M. De Blasi

46 PLASMINOGEN ACTIVATOR ACTIVITY IN BUFFALO IN VITRO MATURED OOCYTES AFTER VITRIFICATION-WARMING

Plasminogen activators (PA) are proteolytic enzymes that convert plasminogen into plasmin. Plasmin is involved in physiological processes such as ovulation (Liu 2004 Front. Biosci. 9, 3356–3373), cumulus cell layer expansion (Liu et al. 1986 Endocrinology 119, 1578–1587), oocyte maturation (Dow et al. 2002 Biol. Reprod. 66, 1413–1421) and fertilization (Huarte et al. 1993 Dev. Biol. 157, 539–546). Although the interest is increasing, buffalo oocyte cryopreservation is still inefficient, especially in terms of blastocyst development after IVF. The aim of the present study was to evaluate whether exposure to cryoprotectants and the vitrification procedure affect plasminogen activator activity (PAA) in buffalo in vitro-matured oocytes. A total number of 300 cumulus-oocyte complexes over 5 replicates were selected and in vitro-matured. Cumulus-oocyte complexes were mechanically stripped of their cumulus cells by gentle pipetting, washed and divided into 3 groups (20 oocytes/group, over 5 replicates). The control group consisted of fresh in vitro-matured oocytes. In the vitrification group, denuded oocytes were first exposed to 10% ethylene glycol (EG) + 10% dimethyl sulfoxide (DMSO) for 3 min, then to 20% EG + 20% DMSO and 0.5 M sucrose, loaded on cryotops and plunged into liquid nitrogen within 25 s. Subsequently, oocytes were warmed in a 1.25 M sucrose solution for 1 min and then in decreasing concentrations of sucrose (0.625 M, 0.42 M and 0.31 M) for 30 s each. In order to test cryoprotectant effects, oocytes were simply exposed to the vitrification and warming solutions (toxicity group). Surviving oocytes were extracted by a fine needle, centrifuged at 4000 rpm for 10 min and the supernatant was mixed with the reaction solution: TRIS-HCl 0.1 M, homologous plasminogen, the chromogenic substrate for plasmin S-2251 and incubated at 37°C for 30 min. The PAA levels were measured by a spectrophotometer (405 nm) expressed as Abs/20 oocytes. The data were analysed by the Kruskal-Wallis nonparametric test. Low levels of PAA were detected in the denuded oocytes of the control, toxicity and vitrification groups. No significant differences in mean PAA values were observed among the 3 experimental groups (0.017 ± 0.001, 0.018 ± 0.002 and 0.017 ± 0.001 units, in the control, toxicity and vitrification groups, respectively). In conclusion, cryoprotectants and the vitrification procedure do not affect the proteolytic activity linked to plasmin in in vitro-matured buffalo oocytes. The results show that the vitrification/warming procedure does not exert an effect on in vitro-matured buffalo oocytes in terms of PAA generation, a parameter that plays an important role in fertilization and in vitro embryo development. Further studies are needed to identify factors affecting the efficiency of oocyte cryopreservation.

322 EFFECT OF PLASMIN ON ACROSOME REACTION OF BUFFALO (BUBALUS BUBALIS) SPERMATOZOA IN VITRO

Fertilization is a critical step of the in vitro embryo production (IVEP) technology in buffalo. It is known that proteolytic enzymes are involved in different steps of the fertilization process; among these, a critical role may be played by the plasminogen activator-plasmin system. It has been demonstrated that plasmin, the active enzyme of this system, induces acrosome reaction (AR) in bull spermatozoa (Taitzoglou IA et al. 2003 Andrologia 35, 112-116). The aim of this study was to investigate the effect of plasmin on the ability of buffalo sperm to undergo the AR. Frozen- thawed sperm from 4 buffalo bulls were treated by swim-up and incubated with 0.01 mM heparin for 4 h. At 0, 2, and 4 h, aliquots of spermatozoa were exposed for 10 min to 60 μg mL-1 of lysophosphatidylcholine (LPC), as positive control, and to 0.01 μg mL-1 of plasmin. This concentration was chosen after a preliminary dose-response trial. Another sample from each treatment was incubated with IVF medium (negative control). After 10 min, sperm motility was evaluated and sperm were fixed in 37% formaldehyde and stained with trypan blue-Giemsa for subsequent microscopic examination. The total number of sperm counted, over 3 replicates, was 1269 for the negative control, 1293 for LPC, and 1238 for plasmin, equally distributed among incubation times. Differences among groups were analyzed by chi-square test. After swim-up, acrosomal loss was observed only in 4% of the sperm. The addition of 0.01 μg mL-1 of plasmin for 10 min to buffalo spermatozoa at time 0 significantly (P < 0.01) enhanced (23%) AR compared with the control (7.8%), with the same efficiency of LPC (17.1%). After 2 h of incubation with heparin, both plasmin and LPC increased the AR compared to the control (24.4, 20.1, and 14.0%, respectively; P < 0.01). After 4 h, plasmin gave higher percentages of AR (27.2%) compared to both the control (21.0%; P < 0.05) and LPC (19.2%; P < 0.01). Another interesting result is the improved motility recorded with plasmin compared to both the control and LPC groups at 2 h of incubation (90, 75, and 75%, respectively; P < 0.05) and at 4 h of incubation (75, 60, and 60%, respectively; P < 0.05). Finally, no differences in sperm viability were observed between plasmin and the control, whereas a decreased viability was found when LPC was used at 0 h (96.2, 95.0, and 89.0%, respectively, for plasmin, control, and LPC; P < 0.05), at 2 h (85.0, 87.5, and 77.0%, respectively, for plasmin, control, and LPC; P < 0.01), and at 4 h (85.0, 93.3, and 81.1%, respectively, for plasmin, control, and LPC; P < 0.01). In conclusion, we found that addition of plasmin to capacitated sperm increases the percentage of acrosome-reacted spermatozoa and improves motility. Our results suggest that plasmin may play a role in events surrounding fertilization and suggest to evaluate in further studies whether the addition of plasmin during IVF improves the IVEP efficiency in buffalo.