• 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.

• Clinical and Experimental Reproductive Medicine
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• v.18 no.1
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• pp.63-71
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• 1991

To establish the normal fertile range in the results of the sperm zona-free hamster ova penetration assay (SPA) in Korean male, SPA using the low temperature ($4^{\circ}C$) capacitation in TEST-yolk buffer (TYB) was performed in 67 fertile and 26 infertile men. Sperm parameters in routine semen analysis were also checked and compared with the results of SPA. Sperm concentration, motility and motility index (MI) were significantly higher in fertile group compared with infertile group: $96.0{\pm}46.6$ vs $43.6{\pm}31.9{\times}10^6/ml$, $65.5{\pm}14.8%$ vs $45.8{\pm}23.6%$ and $46.31{\pm}13.29$ vs 27.40{\pm}17.98$, respectively. In fertile group, the hamster ova penetration rate (PR) was $98.5{\pm}5.0%$ (80%-100%), and the penetration index (mean penetrations per ovum, PI) was $9.59{\pm}6.35$(3.1-29.0). All the fertile men showed PI>3.0. In infertile group, PR was $24.6{\pm}24.8%$ (0%-70%), and PI was $0.40{\pm}0.42$ (0-1.3). Both PR and PI were significantly lower in infertile group. There was a significant correlation beween PI and sperm motility or MI, respectively, in fertile group whereas there was no correlation in infertile group. These data suggest that SPA using the low temperature capacitation in TYB can be a valuable diagnostic tool for the assessment of male fertility in vitro and provide an important supplement to the traditional tests of sperm quality.

• Korean Journal of Veterinary Service
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• v.26 no.4
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• pp.329-337
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• 2003

The functional role of the cumulus cells on sperm penetration and polyspermy during in vitro fertilization was examined. The penetration rate was significantly higher(p<0.01) in oocytes with(61%) than without(25%) cumulus cells. No significant differences, however, was observed in polyspermy. When the hyaluronidase was supplemented to the fertilization medium with different concentrations, penetration rates in oocytes with cumulus cells were higher than oocytes without cumulus cells at 0(61 vs 34% ; p<0.05), 0.01(56 vs 35% ; p<0.05), 0.1(66 vs 30% ; p<0.05) and 1.0 mg/$m\ell$(39 vs 27%). On the other hand, the polyspermy rates were lower oocytes without than with cumulus cells, and had a tendency to decrease with high concentrations of hyaluronidase. In another experiment, the penetration and polyspermy rates had a tendency to increase as time of sperm-oocytes culture was prolonged. At 16 and 20hrs after insemination, the penetration rates were significantly higher(p<0.05) in oocytes with(48 and 62% for 16 and 20hrs) than without(25 and 31% for 16 and 20hrs) cumulus cells in medium with hyaluronidase. However, the polyspermy rates were significantly(p<0.05) lower in oocytes without(3 and 16%) than with(37 and 48%) cumulus cells at 16 and 20hrs after insemination. In cumulus-free oocytes inseminated in medium with or without hyaluronidase at different concentrations of cumulus cells, the penetration rates were significantly(p<0.05) higher in medium with than without hyaluronidase at different concentrations of cumulus cells. The proportions of polyspermy were lower in medium without than with hyaluronidase at 0 (10 vs 0%), 10$^2$(25 vs 0%), 10$^4$(24 vs 14%) and 10$\^$6/(29 vs 10% ; p<0.05) cumulus cells/ml. These results suggest the advantage of culture in medium with cumulus cells and denuded oocytes to inhibit polyspermy with no decrease in the penetration rates during the fertilization in vitro in the porcine.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2001.05b
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• pp.4-16
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• 2001

Sperm competent for fertilization can become capacitated, bind to the zona pellucida (ZP)of an egg in a specific manner, and complete acrosomal exocytosis. Failure to carry out these functions results in infertility. Although the interactions between the ZP and the plasma membrane overlying the sperm acrosome have been considered important for sperm-egg recognition and signalling recent results have prompted a reassessment of current paradigms concerning these interactions. In this review, we're going to discuss about the roles of the acrosomal matrix, the particulate component of the acrosomal contents, in fertilization. The general hypothesis is that acrosomal exocytosis leads to the exposure of acrosomal matrix proteins that become de facto extracellula matrix(ECM) on the surface of the sperm head, and that the dynamic interactions of this newly-exposed sperm ECM with the egg ECM (the ZP) govern sperm-egg recognition and sperm penetration of the ZP. Informations from these experiments may provide new ways to address the poor ZP binding of sperm from some human infertility patients and may offer new avenues for contraception through the disruption of purposeful sperm-ZP binding.

• Asian-Australasian Journal of Animal Sciences
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• v.17 no.2
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• pp.164-167
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• 2004

This study was carried out to investigate in vitro fertilization and development of in vitro matured pig oocytes inseminated with the Duroc boar sperm by different sperm washing media after thawing of the 5 ml frozen straws. Immature follicular oocytes (30-40) were transferred into each well of a Nunc 4-well multidish containing $500{\mu}l$ mTCM199 maturation medium. The sperm rich portion of ejaculates was collected into a 250 ml insulated vacuum bottle and gradually cooled 22 to $24^{\circ}C$ over a 2 h period. Semen was centrifuged at 800 g for 10 min and the seminal plasma discarded. Sperm were esuspended in a lactose-egg yolk and N-acetyl-Dglucosamine (LEN) diluent to contain $1{\times}10^{9}$ sperm/ml and cooled to $5^{\circ}C$ over a 2 h period. Immediately before freezing, semen was rediluted with an equal volume of LEN+4% glycerol and packed into 5 ml straws. After thawing of the 5 ml straw, the 5 ml semen was diluted with 20 ml Beltsville thawing solution (BTS) at room temperature. Oocytes were inseminated with untreated (unwashed and nonpreincubated) or treated sperm (washed two times in BTS, mTLP-PVA and mTBM media, respectively and nonpreincubated) with $2{\times}10^{7}$ sperm concentration. Oocytes were coincubated for 6 h in $500{\mu}l$ mTBM fertilization. At 6 h after IVF, oocytes were transferred into $500{\mu}l$ NCSU-23 culture medium for further culture of 6 h. Sperm penetration, polyspermy and male pronuclear formation of oocytes at 12 h after IVF and developmental ability of oocytes at 48 h after IVF were evaluated. Sperm penetration rate, male pronuclear formation and rate of cleaved embryos were higher in the BTS, mTLP-PVA and mTBM treatments than the unwashed treatment (p<0.05). The rate of blastocysts from the cleaved oocytes (2-4 cell stage) were higher in the mTLP-PVA treatment than in the unwashed, BTS and mTBM treatments. In conclusion, we recommend the washing of frozen-thawed sperm with mTLP-PVA medium before in vitro fertilization of oocytes in mTBM medium.

• Journal of Animal Reproduction and Biotechnology
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• v.38 no.2
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• pp.70-76
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• 2023

Background: Despite considerable technological advancements, polyspermy remains a significant challenge in in vitro fertilization (IVF) procedures in pigs, disrupting normal embryonic development. Here, we aimed to determine whether optimal fertilization conditions reduce the polyspermy incidence in pigs. Methods: In vitro-matured oocytes were co-incubated with sperm according to a modified two-step culture system. Results: In the first experiment, oocytes were briefly co-incubated with sperm, washed in IVF medium, and then moved to fresh IVF medium for 5 or 6 h. Although the 6 h sperm-free cultured group had a higher penetration rate than the 5 h cultured group, the polyspermy rate significantly increased in the 6 h sperm-free cultured group. The gamete co-incubation period was either 20 or 40 min. The 40 min cultured group had a higher rate of blastocyst formation and number of total cells in blastocysts than the 20 min cultured group. In experiment 2, oocytes were inseminated with sperm separated by Pecroll treatment. Percoll treatment increased the rate of oocyte penetration and blastocyst formation compared to the control. In experiment 3, fertilized oocytes were cultured in 25 µL microdroplets (10 gametes/drop) or 500 µL (100 gametes/well) of culture medium in 4-well plates. The large volume of medium significantly reduced the number of dead oocytes and increased the rate of blastocyst formation compared to the small volume. Conclusions: Collectively, these results demonstrate that various fertilization conditions, including modified co-culture period, active sperm separation, and culture medium volume, enhance fertilization efficiency and subsequent embryonic development by decreasing polyspermy occurrence.

• Asian-Australasian Journal of Animal Sciences
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• v.15 no.2
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• pp.172-178
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• 2002

The present study examined whether treatment of in vitro matured pig oocytes with calcium ionophore (A23187) could prevent polyspermic penetration in vitro. When oocytes cultured for maturation for 33, 36 or 44 h were subsequently treated with $50{\mu}M$ A23187 in medium with fetal calf serum (FCS) for 1, 2 and 3 h and then cultured for 12 h without spermatozoa, virtually no activation occurred. In the absence of FCS, however, 31-42, 45-49 and 56-64% of oocytes were activated, respectively. When oocytes treated with $50 {\mu}M$ A23187 in medium with FCS for 3 h were inseminated in vitro, the penetration rates (14-57%) were lower (p<0.01) with a higher (p<0.01) incidence (35-67%) of monospermy compared with untreated oocytes (69-80% penetration and 15-17% monospermy). However, sperm penetration was completely blocked in all oocytes treated with A23187 in the absence of FCS. When oocytes matured for 33 h were treated with different concentrations of A23187 for 3 h and inseminated in vitro, the penetration rate did not change but there was an increased incidence (p<0.05) of monospermy at $10-20{\mu}M$ and $2.5-5{\mu}M$ A23187 in the presence and absence of FCS, respectively, compared with at $0{\mu}M$ A23187. With these lower concentrations of A23187, treatment of oocytes for at least 60 and 30 min in the presence and absence of FCS, respectively, was required to increase the incidence of monospermy without reducing penetration rate. These results indicate that a high concentration ($50{\mu}M$) of A23187 in medium without FCS, but not in medium with FCS, stimulated in vitro matured pig oocytes to induce parthenogenetic activation and a complete block to sperm penetration in vitro. However, treatment of oocytes with lower concentrations of A23187 ( $10-20{\mu}M$ and $2.5-5{\mu}M$) both in the presence and absence of FCS maintained sperm penetration in vitro and increased the incidence of monospermy.

• Asian-Australasian Journal of Animal Sciences
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• v.17 no.10
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• pp.1369-1373
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• 2004

The percentages of sperm motility and normal acrosome on the liquid boar semen diluted and preserved at $4^{\circ}C$ with lactose hydrate, egg yolk and N-acetyl-D-glucosamine (LEN) diluent were significant differences according to preservation day and incubation time, respectively. The sperm motility steadily declined from 96.9% at 0.5 h incubation to 78.8% at 6 h incubation at 1 day of preservation. However, the sperm motility rapidly declined after 4 day of preservation during incubation. The normal acrosome steadily declined from 93.3% at 0.5 h incubation to 73.8% at 6 h incubation at 1 day of preservation. However, the normal acrosome rapidly declined after 3 day of preservation during incubation. The rates of sperm penetration and polyspermy were higher in 5 and $10{\times}10^6$ sperm/ml than in 0.2 and $1{\times}10^6$ sperm/ml. Mean numbers of sperm in penetrated oocyte were highest in $10{\times}10^6$ sperm/ml compared with other sperm concentrations. The rates of blastocysts from the cleaved oocytes (2-4 cell stage) were highest in $1{\times}10^6$sperm/ml compared with other sperm concentrations. In conclusion, we found out that liquid boar sperm stored at $4^{\circ}C$ could be used for in vitro fertilization of pig oocytes matured in vitro. Also, we recommend $1{\times}10^6$sperm/ml concentration for in vitro fertilization of pig oocytes.

• Journal of Embryo Transfer
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• v.15 no.1
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• pp.85-94
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• 2000

These experiments were conducted to examine the effects of theophylline, pentoxifylline and heparin on frozen-thawed Hanwoo sperm for enhancing motility and viability of sperm. Frozen-thawed semen collected from one bull was treated in TALP(tyrode-albuminlactate-pyruvate) containing varous concentrations of theophylline and pentoxifylline. After incubated at 5% CO2 in air atmosphere for 6 hours, the motility of sperm after the treatments was characterized by CASA(computer aided semen analysis) system. When monitored notility(MOT) and curvilinear velocity(VCL), theophylline and pentoxifylline exerted their optimal action at the concentration of 30 mM and 3 mM, respectively. No difference of sperm motility was observed when the sperm was treated with both substances compared with a single treatment of each substance. Comparison was then made for evaluating the effect of theophylline and / or pentoxiophylline on the motility and viability of significant treatment effects of each substance, high MOT and VCL values were detected in sperm treated with theophylline. In the case of sperm viability examined by an eosin-nigrosin staining, however, a significant decrease was found after the combined treatment of theophylline+pentoxyphilline than after the treatment with heparin alone or no treatment(P<0.05). In conclusion, theophylline, pentoxiphylline or heparin can be used for enhancing the motional characteristics and viability of frozen thawed Hanwoo semen. Considering characteristics of these substances, theophyline may be useful in the artificial insemination system, which requires vigorous sperm motility. While, heparin supporting sperm viability in vitro can be effectively used for improving in vitro-fertilization system.

• Clinical and Experimental Reproductive Medicine
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• v.30 no.1
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• pp.105-109
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• 2003

Objective s: To assess the fertilizing capacity using sperm penetration assay (SPA) to predict the outcome of the in vitro fertilization-embryo transfer (IVF-ET) outcome. Materials and Methods: Semen samples were provided by 129 patients undergoing IVF. We attempted to correlate the extent of sperm penetration under enhanced SPA protocol with the results of fertilization, cleavage, preimplantation embryo development, and pregnancy. Results: Univariate analysis demonstrated a statistically significant correlation between fertilizing capacity and motility, kinetics, fertilization, cleavage and embryo development, and pregnancy rate. By logistic regression analysis, fertilizing capacity was found to be the only variable that was statistically significant with respect to pregnancy rate. Fertilizing capacity, cleavage rate and pregnant rate were significantly higher in pregnant group. However, the fertilization rates was comparable with both group. Conclusions: Lower fertilizing capacity could denote a poorer prognosis for establishing a pregnancy, even after satisfactory fertilization rate is achieved.