• Korean Journal of Animal Reproduction
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• v.17 no.2
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• pp.93-101
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• 1993

These experiments were carried out to investigate whether zona hardening affect the efficiency of in vitro fertilization in mouse oocytes. The soluble properties for zona pellucida of oocytes matured in vivo, aged oocytes, and ovarian oocytes matured in vitro have been analyzed with proteolytic enzyme, 3mg/ml of $\alpha$-chymotrypsin. The mean solubility(t50) for the zona of unfertilized oocytes, oocytes not fertilized at the first inseminati and in vitro produced zygotes were 10.1, 20.3 and 32.3min., respectively. The t50 for zona lysis of fertilized oocytes was significantly difference than those observed for unfertilized oocytes and oocytes not fertilized at the first insemination(P<0.01). In addition, the t50 of zona in ovulated oocytes with and without cumulus cells incubated for 0, 3, 6, 9 and 12 hr in vitro, t50 were 13.9, 11.1, 20.7 and 28.0min., and 22.3, 21.0, 30.0 and 33.5min., respectively. In these experiments, the zona pellucida showed a gradual increase in resistance to dissolution by $\alpha$-chyjotrypsin with in vitro aging for more than 6 hrs. This effect was greater in cumulus-free as compared to cumulus-intact oocytes. Finally, in cumulus-intact and cumulus-free ovarian oocytes matured for 0, 5, 10 and 15 hr in vitro the t50 of zona pellucida were 3.0, 10.6, 18.4 and 24.5 min., and 3.0, 14.0, 26.2 and 32.0 min., respectively. Clear differences in solubility between the zona pellucida of oocytes matured in vivo and in vitro. This data were found suggest that under in vitro conditions there is a gradual change in the soluble properties of the zona pellucida, particularly in the absence of the cumulus cells.

• Clinical and Experimental Reproductive Medicine
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• v.29 no.3
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• pp.179-186
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• 2002

Objective: Lectins are cell-agglutinating and sugar specific proteins or glycoproteins of non-immune origin that precipitate glycoconjugates having saccharides of appropriate complementarity. Because of these properties, plant lectins have been used to help characterize the carbohydrate moieties of glycoproteins in the zona pellucida (ZP) of several mammalian species including pigs. Treatment of oocytes with various lectins blocks sperm binding to the ZP in various mammalian species. This study was undertaken to examine the distribution of sugar residues in the ZP of pig oocytes matured in vitro and the ability of spermatozoa to bind to ZP and in vitro penetration in oocytes treated with fluorescein isothiocyanate (FITC)-labelled lectins. Materials and Methods: The lectins of Banderiaea simplicifolia (BS-II, bind to $\beta$-D-N-acetylglucosamine), Canavalin ensiformis (Con A, bind to $\alpha$-D-Mannose), Lens culinaris (LCA, bind to a-D-Mannose), Ricinus communis (RCA-I, bind to $\beta$-D-Galactose) and Ulex europaeus (UEA-I, bind to $\alpha$-L-Fucose) were examined for spermatozoa penetration, binding capacity to ZP and distribution of lectins. Results: The penetration rates were significantry (p<0.05) higher in control oocytes (63%) than those treated with all lectins, but penetration rates ($40{\sim}49%$) were simililar in group treated with lectins. The incidence of monospermy was similar in oocytes untreated and UEA-I, but it was higher in oocytes treated with BS-II, Con A, RCA-I and LCA. The porcine oocytes cultured for 48 h in TC-199 medium were freed from cumulus cells and treated for 30 min with fluorescein isothiocyanate-labelled lectins. When examined under fluorescein illumination, higher (p<0.001) proportions of oocytes showed fluorescein of zona pellucida after treatment with Con A (93%), LCA (93%) and RCA-I (100%) than BS-II (37%) and UEA-I (50%). All of the oocytes treated with RCA-I exhibited strong fluorescein in the outer region of the zona pellucida while those treated with LCA exhibited strong fluorescein throughout the zona pellucida. BS-II bounded mainly to the outer region and UEA-I bounded mainly to the inner region of the zona pellucida, with either strong or weak fluorescein. At 120 min after insemination in vitro, fewer spermatozoa were bound to the zona pellucida of the oocytes treated with BS-II, Con-A and RCA-I. Of the lectins, Con A most inhibited sperm binding. Conclusions: These results suggest that $\beta$-D-Galactose residues in the porcine zona pellucida may act as primary sperm receptors and inducers of the sperm acrosome reaction and these sugar residues may be involved in the block to polyspermy.

• Korean Journal of Animal Reproduction
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• v.15 no.2
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• pp.103-107
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• 1991

This study was carried out to investigate the survival rate in vitro culture after frozen-thawed to used DMSO(dimethyl sulfoxide), glycerol and ethylene glycol of cryoprotective agents at the zona pellucida removed and encased into alien bisected embryo of the mouse early embryos. The results obtained from this study were as follows : 1. The survival rate of in vitro culture after frozen-thawed to used cryoprotective agents of three kinds at the zona pellucida removed bisected morula was 46.6%, 35.8% and 27.3%, total or mean were 36.6%, respectively. 2. The survival rate of in vitro culture after frozen-thawed to used cryoprotective agents of three kinds at the encased into alien bisected morula was 70.6%, 65.3% and 66.4%, total or mean were 67.4%, respectively. 3. The survival rate of in vitro culture after frozen-thawed to used cryoprotective agents of three kinds at the zona pellucida removed bisected blastocysts was 50.4%, 36.7% and 30.4%, total of mean were 39.2%, respectively. 4. The survival rate of in vitro culture after frozen-thawed to used cryoprotective agents of three kinds at the encased into alien bisected blastocysts was 71.1%, 66.7% and 63.9%, total or mean were 67.2%, respectively.

• Korean Journal of Animal Reproduction
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• v.20 no.3
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• pp.233-238
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• 1996

This study was conducted to investigate the effects of cryoprotective agents and thawing temperature on the survival rate of the bisected embryos of mouse. The results of this study were summairzed as follows: 1. In in vitro culture after frozen with DMSO, glycerol, ethylene glycol or propanediol and thawed, the rates of normally developed bisected morula which was denuded were 31.7, 39.1, 28.0 and 23.1%, respectively. 2. The survival rates of bisected morula encased into the zona pellucida in in vitro culture after frozen with DMSO, glycerol, ethylene glycol or propanediol and thawed were 72.4, 68.7, 64.0 and 59.5%, respectively. 3. The survival rates of bisected denuded blastura in in vitro culture after frozen with DMSO, glycerol, ethylene glycol or propanediol and thawed were 48.3, 44.8, 32.1 and 28.6%, respectively. 4. The survival rates of bisected blasturae encased into the zona pellucida in in vitro culture after frozen with DMSO, glycerol, ethylene glycol or propanediol and thawed were 73.6, 67.4, 53.0 and 49.1%, respectively. 5. In in vitro culture after frozen with DMSO, glycerol, ethylene glycol or propanediol and thawed at room temperature, the rates of the normally developed bisected morula which was encased into the zona pellucida were 67.1, 62.3, 57.7 and 53.0%, respectively, and those of the bisected blasturae encased into the zona pellucida were 70.8, 65.4, 56.6 and 52.1%, respectively. 6. The survival rates of bisected morula which was encased into the zona pellucida in in vitro culture after forzen with DMSO, glycerol, ethylene glycerol or propanediol and thawed at 37$^{\circ}C$ were 74.3, 71.3, 63.9 and 57.4%, respectively, and those of bisected blastura encased into zona pellucida were 76.0, 69.1, 61.1 and 56.1%, respectively.

• Korean Journal of Animal Reproduction
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• v.15 no.2
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• pp.97-101
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• 1991

This study was carried out to investigate the survival rate of in vitro culture after frozenthawed, to used DMSO(dimethyl sulfoxide), glycerol and ethylene glycol of cryorpotective agents at the zona pellucida removed and intact on the morulae and blastocysts. The results obtained from this study were as follows : 1. The survival rate of in vitro culture after frozen-thawed to used cryoprotective agents of three kinds at the morulae was 86.0%, 87.1% and 83.3%, total or mean were 85.5%, respectively. 2. The survival rate of in vitro culture after frozen-thawed to used cryoprotective agents of three kinds at the zona pellucida removed morulae was 53.2%, 42.3% and 37.5%, total or mean were 44.3%, respectively. 3. The survival rate of in vitro cultrue after frozen-thawed to used cryoprotective agents of three kinds at the blastocysts was 89.4%, 86.2%, total or mean were 86.7%, respectively. 4. The survival rate of in vitro culture after frozen-thawed to used cryoprotective agents of three kinds at the zona pellucida removed blastocysts was 55.8%, 51.6% and 40.6%, total or mean were 49.3%, respectively.

• International Journal of Oral Biology
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• v.32 no.3
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• pp.109-112
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• 2007

Somatic cells nuclear transfer (SCNT) is a useful tool in studies of developmental biology and animal cloning. However, SCNT experiments only are allowed to skilled technical experts. In this experiment, laser-assisted zona pellucida piercing tool (LASER) was applied in murine SCNT. LASER minimized the use of piezo-driven micromanipulator (PIEZO), reducing chances of problems caused by PIEZO pulses. LASER reduced time that took to pierce zona pellucida in removal of nucleus from oocyte and somatic cell injection, which might have taken longer time with PIEZO. Time and difficulties that took researcher of equivalent skilled for their experiments were decreased with LASER, and this might affect the improvement of embryonic development. (LASER, 6.2% versus PIEZO, 2.9%; P<0.05). Thus, these data support that the use of LASER can be used for zona pellucida piercing in murine SCNT program as an alternative to PIEZO.

• Korean Journal of Animal Reproduction
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• v.18 no.1
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• pp.31-37
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• 1994

This study carried out to investigate the effects of cryoprotectants in the medium on the survival rate of rapidly frozen porcine bisected demi-embryos. The porcine bisected demi-embryos following dehydration by cryoprotectants containing sucrose were directly plunged into liquid nitrogen and thawed in 3$0^{\circ}C$ water bath. Survival rate was defined as development rate on in vitro culture or FDA-test. The results are summarized as follows : 1. The survival rates of without-zona pellucida embryos and 2 blastomeres porcine embryos were 10.0 and 7.1%, respectively. The rate of unfrozen blastomeres (20.00%) was significantly higher than that of non-frozen oocytes. 2. The survival rates of with and without-zona pellucida of bisected porcine embryos by micromanipulator were 20.0 and 14.3%, respectively. 3. The developmental rates of with and without-zona pellucida of bisected poricine embryos by micromanipulator were 13.3 and 7.1%, respectively.

• Clinical and Experimental Reproductive Medicine
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• v.24 no.2
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• pp.187-192
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• 1997

The survival rate and motility recovered after cryopreservation of testicular spermatozoa in testicular sperm extraction (TESE)-ICSI program is low. The purpose of this study was to assess the availability and efficiency of mouse empty zona pellucida in cryopreserving human TESE spermatozoa. Mouse empty zonae pellucidae were obtained by extraction of cytoplasm with or without cytochalasin B treatment. Motile sperm from proven-fertile donor and two azoospermic patients after TESE were individually inserted into empty zona pellucida and cryopreserved. Two to five days after cyropreservation, the frozen sperm were thawed and the rates of recovery and motility were observed. The ooplasmic extraction rates of control (N=80) and cytochalasin B treated oocytes (N=80) were 94.0% and 96.2%, respectively (p>0.05). The post-thaw recovery rates of spermatozoa and rates of motility recovery of ejaculate (N=70) and testicular (N=70) sperm were 97.1%, 97.1% and 95.7%, 94.3%, respectively (p>0.05). The results of this study showed that the mouse zone pellucida is useful for cryostorage of single testicular spermatozoa.

• Clinical and Experimental Reproductive Medicine
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• v.25 no.2
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• pp.123-128
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• 1998

Implantation rates remain low following human in vitro fertilization (IVF). Suboptimal culture conditions may limit the ability of embryos to hatch as blastocysts, and artificial opening of the zona pellucida has been proposed as a means to promote subsequent hatching (assisted hatching). In this study, assisted hatching (AH) by zona drilling using acidic Tyrode's solution was performed in 320 patients, due to their age of more than 38 years (group A), the thick zona pellucida (group Z; $ZP\geq0.18{\mu}m$), and failures in implantation more than 3 times in previous IVF-ET trial (group P). This study was designed firstly, to study the effects of AH on the outcomes of IVF-ET according to the indications and secondly, to verify the appropriate application of AH. The results were as follows; 1. There was no difference in pregnancy rate between AH group (26.6%) and non-AH group (26.5%). 2. Assisted hatching (AH) showed significantly higher pregnancy rate of the patients with thick zona pellucid a than those of the patients with age factor and with the history of repeated implantation failure. But in the patients with age factor only, AH resulted in higher pregnancy rate. 3. Interestingly, the patients with complex factors including zona factor (Z: 33.9%; ZA: 30.4%; ZP: 31.6%; ZAP: 21.4%) showed higher pregnancy rates than other complex factors excluding zona factor (A: 24.4%, P: 0%; AP: 10.8%). From these results, AH is more helpful to the patients with thick zona pellucida rather than patients with older age and/or previous repeated implantation failure.

• Clinical and Experimental Reproductive Medicine
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• v.21 no.1
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• pp.99-110
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• 1994

One consequence of fertilization in mammals is an increased resistance of the zona pellucida (ZP) to proteases and various chemical reagents. This phenomenon has been called 'zona pellucida hardening' (ZPH), and it is generally accepted that it is caused by the secretory products of cortical granules released by the egg at fertilization. ZP of mouse oocytes maturing in vitro in a chemically defined medium becomes progressively more resistant to solubilization by chymotrypsin ("Spontaneous" ZP hardening). In the present study, it was aimed to find the specificity of spontaneous ZPH in relation to its possible relevance to the cortical reaction and the physiological block to polyspermy. When a maturation inhibitors, cAMP analog(dbcAMP) and phosphodiesterase inhibitor (IBMX) was added to culture medium, it prevent spontaneous ZPH of mouse oocyte during in vitro culture. Thus spontaneous ZPH requires GVBD, since it is prevented by those agents, which inhibit GVBD in vitro. However, culture for 3 hours in the presence of PMA(lOng/ml), a protein kinase C activator, resulted in ZPH without GVBD, thus suggesting that ZPH may be regulated independently apart from the event of GVBD. Pretreatment of mouse oocyte with FBS result in partially inhibitory effect on subsequent spontaneous ZPH. Induction of GVBD in vivo had a inhibitory effect on the spontaneous ZPH, but subsequent spontaneous ZPH. Induction of GVBD in vivo had a inhinbitory effect on the spontaneous ZPII, but had no inhibitory effect on PMA-induced ZPH. Treatment with a microfilament formation blocker(cytochalasin-B) at 1${\mu}g$/ml concentration, resulted in the excellent inhibitory effect on spontaneous ZPH. However cytochalasin-B did not inhibit PMA-induced ZPH. Thus this suggesting that spontaneuse ZPH had a different mechanism from PMA-induced ZPH.