrimental for successful

As in human (Fusi *et al.* 1994), Vn also appears to be an intrinsic protein of bovine sperm cells (Fig.9*a-b*). After fixation and permeabilization, practically all sampled spermatozoa did show fluorescence when applying indirect immunofluorescence and flow cytometry. The observed shift in fluorescent pattern after LPC treatment suggests that the Vn sequestered inside the sperm head is exposed at the sperm cell surface after acrosomal reaction (Fig.9*c*). With respect to the biological relevance of the moderate increase of Vn expression in unfixed sperm cells, we have to note that LPC treatment does not induce acrosomal reaction in all spermatozoa of the samples. Only penetration of the sperm cells through the ZP leads to nearly 100 % acrosome reaction. LPC treatment typically induces an increase in the percentage of acrosome reacted sperm ranging from about 26% to 38% (compared to a negative control group) in frozen-thawed bovine semen (O'Flaherty *et al*. 2005). With respect to the expression of the αv subunit of the Vn receptor, all fixed and permeabilized sperm cells seemed to display fluorescence at their apical surface, irrespective of their functional state (NT, CAP or AR). However, incubation of the same fixed sperm fractions with heatinactivated rabbit serum (instead of the primary rabbit polyclonal antibody to integrin subunit αv) appeared to induce a similar fluorescent pattern in NT and CAP sperm cells (data not shown). Some degree of non-specific binding of the secondary FITC-labeled antibody should therefore be acknowledged. Nevertheless, the finding that AR sperm cells did not show non-specific fluorescence (when using heat-inactivated rabbit serum) indicates that there is specific binding of the primary anti-integrin subunit αv antibody as well. In addition, the equivalent unfixed, not permeabilized, sperm fractions displayed a different pattern. Only the AR fraction was visually fluorescent, which also supports the specificity of the primary antibody to integrin subunit αv. The present findings, including a clear quantitative increase in fluorescence in unfixed AR spermatozoa measured by means of flow cytometry, are in accordance with the work of Fusi *et al.* (1996*a*) stating that α<sup>v</sup> expression was maximal following ionophore-exposure, used as inducer of the acrosome reaction. They assumed that αv may be located on the inner acrosomal membrane, which becomes accessible to anti-integrin subunit antibody both during the acromose reaction and

A fascinating observation was the abundant presence of endogenous Vn at the exterior side of the ZP including fluorescent spurs penetrating the ZP. Probably, the glycoprotein is bound to the αvβ3 integrin, of which the αv subunit was also detected at the level of the ZP in a similar pattern. These fluorescent signals are assumed to be remnants of molecules present at the cell surface of corona radiata cells which have been shown to have cellular projections traversing the ZP and terminating upon the oolemma (Tanghe *et al.* 2002). Integrin expression has been observed in bovine cumulus cells before (Sutovsky *et al.* 1995), and

following permeabilization of capacitated spermatozoa.

fertilization.

ZP. When supplementing low concentrations of Vn (100 nM), sperm penetration of COCs was enhanced, possibly through the increased sperm-ZP binding. Compared to 500 nM Vn, such low doses did not affect sperm membrane integrity and did not have the same impact on progressive sperm motility. Possibly, the forward progression of these ZP bound sperm cells was still sufficient for proper penetration.

A reversible dual binding function connecting both the male and female gamete – as suggested in human by Fusi *et al.* (1996*b*) – is even more plausible in the bovine species, since ruminant Vn apparently displays two integrin binding RGD sequences, in contrast to only one RGD site in human Vn (Suzuki *et al.* 1985; Mahawar & Joshi 2008). Furthermore, the presumed reversible dual binding function could additionally be exerted through spontaneous multimerization of several Vn molecules as described by Stockmann *et al.* (1993). The C-terminal half of the molecule comprises two hemopexin-like domains, able to interact with the acidic residues of the connecting segment of the same molecule, consequently allowing intramolecular and intermolecular linking to form Vn polymers (Royce & Steinmann 2002).

Vitronectin supplementation to the fertilization medium did not lead to a statistically significant inhibition of the sperm-oolemma binding in our experiment. Fusi *et al.* (1996*b*) previously suggested that Vn was well suited to play a significant role in human sperm-egg adhesion. These authors found a promotion of oolemmal adherence of spermatozoa, following addition of Vn to the medium over a certain concentration range. Supplementation of Vn enhanced oolemmal adherence of spermatozoa over a concentration range of 2.2 nM to 100 nM (Fusi *et al.* 1996*b*). Higher Vn concentrations – like in the present study – reduced the number of spermatozoa adhering to the egg (Fusi *et al.* 1996*b*), possibly due to Vn-mediated sperm aggregation within the culture dish. During the sperm incubation experiment, substantial head-to-head agglutination was observed after 4 h incubation in the presence of 100 nM and the agglutination was even more distinct when supplementing 500 nM Vn. Nevertheless, we could merely detect a slight – statistically insignificant – decrease in sperm-oolemma binding during bovine IVF when 500 nM Vn was supplemented to the fertilization medium. However, the physiological relevance of the number of sperm cells bound to a ZP-free oocyte is debatable (Talbot *et al.* 2003). The underlying assumption is that spermatozoa are bound to the oolemma by a mechanism that can result in sperm-egg fusion. This *in vitro* assay may – though – include a heterogeneous population of bound sperm cells, including non-physiologically bound sperm, besides the specific population of physiologically relevant sperm that are tethered or docked before fusion. Some of the sperm cells may well be bound via interactions that will not result into fusion. Acrosome-intact sperm are – for instance – known to be able to bind to ZP-free oocytes, but not to fuse. If the non-specifically bound sperm fraction outnumbers the specifically bound fraction, variations in the number of physiologically relevant bound sperm will not be measured (Talbot *et al.* 2003).

In the present experiment, Vn supplementation inhibited 42.9% of sperm-oolemma fusion. Accordingly, Fusi *et al.* (1996*b*) observed a decrease in the number of penetrating sperm cells in human oocytes in the presence of increasing Vn concentrations starting from 100 nM.

Furthermore, a dual (concentration-dependent) effect of exogenous Vn supplementation on bovine IVF was observed. Low Vn concentrations (10 nM – 100 nM) appeared to enhance

ZP. When supplementing low concentrations of Vn (100 nM), sperm penetration of COCs was enhanced, possibly through the increased sperm-ZP binding. Compared to 500 nM Vn, such low doses did not affect sperm membrane integrity and did not have the same impact on progressive sperm motility. Possibly, the forward progression of these ZP bound sperm

A reversible dual binding function connecting both the male and female gamete – as suggested in human by Fusi *et al.* (1996*b*) – is even more plausible in the bovine species, since ruminant Vn apparently displays two integrin binding RGD sequences, in contrast to only one RGD site in human Vn (Suzuki *et al.* 1985; Mahawar & Joshi 2008). Furthermore, the presumed reversible dual binding function could additionally be exerted through spontaneous multimerization of several Vn molecules as described by Stockmann *et al.* (1993). The C-terminal half of the molecule comprises two hemopexin-like domains, able to interact with the acidic residues of the connecting segment of the same molecule, consequently allowing intramolecular and intermolecular linking to form Vn polymers

Vitronectin supplementation to the fertilization medium did not lead to a statistically significant inhibition of the sperm-oolemma binding in our experiment. Fusi *et al.* (1996*b*) previously suggested that Vn was well suited to play a significant role in human sperm-egg adhesion. These authors found a promotion of oolemmal adherence of spermatozoa, following addition of Vn to the medium over a certain concentration range. Supplementation of Vn enhanced oolemmal adherence of spermatozoa over a concentration range of 2.2 nM to 100 nM (Fusi *et al.* 1996*b*). Higher Vn concentrations – like in the present study – reduced the number of spermatozoa adhering to the egg (Fusi *et al.* 1996*b*), possibly due to Vn-mediated sperm aggregation within the culture dish. During the sperm incubation experiment, substantial head-to-head agglutination was observed after 4 h incubation in the presence of 100 nM and the agglutination was even more distinct when supplementing 500 nM Vn. Nevertheless, we could merely detect a slight – statistically insignificant – decrease in sperm-oolemma binding during bovine IVF when 500 nM Vn was supplemented to the fertilization medium. However, the physiological relevance of the number of sperm cells bound to a ZP-free oocyte is debatable (Talbot *et al.* 2003). The underlying assumption is that spermatozoa are bound to the oolemma by a mechanism that can result in sperm-egg fusion. This *in vitro* assay may – though – include a heterogeneous population of bound sperm cells, including non-physiologically bound sperm, besides the specific population of physiologically relevant sperm that are tethered or docked before fusion. Some of the sperm cells may well be bound via interactions that will not result into fusion. Acrosome-intact sperm are – for instance – known to be able to bind to ZP-free oocytes, but not to fuse. If the non-specifically bound sperm fraction outnumbers the specifically bound fraction, variations in the number of physiologically relevant bound

In the present experiment, Vn supplementation inhibited 42.9% of sperm-oolemma fusion. Accordingly, Fusi *et al.* (1996*b*) observed a decrease in the number of penetrating sperm cells in human oocytes in the presence of increasing Vn concentrations starting from 100 nM.

Furthermore, a dual (concentration-dependent) effect of exogenous Vn supplementation on bovine IVF was observed. Low Vn concentrations (10 nM – 100 nM) appeared to enhance

cells was still sufficient for proper penetration.

sperm will not be measured (Talbot *et al.* 2003).

(Royce & Steinmann 2002).

sperm penetration, whereas a negative influence was noted in the presence of high concentrations (500 nM – 1 µM). This inhibitory effect may well be – at least partially – due to compromised membrane integrity. Furthermore, the observed substantial twofold decrease in progressive motility in the presence of 500 nM Vn (compared to the control) should most likely be considered as an important factor contributing to the inhibitory effect of Vn supplementation on sperm penetration during IVF. Combining these results with the explicit head-to-head agglutination noted when incubating sperm with 500 nM of Vn, high concentrations of Vn should obviously be regarded as detrimental for successful fertilization.

As in human (Fusi *et al.* 1994), Vn also appears to be an intrinsic protein of bovine sperm cells (Fig.9*a-b*). After fixation and permeabilization, practically all sampled spermatozoa did show fluorescence when applying indirect immunofluorescence and flow cytometry. The observed shift in fluorescent pattern after LPC treatment suggests that the Vn sequestered inside the sperm head is exposed at the sperm cell surface after acrosomal reaction (Fig.9*c*). With respect to the biological relevance of the moderate increase of Vn expression in unfixed sperm cells, we have to note that LPC treatment does not induce acrosomal reaction in all spermatozoa of the samples. Only penetration of the sperm cells through the ZP leads to nearly 100 % acrosome reaction. LPC treatment typically induces an increase in the percentage of acrosome reacted sperm ranging from about 26% to 38% (compared to a negative control group) in frozen-thawed bovine semen (O'Flaherty *et al*. 2005). With respect to the expression of the αv subunit of the Vn receptor, all fixed and permeabilized sperm cells seemed to display fluorescence at their apical surface, irrespective of their functional state (NT, CAP or AR). However, incubation of the same fixed sperm fractions with heatinactivated rabbit serum (instead of the primary rabbit polyclonal antibody to integrin subunit αv) appeared to induce a similar fluorescent pattern in NT and CAP sperm cells (data not shown). Some degree of non-specific binding of the secondary FITC-labeled antibody should therefore be acknowledged. Nevertheless, the finding that AR sperm cells did not show non-specific fluorescence (when using heat-inactivated rabbit serum) indicates that there is specific binding of the primary anti-integrin subunit αv antibody as well. In addition, the equivalent unfixed, not permeabilized, sperm fractions displayed a different pattern. Only the AR fraction was visually fluorescent, which also supports the specificity of the primary antibody to integrin subunit αv. The present findings, including a clear quantitative increase in fluorescence in unfixed AR spermatozoa measured by means of flow cytometry, are in accordance with the work of Fusi *et al.* (1996*a*) stating that α<sup>v</sup> expression was maximal following ionophore-exposure, used as inducer of the acrosome reaction. They assumed that αv may be located on the inner acrosomal membrane, which becomes accessible to anti-integrin subunit antibody both during the acromose reaction and following permeabilization of capacitated spermatozoa.

A fascinating observation was the abundant presence of endogenous Vn at the exterior side of the ZP including fluorescent spurs penetrating the ZP. Probably, the glycoprotein is bound to the αvβ3 integrin, of which the αv subunit was also detected at the level of the ZP in a similar pattern. These fluorescent signals are assumed to be remnants of molecules present at the cell surface of corona radiata cells which have been shown to have cellular projections traversing the ZP and terminating upon the oolemma (Tanghe *et al.* 2002). Integrin expression has been observed in bovine cumulus cells before (Sutovsky *et al.* 1995), and

Vitronectin and Its Receptor (Integrin αvβ3) During Bovine Fertilization *In Vitro* 519

indicated a reversible dual binding interaction between the fibronectin ligand and corresponding receptors on both (acrosome reacted) sperm cell and oolemma, initiating

Bearing in mind the present findings, the following hypothesis can be put forward concerning the interaction of vitronectin during bovine fertilization. Detection of endogenous Vn and integrin subunit αv at the exterior side of the ZP and integrin subunit α<sup>v</sup> on the sperm cell surface, combined with an increased sperm-ZP binding in presence of exogenous Vn, suggests at least some intervention of this glycoprotein in initial sperm-ZP interaction (Fig.9*e*). Since the αv subunit of the Vn receptor was identified on the oolemma, and spermatozoa appeared to express both integrin subunit αv and Vn after acrosomal reaction, this receptor-ligand mechanism may play a role in sperm-oocyte interaction (Fig.9*f*). The inhibitory effect of exogenously supplemented Vn on sperm penetration of ZPfree oocytes could then be explained by the competition between the exogenous Vn and the Vn liberated from the sperm cell following the acrosome reaction. Further research is required to distinguish the dual effect of low versus high concentrations of exogenous Vn

Alberts, B.; Bray, D; Lewis, J.; Raff, M.; Roberts, K. & Watson, J. D. (1994). Cell Junctions, Cell

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interactions: analysis of roles of egg integrins and the mouse sperm homologue of

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**5. References** 

1460-2407

Fig. 9. Hypothetical model concerning the function of endogenous vitronectin (Vn) during bovine IVF. (A) non-treated sperm cell, (B) capacitated sperm cell, (C) acrosome-reacted sperm cell, (D) capacitated spermatozoa traversing the cumulus oophorus, (E) spermatozoa binding the zona pellucida through endogenous oocyte Vn, (F) sperm-oocyte interaction through endogenous sperm Vn liberated after acrosome reaction.

these transmembrane receptors are assumed to be easily ripped out of the cell with bits of attached membrane, when internal anchorage with the cytoskeleton is disturbed (Alberts *et al.* 1994). This disconnection may have occurred through the mechanical force exerted on the cell by vortexing. Since integrin subunit αv was already observed to some extent in CAP spermatozoa (69.4% of that sperm fraction), it could be speculated that capacitated sperm cells reversibly bind to the endogenous Vn at the level of the ZP resulting in penetration of this acellular egg vestment. The finding that low concentrations of exogenously supplemented Vn improve sperm penetration during bovine IVF, might be attributed to an additional reversible dual adhesion function exerted by this molecule. A similar function could be exerted by the endogenous Vn located at the level of the extracellular cumulus matrix, in this case assisting the sperm cell in traversing the cumulus oophorus (Fig.9*d*). In order to elucidate the beneficial effect of low Vn concentrations on sperm penetration, further studies are required.

Since integrin subunit αv was present at the oolemma of ZP-free bovine oocytes and spermatozoa express Vn at their surface after acrosomal reaction, this receptor-ligand after might play a role in sperm-oocyte interaction. To confirm this hypothesis additional experiments investigating whether supplementation of low Vn concentrations to an IVF system with ZP-free oocytes and acrosome reacted sperm effectively inhibit sperm penetration are necessary. Previously, detection of fibronectin (another extracellular matrix glycoprotein) underneath the ZP together with observed expression of the α5 subunit of its corresponding receptor on oolemma and acrosome reacted sperm cell surface already indicated a reversible dual binding interaction between the fibronectin ligand and corresponding receptors on both (acrosome reacted) sperm cell and oolemma, initiating bovine sperm-egg binding (Thys *et al.* 2009*b*).

Bearing in mind the present findings, the following hypothesis can be put forward concerning the interaction of vitronectin during bovine fertilization. Detection of endogenous Vn and integrin subunit αv at the exterior side of the ZP and integrin subunit α<sup>v</sup> on the sperm cell surface, combined with an increased sperm-ZP binding in presence of exogenous Vn, suggests at least some intervention of this glycoprotein in initial sperm-ZP interaction (Fig.9*e*). Since the αv subunit of the Vn receptor was identified on the oolemma, and spermatozoa appeared to express both integrin subunit αv and Vn after acrosomal reaction, this receptor-ligand mechanism may play a role in sperm-oocyte interaction (Fig.9*f*). The inhibitory effect of exogenously supplemented Vn on sperm penetration of ZPfree oocytes could then be explained by the competition between the exogenous Vn and the Vn liberated from the sperm cell following the acrosome reaction. Further research is required to distinguish the dual effect of low versus high concentrations of exogenous Vn during bovine IVF.

#### **5. References**

518 A Bird's-Eye View of Veterinary Medicine

Fig. 9. Hypothetical model concerning the function of endogenous vitronectin (Vn) during bovine IVF. (A) non-treated sperm cell, (B) capacitated sperm cell, (C) acrosome-reacted sperm cell, (D) capacitated spermatozoa traversing the cumulus oophorus, (E) spermatozoa binding the zona pellucida through endogenous oocyte Vn, (F) sperm-oocyte interaction

these transmembrane receptors are assumed to be easily ripped out of the cell with bits of attached membrane, when internal anchorage with the cytoskeleton is disturbed (Alberts *et al.* 1994). This disconnection may have occurred through the mechanical force exerted on the cell by vortexing. Since integrin subunit αv was already observed to some extent in CAP spermatozoa (69.4% of that sperm fraction), it could be speculated that capacitated sperm cells reversibly bind to the endogenous Vn at the level of the ZP resulting in penetration of this acellular egg vestment. The finding that low concentrations of exogenously supplemented Vn improve sperm penetration during bovine IVF, might be attributed to an additional reversible dual adhesion function exerted by this molecule. A similar function could be exerted by the endogenous Vn located at the level of the extracellular cumulus matrix, in this case assisting the sperm cell in traversing the cumulus oophorus (Fig.9*d*). In order to elucidate the beneficial effect of low Vn concentrations on sperm penetration,

Since integrin subunit αv was present at the oolemma of ZP-free bovine oocytes and spermatozoa express Vn at their surface after acrosomal reaction, this receptor-ligand after might play a role in sperm-oocyte interaction. To confirm this hypothesis additional experiments investigating whether supplementation of low Vn concentrations to an IVF system with ZP-free oocytes and acrosome reacted sperm effectively inhibit sperm penetration are necessary. Previously, detection of fibronectin (another extracellular matrix glycoprotein) underneath the ZP together with observed expression of the α5 subunit of its corresponding receptor on oolemma and acrosome reacted sperm cell surface already

through endogenous sperm Vn liberated after acrosome reaction.

further studies are required.


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**25** 

 *France*

**Analysis of 3'UTR of** *Prnp* **Gene in Mammals: Possible Role of Target Sequences of miRNA** 

**for TSE Sensitivity in Bovidae and Cervidae** 

Transmissible spongiform encephalopathies (TSEs) or prion diseases are neurodegenerative diseases with an inexorably fatal outcome. They affect both human and other mammals, and are especially frequently found in Bovidae and Cervidae families (Aguzzi & Sigurdson, 2004). In the first family, sheep and goats can spontaneously develop the prion disease or scrapie (Grenn et al., 2008), whereas cattle only develop a disease, known as Bovine Spongiform Encephalopathy (BSE), after a contact with the infectious prion protein (Ducrot et al., 2008). In the second family, the chronic wasting disease (CWD) has been described in mule deer and elk. Besides these two Ruminant families, TSE was also retrieved in Carnivora, as Felidae (feline spongiform encephalopathy) and Mustelidae (transmissible mink encephalopathy) (Miller et al., 2008; Sigurdson & Miller, 2003). All these diseases are characterized by the accumulation of PrPSc, the abnormally folded isoform of the cellular

The cellular prion protein is a glycosylphosphatidylinositol anchored glycoprotein of 256 amino acids in sheep (Bovidae, subfamily Caprinae) and Cervidae. The N-ter of the protein is mainly unstructured while the C-terminal domain is globular. The C-ter domain is highly structured and is stabilized by an intramolecular disulfide bound. It contains three -helices and a short -sheet. TSE is a conformational disease, where change from PrPC to PrPSc involves an increase in -sheet content from 3% to 40%, and a decrease in -helical structure from 40% to 30% that modify irreversibly the protein folding (Cohen et

In a previous work, we showed that the ARQ/ARQ genotype is rather sensitive to the TSE in sheep and goat but protective in pig and rabbit, known as resistant species [Martin et al., 2009]. Otherwise, several studies point out that the expression level of the prion gene could modulate the onset of TSE, particularly in cattle (Sander et al., 2004-2006; Haase et al., 2007). The involvement of the 5' promoter region has been investigated in sheep (Saunders et al., 2009) and cattle (Xue et al., 2008) and it appears that polymorphism in this region could induce different responses to scrapie (Marcos-Carcavilla et al., 2008) and BSE (Brunelle et al., 2008). The analysis of a 23 pb insertion/deletion polymorphism in German and Swiss breed cattle revealed that the deletion is associated to a higher expression level, more frequently

prion protein (PrPC) in the brain (Prusiner 1998; Norrby 2011).

al., 1998; Smirnovas et al., 2009).

**1. Introduction** 

Daniel Petit, Jean-Michel Petit and François Gallet

*UGMA, UMR 1061 INRA/University of Limoges* 

