Mouse Zona Pellucida Mechanically Hardens in the Amount of Cortical Granule Exudates Dependent Manner.

The alteration of zona pellucida (ZP) following fertilization is due to the effects of the cortical granules (CG) exudates. Recently, it was demonstrated that the fertilization is also resulted in ZP mechanical hardening in pig, mouse and human. The objective of this study is to investigate the correlation of mechanical ZP hardening and the amount of CG release in mouse egg. The elastic modulus of ZP was measured using a Micro Tactile Sensor (MTS) which is an ultrasonic resonator based contact impedance sensor. MTS was fabricated using a thin glass needle connected to a piezo-electric ceramic material. The sample was pressed against the MTS at fixed speed under a computer controlled micromanipulation system. The amount of frequency change at the time of penetration was recorded using a computer program. Measurement of gelatin samples of known concentration (2,4,6%) was used to determine the basic characteristic of the MTS and to make a standard curve. Since the measurement was done with MTS indentation less than five microns and the ultrasound radiated from the tip of the MTS was very weak, the measurement was damageless and did not induce the cortical reaction. The methods of retrieval of mature oocytes (MII) and pronuclear (PN) stage of embryos were largely based on those described previously. MII oocytes immediately after collection (MII group), MII oocytes after vitrification/melting (MII vitri. group), MII oocytes with 8% ethanol activation for five minutes (MII ethanol group) and PN embryos at 16 hrs after mating (PN group) were used for measurement of ZP elasticity, respectively, as experimental model of intact (CG not released), partially activated (CG partially released), parthenogenetically activated (CG fully but artificially released) and naturally activated (CG fully and naturally released) eggs, respectively. The elastic modulus of MII, MII vitri., MII ethanol and PN group was 7.10 ± 4.26 kPa (n=26), 9.70 ± 5.13 kPa (n=22), 10.8 ±3.45 kPa (n=16) and 22.3 ± 7.33 kPa (n=26), respectively. ZP of PN group significantly hardened following fertilization (3.1 times) as previously reported. Ethanol activation resulted in ZP hardening significantly (1.5 times) but not so much as natural fertilization, indicating that the artificial CG release following ethanol activation was not enough to harden the ZP as natural fertilization. Vitrification/melting also caused ZP hardening but not significantly, indicating that the partial CG release slightly hardened the ZP. That is, it was indicated that the mechanical ZP hardening was dependent on the amount of CG released into perivitelline space.