• 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.

• Journal of Embryo Transfer
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• v.11 no.1
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• pp.71-80
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• 1996

The envelope of the rnannnalian oocyte plays crucial roles in sperm-oocyte interactions by providing sperm receptors, inducing acrosome reaction and preventing polyspermy. Understanding of properties of the zona pellucida (ZP) is essential for the artificial control of fertility in mammals. This study was carried out to produce and characterize monoclonal antibodies(MAbs) to porcine ZP proteins. Approximately 8,000 ZPs were obtained from follicular oocytes and dissolved in 40$\mu$l of double distilled water. Following immunization through foot-pad injections of Balb /c mice with a ZP solution, the popliteal lymph nodes were recovered at 2 weeks after the last injection. Hybridoma cell lines were established by fusing lymph node cells with P3X63 myeloma cells through selection using HAT medium and screening by immunofluorescence(IF) microscopy on the isolated ZP. Secreted MAbs were found to consist k chains and different heavy chains as evidenced by isotyping. Some of the MAbs demonstrated high specificity to the ZP in IF. The Mabs also showed positive cross reactivity with hamster and mouse eggs, while negative with bovine eggs. The results implicate that the MAbs can be used not only for identification of functional regions of the ZP, but also for elucidation of mechanisms involved in fertilization of mammals. The MAbs will provide basic information on biochemical anatomy of the ZP as well as can be candidates for the future contraceptive vaccines.

• Clinical and Experimental Reproductive Medicine
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• v.50 no.3
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• pp.154-159
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• 2023

Monospermy occurs in the process of normal fertilization where a single sperm fuses with the egg, resulting in the formation of a diploid zygote. During the process of fertilization, the sperm must penetrate the zona pellucida (ZP), the outer layer of the egg, to reach the egg's plasma membrane. Once a sperm binds to the ZP, it undergoes an acrosomal reaction, which involves the release of enzymes from the sperm's acrosome that help it to penetrate the ZP. Ovastacin is one of the enzymes that is involved in breaking down the ZP. Studies have shown that ovastacin is necessary for the breakdown of the ZP and for successful fertilization to occur. However, the activity of ovastacin is tightly regulated to ensure that only one sperm can fertilize the egg. One way in which ovastacin helps to prevent polyspermy (the fertilization of an egg by more than one sperm) is by rapidly degrading the ZP after a sperm has penetrated it. This makes it difficult for additional sperm to penetrate the ZP and fertilize the egg. Ovastacin is also thought to play a role in the block to polyspermy, a mechanism that prevents additional sperm from fusing with the egg's plasma membrane after fertilization has occurred. In summary, the role of ovastacin in monospermic fertilization is to help ensure that only one sperm can fertilize the egg, while preventing polyspermy and ensuring successful fertilization.

• Clinical and Experimental Reproductive Medicine
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• v.27 no.1
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• pp.23-29
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• 2000

Objective: Zona pellucida (ZP) has been thought to be the barrier of egg to sperm penetration before and after fertilization. The phenomenon of ZP hardening has been considered as a post-fertilization event until now, and it is generally accepted that it is caused by the secretory products of cortical granules released during the cortical reaction. Hardening of ZP could occur "spontaneously" in mammalian oocytes in standard culture conditions, and that it is probably not a consequence of cortical reaction. The purpose of our study was to investigate the effect of human amniotic fluid (HAF) on nuclear maturation (NM) and fertilization ability of mouse immature oocytes. Methods: HAF was obtained from patients undergoing amniocentesis at $16{\sim}20$ weeks of gestation. HAF from five to ten patients was centrifuged and the supernatants was pooled. Cumulusenclosed mouse immature oocytes were incubated in the medium containing HAF, and examined to confirm NM and fertilization. Female ICR mice (about 3 weeks old) were stimulated with 7.5 IU PMSG. Immature oocytes were isolated at $48{\sim}52$ hrs post PMSG injection and cultured in TCM-199 supplemented with 20% HAF for 18 hrs. FBS was used as a control for the examination. Matured oocytes (MII) were fertilized with sperms collected from the epididymis of male mice (over 10 weeks old). Fertilization was in conducted T6 medium containing 15 mg/ml BSA, and confirmed at 6 hrs post-insemination. Fertilization rate was assessed in zona-intact or zona-free oocytes (denuded by trypsin). Evaluation of NM and fertilization was carried out by rapid staining method. ZP hardening was evaluated by incubating cumulus cell-free mature oocytes in 0.001% chymotrypsin at $37^{\circ}C$ for 10 min. Results: There was no significant difference between the effects of HAF (86.6%) and FBS (87.7%) supplements on NM of immature oocytes. When maturation medium was supplemented with HAF, total fertilization rates (7%) were significantly lower (p<0.01) than that of FBS (85.1%). In HAF group, fertilization rate was increased (p<0.01) in zona-free oocytes (7% versus 100%). The resistance of mouse oocyte ZP to digestion by chymotrypsin after maturation in vitro was significantly higher (p<0.01) in HAF group (86.7%) than in FBS (6.7%). To culture oocytes in FBS were very effective in preventing ZP hardening. However cultured oocytes in HAF showed high rate of ZP hardening (p<0.01). Conclusions: These results suggest that HAF can be used as a supplement for the NM of mouse immature oocytes in vitro. However, HAF induces spontaneous hardening of ZP of mouse immaure oocytes during maturation in vitro.

• Asian-Australasian Journal of Animal Sciences
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• v.22 no.7
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• pp.962-968
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• 2009

The aim of this study was to compare the effects of oviductal fluid, porcine zygote medium (PZM)-3, PZM-4 and PZM-5, and modified PZM-5 culture media, and determine the effects of zona pellucida (ZP) removal on the development of nuclear transfer (NT) embryos. There were no significant differences in the rates of fusion and cleavage among the five different oviductal fluid concentrations. However, the rates of blastocyst formation and the cell numbers per blastocyst were high in the embryos at the 14 and 28 $\mu{g}$/ml concentrations of oviductal fluid compared to the 0, 56 and 100 $\mu{g}$/ml concentrations. The rates of cleavage and blastocyst formation, and the cell numbers per blastocyst were higher in the PZM-3, PZM-5 and modified PZM-5 media than in the PZM-4 medium. However, there were no significant differences in the fusion rates of oocytes among the four culture media. The cell numbers per blastocyst in the embryos without ZP were significantly greater than those with ZP. However, there were no significant differences in the rates of fusion, cleavage and blastocyst formation between the embryos with and without ZP. In conclusion, we improved blastocyst development and the quality of NT embryos by replacing PVA with 3 mg/ml of BSA in PZM-5 medium and supplementing the PZM-5 medium with 14 $\mu{g}$/ml oviductal fluid. The NT embryos produced by the zona-free NT method had a high rate of blastocyst formation in the modified PZM-5 medium.

• 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.

• Journal of Embryo Transfer
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• v.23 no.1
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• pp.1-4
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• 2008

This study was performed to investigate the effect of laser-assisted hole in the zona pellucida (ZP) of frozen-thawed ICR mouse embryos on the process of hatching that is critical for expanded blastocysts to implant into endometrium, Vitrification medium, composed of ethylene glycol and sucrose supplemented with 7.5% (w/v) PVP, was used to freeze $2{\sim}4$ cell stage embryos recovered from oviducts of superovulated and mated female mice before storing them in $LN_2$. Right after thawing them, a laser beam was shot to make a hole in ZP followed by culturing in KSOM for $96{\sim}120\;hr$ and examining development to blastocyst and hatching every 12 hr. Laser-treated embryos showed significantly higher hatching rate compared to control (92.9% vs. 22.1%, p<0.05). From around Day 4, blastocysts developed from laser-treated embryos started hatching while the blastocysts of control group failed to hatch showing a lot of shrinkage. This study shows that a laser-assisted hole in ZP improves the hatching rate of blastocysts developed from frozen-thawed, in vitro cultured ICR mouse embryos.

• Journal of Embryo Transfer
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• v.26 no.2
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• pp.111-115
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• 2011

In general, zona pellucida (ZP) of the blastocyst has to be removed first, then either isolated the inner cell mass (ICM) or ZP-removed whole blastocyst, which is then cultured on the feeder layer to induce ICM outgrowth for the generation of embryonic stem cells (ESC). However, it is unclear whether ICM isolation before seeding on feeder layer is beneficial or not because the interaction between ICM and trophoblasts may affect cellular growth and/or pluripotency during the culture on the feeder. In the present study, two ZP removal methods (mechanically by splitting with a 28-gauge needle versus chemically by the treatment of acid-Tyrode's solution) and two ICM isolation methods (ZP-free whole blastocyst seeding versus mechanical isolation of ICM) were evaluated for the efficient isolation and culture of putative parthenogenetic bovine ESC. The number of maintained outgrown colonies was counted in each experimental group. As the result, mechanical removal of ZP with a needle and followed by whole ZP-free blastocyst seeding on feeder cells tended to attach more on the feeder layer and resulted in more outgrown colonies with its simple and less time-costing benefits. Currently we are generating ESC lines in HanWoo cattle by using this method for initial outgrowth of the parthenogenetic bovine blastocysts.

• 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.24 no.1
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• pp.1-11
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• 1997

It is well known that the zona pellucidae of mouse oocytes become "hardened" when they are allowed to mature in vitro in the absence of serum components. To see if oocytes already undergone meiotic resumption in vivo exhibit similar zona hardening, hardening of ZP of cumulus-enclosed oocytes(CEOs) was examined after culture in vitro since their release from follicles various hours after hCG injection. When CEOs matured in vivo for 3h or longer were subjected to culture in vitro for 14h with BSA alone, zona hardening was significantly reduced compared to those cultured in vitro from the begining of maturation. However, when CEOs matured in vivo for 5h were freed from cumulus cells and then cultured in vitro with BSA alone, little reduction of zona hardening was observed. Preincubation of CEOs for 5h with fetuin, one of the well known inhibitor of in vitro zone hardening, did not prevent zona hardening during its subsequent culture of CEOs for 14h without fetuin. However, when CEOs precultured with both fetuin and PMSG for 5h and then further cultured with BSA alone for 14h, zona hardening was dramatically reduced. Under these conditions, the expansion of cumulus cell was observed. In addition, CEOs cultured with both BSA and dbcAMP to prevent their meiotic resumption showed a significant increase of zona hardening. Whether the observed zona hardening was correlated with the conversion of ZP2 to $ZP2_{f}$ was examined. Zona pellucida, isolated from CEOs matured for 5h in vivo and then further cultured with BSA alone was subjected to SDS-PAGE. Most of ZP2 molecules from these CEOs did not undergo conversion from ZP2 to $ZP2_{f}$. From these results, it is concluded that CEOs undergone meiotic resumption in vivo do not exhibit zona hardening when they were subsequently cultured in vitro without serum components. It appears that cumulus cells play an important role in this phenomenon.