Following gamete membrane fusion or artificial oocyte activation, cortical granules undergo exocytosis and the released content modifies the zona pellucida (ZP), preventing polyspermy. The specific cortical granule-derived proteins responsible for these post-fertilization events are not fully characterized. Calreticulin, a highly conserved ubiquitous protein of 60 kDa, was exocytosed from activated hamster eggs (Muñoz-Gotera et al. 2001 Mol. Reprod. Dev. 60, 405–413). Preliminary results from our laboratory have shown that calreticulin is located in the cortical area of pig oocytes (data not shown). This study was designed to test whether calreticulin is exocytosed after oocyte activation with calcium ionophore. Immature cumulus–oocyte complexes from Landrace × Large White gilts were in vitro matured for 44 h in an NCSU-37 medium. After maturation, the oocytes were stripped of cumulus cells and their ZP were removed with 0.5% pronase in Ca2+-free PBS. After washing, the ZP-free oocytes were incubated with calcium ionophore A23187 (6.5 μM) for 2min, transferred to a 100-μL droplet of exudate medium (Romar et al. 2011 Reprod. Fertil. Dev. 23, 221 abst) and incubated at 38.5°C, 5% CO2 and saturated humidity for 30 min. After incubation, the medium containing the oocyte exudate (n = 1000) was carefully aspirated and run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE). The gel was then electro transferred onto a polyvinylidene fluoride (PVDF) membrane, incubated with an anti-calreticulin rabbit polyclonal antibody (1:1000) and finally conjugated to horseradish peroxidase (1:20 000) for 1 h with a monoclonal anti-rabbit IgG. Membrane visualization was accomplished using the ECL plus method and Typhoon 9410. A control group was performed with exudate collected from non-activated ZP-free oocytes. To verify cortical reaction and calreticulin exocytosis, an aliquot of activated ZP-free oocytes (n = 18) were fixed (3.7% paraformaldehyde for 30 min), permeabilized (0.1% Triton X-100 for 10 min), incubated with anti-calreticulin antibody (1:10 for 1 h) and conjugated to tetramethyl rhodamine isothiocyanate (1:400 for 1 h) with an anti-rabbit IgG. Finally, samples were incubated with peanut agglutinin conjugated to fluorescein isothiocyanate (10 μg mL–1 for 30 min), mounted and examined under a confocal microscope. No statistical analysis was made because the observations were purely qualitative. A Western blot analysis showed an immunoreactive band of ∼60 kDa, consistent with the expected size of calreticulin, in the lane containing the exudate from activated oocytes. No band was observed in the lane with the exudate collected from non-activated oocytes. Observation under confocal microscopy showed no PNA or anti-calreticulin fluorescence in the cortical region, indicating that the activated pig oocytes displayed full cortical reaction and calreticulin exocytosis during incubation time. These results show that calreticulin protein is exocytosed after the chemical activation of ZP-free pig oocytes as well as the disappearance of the cortical granule monolayer. The possible role of calreticulin on preventing polyspermy should be further investigated.
Supported by MEC and FEDER (AGL2009-12512-C02-01) and CONACYT (0105961/I0110/194/09).
Zona pellucida in the process of fertilization and mammal embryonal development
