
TY  - JOUR
A1  - Grullón, L. 
A1  - Cánovas, S. 
A1  - Matás, C. 
A1  - Romar, R. 
A1  - Coy, P. 
T1  - 204 AMINO-REACTIVE CROSSLINKER BIS(SULFOSUCCINIMIDYL)SUBERATE INDUCES ZONA PELLUCIDA HARDENING AND REDUCES PENETRATION IN PIG <i>IN VITRO</i> FERTILIZATION
JF  - Reproduction, Fertility and Development
JO  - Reprod. Fertil. Dev.
Y1  - 2007
VL  - 20
IS  - 1
SP  - 181
EP  - 181
KW  - 
UR  - https://doi.org/10.1071/RDV20N1AB204 
N2  - Zona pellucida (ZP) hardening is considered to be the final step in the prevention of polyspermy during fertilization in mammals. However, unfertilized pig oviductal oocytes show a resistance of hours or days to pronase digestion (Broermann et al. 1989 J. Anim. Sci. 67, 1324&#8211;1329). We previously demonstrated that the amino-reactive crosslinker DSP is effective in inducing ZP hardening and improves the monospermy levels at pig IVF (Coy et al. 2007, Reprod. Dom. Anim., in press). In this study, a different chemical crosslinker, BS3 [bis(sulfosuccinimidyl) suberate], which also forms stable amide bonds among proteins, was used to evaluate its effect on ZP digestion time, penetration, male pronuclear formation and monospermy percentages, and the mean number of sperm per oocyte. In experiment 1, porcine in vitro-matured oocytes (n = 300) were incubated for 30 min at 0, 0.06, 0.30, or 0.60 mg mL&#8211;1 of BS3 in TALP medium and assessed for ZP digestion time in 0.5% pronase solution. The results (analyzed by ANOVA in all the experiments) showed a significant (P  &#8804; 0.01) dose-dependent increase in ZP hardening, from 69.0 s in the control to 426.3, 2028.3, and 2979.2 s, respectively, for the different BS3 concentrations. In experiment 2, oocytes (n = 473) were fertilized in vitro after no treatment or treatment with BS3 at 0.06, 0.30, and 0.60 mg mL&#8211;1. Fresh ejaculated spermatozoa were selected by Percoll® gradient 45:90. Oocytes were inseminated with 105 sperm mL&#8211;1, which resulted in high penetration and polyspermy percentages in the control group (83.1 and 89.9%, respectively). However, for the BS3-treated oocytes, significant differences compared with the control group (P  &#8804; 0.001) were observed in all 3 groups, showing penetration percentages of 22.2, 18.1, and 21.5%, respectively, and monospermy percentages of 100, 88.2, and 95.0%, respectively. The mean numbers of sperm per oocyte were 1.0, 1.1, and 1.05 for the BS3 groups, which were significantly different from 5.0 for the control group. In conclusion, BS3 can be used to induce ZP hardening in the pig and regulate polyspermy in IVF systems, although additional experiments are necessary to find the optimal concentration to improve the penetration percentages with high levels of monospermy. Granted by MEC and FEDER (AGL2006-03495). 
ER  -

