• 한국동물위생학회지
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• 제26권4호
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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.

• 한국가축번식학회지
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• 제20권4호
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• pp.413-421
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• 1997

체외에서 포유동물의 난자와 정자의 배양에 관한 연구는 난자의 성숙과정과 수정현상에 대한 많은 새로운 정보를 제공하였다. 동시에 체외수정의 연구로부터 얻은 결과는 또다른 의문을 제기하였다. 특히 동결융해정액을 이용한 돼지체외수정의 경우 낮은 정자의 침입율과 전핵형성율 및 높은 다정자침입(polyspermy)율은 아직도 해결해야할 문제점으로 남아있다. 돼지난자의 성숙에 관한 연구의 성과는 수정후 낮은 전핵형성율을 개선시켰으나 타동물종에 비하면 아직도 매우 낮게 나타나고 있다. 한편 동결정액의 처리를 위하여 caffeine이나 Ca2+와 같은 물질을 수정용 배지내에 첨가하는 등 수정능력획득의 유기를 위하여 여러 가지 방법이 연구되고 있지만 정자의 침입율은 아직도 낮고, polyspermy의 발생율은 높게 나타내고 있다. 따라서 정자의 침입율을 향상시키고 polyspermy를 억제하기 위하여 난관세포와의 공동배양, 난포액을 첨가한 배양액 내에서 정자의 전배양 및 정자농도의 조절은 매우 효과적인 방법으로 이용되어왔다. 그러나 수정란의 체외생산성 향상과 이와 관련된 연구를 보다 효과적으로 수행하기 위해서는 위에서 지적한 문제에 영향을 미치는 요인에 대한 보다 근본적인 이해가 요구된다.

• Animal cells and systems
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• 제9권2호
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• pp.57-64
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• 2005

Cortical reaction and polyspermy block are well defined in most marine invertebrates. In Urechis species, the function of cortical granules (CGs) is not yet known, and there is controversy on whether the cortical reaction occurs, or the fertilization envelope (FE) is attributed to CG releases or functions to prevent polyspermy. This study was carried out to determine the cortical reactions and functions of the FE in Urechis unicinctus. Artificial insemination of the eggs revealed that CG release occurred to give rise to perivitelline space (PS) into the final FE. Both PS and final FE effectively blocked polyspermy. The final FE was accomplished within 10 min after sperm-egg initial binding. No massive release of CGs occurred within the early phase of 5 min after the initial binding, initially and the PS seemed to playa role to prevent polyspermy. The CG massively released its content into the PS in late phase of FE formation, and differentiated PS into five intermediate layers. The layers opened into each other by anastomosis, so that the final FE consisted of two layers, the inner layer ($15{\mu}m$ in thickness) and the outer layer ($1{\mu}m$ in thickness). The outer layer derived from vitelline layer and the inner layer consisted of PS and CG secretions. Immunofluorescence and confocal laser microscopy revealed that the spermatozoon took up residence in the egg cortex during FE formation and successive meioses of the fertilized egg. These results suggest that both PS and final FE of U. unicinctus were equivalent to the early and late block, respectively, of other marine animals.

• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2003년도 제3회 국제심포지움 및 학술대회
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• pp.110-110
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• 2003

The high incidence of polyspermic fertilization is one of the major causes lowering the overall efficiency of porcine IVF. The common procedure for IVF involves the co-culture of both gametes in the medium drop, which increases sperm concentration and incidence of polyspermy. Therefore, the present study was carried out to increase the efficiency of porcine IVF by reducing polyspermy using a modified swim-up method. This method modifies conventional swim-up washing by placing oocytes directly at the time of washing. Sperm pellet was prepared in the tube and mature oocytes were placed on cell strainer with $70 \mu m$ pore size (Falcon 2350) at the top of the tube. After insemination, the oocytes were stained for examination. Also, the developmental potential of fertilized embryos was measured to evaluate for the feasibility of this method. While having similar penetration rates in both methods ($86.67 \pm 2.36% to 83.33 \pm 1.36%$), there was a significant reduction of polyspermy in modified swim-up method ($17.50 \pm 1.60%$) compare to the control ($44.1 \pm 3.70%$ (p<0.05). Subsequent culture showed higher rate of blastocyst formation in modified swim-up method (20.44$\pm$0.99%) than the control ($15.73 \pm 3.26%$) (P<0.05), even though there was no significant difference. These results suggest that, by controlling the number of spermatozoa reaching the oocytes, porcine oocytes might be protected from polyspermy in vitro. Also, the developmental potential of the fertilized embryos using this method could be improved by increasing the pool of spermatozoa with better quality. Further optimization of the procedure required to implicate this method in routine porcine IVF.

• 한국가축번식학회지
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• 제22권4호
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• pp.411-417
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• 1998

본 연구는 난관액과 oviductal conditioned medium 이 다정자 침입과 체외배발달에 미치는 효과를 규명하기 위하여 수행하였다. 배양액내 난관액과 oviductal conditioned medium 의 첨가는 다정자침입율과 난자내 침입한 평균정자수를 감소시켰다 (P＜0.05). 정자와 난관액 그리고 oviductal conditioned medium 과 l.5, 3, 4.5 시간 공배양후 첨체반응의 성적은 대조구에 비하여 증가하였다. 체외수정후 체외발달 배양액에 난관액이나 oviductal conditioned medium 을 첨가하여 192 시간동안 배양했을 때 상실배와 배반포배발달율이 난관액과 oviductal conditioned medium 첨가구에서 유의적으로 높았다 (P＜0.05). 이러한 연구결과는 난관액과 OVCM 등 난관 유래물질은 다정자침입율과 난자내 침입한 평균정자수를 감소시키며 체외발달율을 높인다고 사료된다.

• Clinical and Experimental Reproductive Medicine
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• 제50권3호
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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.

• 한국동물생명공학회지
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• 제38권2호
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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.

• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2003년도 제3회 발생공학 국제심포지움 및 학술대회
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• pp.110-110
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• 2003

• 한국수정란이식학회지
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• 제15권1호
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• pp.39-46
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• 2000

This study was conducted to investigate the effect of hormones, protein sources and anti-oxidants on in vitro maturation (IVM) and in vitro fertilization(IVF) of bovine follicular oocytes. The rates of Holstein follicular oocytes classified as grade A and B(50.2% and 33.2%) were higher than those of Hanwoo cattle(40.3% and 32.0%, P<0.05). The cumulus cell expansion rates of oocytes cultured in TCM-199 and Ham's F-10 medium supplemented with 10% FCS and hormones were higher (81.9~87.6%) than those of non-treated groups (74.5~81.7%). The fertilization rates of oocytes cultured in TCM-199 and Ham's F-10 medim supplemented with 10% FCS, 1% BSA and 10% bFF was 53.8~55.0%, 51.4~52.6%, and 47.0~50.0%, respectively. The polyspermy rates was 13.6~14.2%, 10.0~11.1%, and 10.0%, respectively. When the oocytes were cultured in TCM-199 and Ham's F-10 medium with 50${\mu}{\textrm}{m}$ $\alpha$-tocopherol, the fertilization rates was 62.0 and 60.2%, respectively. In the maturation medium added of 100${\mu}{\textrm}{m}$ cysteamine, the fertilization rates was 64.7 and 66.7%, respectively. The fertilization and polyspermy rates of treated groups were higher than those of non-treated group. The results show that hormones, protein sources and anti-oxidants can provide a benefit for in vitro maturation and fertilization of bovine follicular oocytes.

• Clinical and Experimental Reproductive Medicine
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• 제48권4호
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• pp.362-367
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• 2021

Objective: The impact of early mechanical removal of cumulus cells on fertilization and embryonic development is not yet precisely known. This study aimed to investigate the effects of early and late cumulus cell removal on fertilization, polyspermy, embryonic development potential, blastocyst development, and clinical outcomes. Methods: A prospective study was conducted of patients who underwent in vitro fertilization between September 2019 and October 2020. Sibling oocytes were randomly allocated after insemination to early cumulus cell removal at 6 hours (group I) and late cumulus cell removal at 16-18 hours (group II). If total fertilization failure (TFF) was determined to have occurred at early cumulus cell removal, rescue intracytoplasmic sperm injection (ICSI) was performed. Fertilization, embryonic development, and pregnancy outcomes were compared. Results: A total of 912 oocytes were assigned to group I (458 oocytes) and group II (454 oocytes). Fertilization, polyspermy, embryo quality, and pregnancy outcomes were not significantly different between both groups. Rescue ICSI enabled fertilization of 79.2% of the TFF oocytes. Conclusion: Early cumulus cell removal at 6 hours had no significant difference in fertilization, polyspermy, embryo development, or obstetric and perinatal outcomes compared to late removal. Early cumulus cell removal combined with early rescue ICSI may have the potential to help couples with TFF.