Zona pellucida in the process of fertilization and mammal embryonal development

In mammals, oocytes, fertilized eggs and pre-implantation embryos are surrounded by an acellular zona pellucida (zona pellucida – ZP). This structure has a fibro-spongy character but it undergoes constant modifications throughout its existence depending on many internal and external factors. ZP consists of glycoproteins marked as ZP1, ZP2, ZP3 and ZP4, the presence of which is species different. ZP1 and probably ZP4 molecules stabilize the fibrillary skeleton of the zona pellucida formed of ZP2 and ZP3 protein polymers which are ligands for specific spermatozoid receptors. The oligosaccharide chains of ZP3 are responsible for the primary attachment of the male gamete which induces the acrosomal reaction. ZP2 enhances this connection by secondary binding to an acrosome-free spermatozoid. Additionally, oviductal specific glycoprotein 1 which plays a role in interspecific oocyte-sperm interactions, appears around the postovulatory oocyte surrounded by ZP. In addition, this protein modifies the resistance of ZP to the action of proteases released as a result of the cortical reaction during polyspermia block. After fertilization, ZP not only protects the egg and then the embryo until implantation, but also has an embryotrophic effect. Understanding the molecular basics of the structure and properties of ZP can significantly improve animal fertility as well as reproductive rates.

Cortical reaction as an egg quality indicator in artificial reproduction of pikeperch, Sander lucioperca

The aim of this study was to investigate the process of the cortical reaction in eggs of pikeperch, Sander lucioperca (L.), as well as the application of microscopic assessment of this process in egg quality evaluation. The analysis was carried out with eggs obtained from 10 females by artificial reproduction, in which hormonal stimulation with hCG was applied. Subsequently, each sample of eggs (separately from each female fish) was analysed. The analysis included observation of the cortical reaction and the process of egg swelling, and determination of the effect of temperature (12, 14 and 16°C) and the presence of spermatozoa on the cortical reaction. The results indicate that the cortical reaction in pikeperch eggs is quite violent, resulting in visible deformation of eggs between 3 and 5 min after activation. No effect of temperature or the presence of spermatozoa on the cortical reaction was observed. A strong correlation was recorded for the percentage of egg deformations observed and embryo survival rate. The described method of determination of pikeperch egg quality (based on egg deformation rate between 3 and 5 min after activation) may be highly useful, both in scientific research (where high-quality eggs are required) and in hatchery practice.

183 CALRETICULIN, A 60-kDa PROTEIN, IS EXOCYTOSED AFTER CHEMICAL ACTIVATION OF ZONA PELLUCIDA-FREE PIG OOCYTES

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).

182 CHEMICAL ACTIVATION OF ZONA PELLUCIDA-FREE OOCYTES PROVOKES FULL CORTICAL REACTION: AN APPROACH TO STUDY CORTICAL GRANULE-DERIVED PROTEINS IN PIGS

The cortical reaction is a mechanism that prevents polyspermy by cortical granule content being released into the periviteline space, modifying the zona pellucida (ZP). Knowledge about specific cortical granule-derived proteins has progressed slowly because these organelles contain only picogram quantities of proteins. An efficient method for collecting cortical granule content would help in its study; chemical activation of ZP-free oocytes has been successfully used in the murine model (Muñoz-Gotera et al. 2001 Mol. Reprod. Dev. 60, 405–413). Calcium ionophore A23187 is an effective chemical stimulator for provoking the cortical reaction in ZP-intact pig oocytes. However, the commonly used protocol (50 μM for 5min) cannot be employed with ZP-free oocytes because the oolemma is damaged, oocyte lysed and medium contaminated with ooplasm content, which is necessary to reduce the time and ionophore concentration (Romar et al. 2011 Reprod. Fertil. Dev. 23, 221 abst). The objective of this study was to evaluate the efficiency of this activation protocol for provoking the cortical reaction in ZP-free oocytes by assessment with confocal and electron microscopy. 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 pronase. Then, the ZP-free oocytes were incubated with calcium ionophore A23187 (6.5 μM for 2min), transferred to an exudate medium and incubated at 38.5°C, 5% CO2 and saturated humidity for 30 min. Control ZP-free oocytes were incubated without being activated. After incubation, ionophore-treated (n = 10) and control oocytes (n = 18) were used to assess the presence of a cortical granule monolayer. An aliquot was fixed, permeabilized (0.1% Triton), incubated with peanut agglutinin lectin conjugated to fluorescein isothiocyanate (10 μg mL–1 for 30 min) and examined under a confocal microscope. Presence or absence of a cortical granule monolayer at the equator level was recorded. Another aliquot was fixed and processed for electron microscopy observation. The cortical granules in the whole oocytes were counted and results are presented as the mean ± standard error of the mean. No cell lysis was observed in control or activated ZP-free oocytes after treatment and incubation time. The confocal study showed that the activation protocol provokes a full cortical reaction in 100% of A23187-treated oocytes, given that no peanut agglutinin labeling was observed in the cortical area. Presence of a cortical granule monolayer under the oolemma was observed in 100% of control oocytes. Cortical granule release was confirmed by electron microscopy. Control oocytes had 5.90 ± 1.78 cortical granules per 5 μm of oolemma, whereas activated oocytes exhibited a significant reduction (P < 0.05) of up to 0.71 ± 0.20. In conclusion, the presented activation protocol by using ZP-free oocytes is a valid method for provoking a complete cortical reaction and could be employed in the future as an efficient method to collect cortical granule-derived proteins in pig oocytes. Supported by CONACYT (0105961/I0110/194/09), MEC and FEDER (AGL2009-12512-C02-01).