289 EFFECT OF NEURAMINIDASE AND MANNOSIDASE IN THE BOVINE IN VITRO FERTILIZATION

Abstract

Sperm binding to the ZP is considered a carbohydrate- mediated process (Benoff 1997 Mol. Hum. Reprod. 3, 599–637, 827–837, 839–846). In bovine species, evidence that mannose could be a specific residue involved in bovine sperm-ZP binding has been reported (Amari et al. 2001 Mol. Reprod. Dev. 59, 221–226). Recently, Velasquez et al. (2006 Mol. Reprod. Dev. in press) showed that bovine sperm-ZP binding is mediated by α-2,3-linked sialic acid and that the use of a sialidase (neuraminidase) strongly decreased the sperm-ZP binding and fertilization. IVF with oocytes previously incubated with mannosidase or neuraminidase to compare the role played by mannose and sialic residues in the sperm-ZP binding and fertilization was performed. These specific glycosidase enzymes should remove mannose and sialic acid residues in the ZP. The in vitro-matured bovine oocytes were pre-incubated with the following: (i) mannosidase (1, 2, and 10 IU mL^-1), (ii) neuraminidase (1 IU mL^-1), and (iii) no enzyme (control group) in maturation medium (Edwards and Hansen 1996 Biol. Reprod. 55, 341–346) for 1 h. After the enzymatic treatment, the oocytes were rinsed 3 times in fresh fertilization medium and were inseminated in IVF-TALP with frozen bull semen processed by a Percoll gradient at a concentration of 1 × 10⁵ cells mL^-1. The oocytes were co-incubated with spermatozoa for 18–22 h at 38.5°C in a 5% CO₂ atmosphere. Then, the oocytes were washed in PBS twice and fixed in glutaraldehyde. The samples were stained with Hoechst 33342 solution and assessed under an epifluorescence microscope to determine the presence of sperm head in the cytoplasm or pronucleus formation. The data were presented as the mean penetration rate ± SEM, and the variables were analyzed by 1-way ANOVA (SPSS 11.0; SPSS Inc., Chicago, IL, USA). The results showed that neuraminidase treatment (1 IU mL^-1) strongly decreased the penetration percentages from 62% (control group) to 32% (neuraminidase group). When the mannosidase treatment was used, the percentage of penetration was similar to that of the control for both the 1 IU mL^-1 (66%) and the 2 IU mL^-1 (55%) groups. In the group in which 10 IU mL^-1 were used, the oocytes became degenerated, but a control experiment demonstrated that the degeneration was due to the enzyme excipient at that concentration. The neuraminadase results obtained are in accordance with the results obtained by Velasquez et al. (in press). However, mannosidase had no effect, although Amari et al. (2001) reported that mannose could be a specific residue involved in the bovine sperm-ZP binding. Tanghe et al. (2004 Mol. Reprod. Dev. 68, 492–499) showed that the p-aminophenyl derivative of d-mannose affects the sperm–oocyte fusion at the oolema level but not the number of spermatozoa bound to the zona pellucida. When they observed the effect of glycoproteins and carbohydrates on bovine oocyte penetration, sialic acid 10 mM caused 20% inhibition of penetration. According to our results and those previously reported (Tanghe et al. 2004; Velasquez et al. in press), we propose that the sialic acid is a main receptor at the bovine ZP level for bull spermatozoa.

This study was supported by the Spanish Ministry of Education and Science (AGL2006-03495 and BMC 2004-05568) and the Seneca Foundation of the Murcia Regional Government (03018/PI/05).