• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2000년도 수정란의 위생적 처리·검사 및 특수가축의 수정란이식 기술 심포지움
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• pp.64-75
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• 2000

During the last three decades considerable advances has been made in goat embryo production and transfer technology. The Korean native black goat is the most useful domestic ruminant in this country for biological investigation and application because it has a lot of merits such as relatively short generation period(1 vs 2 year for a cow), easy of handling, well adaptation, high fertility, convenient and inexpensive. This article covers the methods of superovulation, estrus synchronization, embryo collection and transfer techniques, pregnancy diagnosis and subsequent pregnancy and kidding rates for the production of transgenic Korean native black goats. More than one hundred goat kids have been produced as a result of our transgenic goat project via microinjection of foreign gene into pronuclei, in vitro culture, transfer of various stages of fresh and frozen-thawed microinjected embryos into oviducts or uteri of recipient does. We have got two transgenic goats carrying a transgene targeting the expression of recombinant human granulocyte colony stimulating factor(hG-CSF) to the mammary gland so far. Since collection and transfer of embryos in this species is usually accomplished by laparotomy, exteriorization of the reproductive tract for surgical embryo collection leads to the formation of post-operative adhesions. Nonsurgical or laparoscopic technique to reduce adhesions from repeated surgeries has great advantages in improving embryo production and transfer especially from valuable donors. We will discuss this later.

• Clinical and Experimental Reproductive Medicine
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• 제22권3호
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• pp.273-278
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• 1995

Mammalian, including human, spermatozoa undergo morphological and physiological changes during sperm maturation. There were, these changes may affect the fertilization and embryonic development. In this study, we examined the pronucleus formation, pronucleus disappearance and embryonic development in the human oocytes fertilized by intracytoplasmic sperm injection (ICSI). The injected spermatozoa were grouped into ejaculated, epididymal and testicular by the collecting region. Among 363 metaphase II injected oocytes, 287(79.1%) oocytes were normally fertilized and displayed two pronuclei. There were no difference in the fertilization rates and in the pronucleus formation and pronucleus disappearance at 16, 20 and 24 hr after ICSI, among the each spermatozoa group. Also, at 64 hr, the appearance of embryonic development was similar. From these results it can be concluded that there was no difference of maturity among the sperm collected from ejaculated, epididymis and testis in the pronucleus formation and embryonic development. Therefore, testicular spermatozoa are successfully used with ICSI in IVF-ET program.

• Molecules and Cells
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• 제20권3호
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• pp.423-428
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• 2005

Immediately after fertilization, a chromatin remodeling process in the oocyte cytoplasm extracts protamine molecules from the sperm-derived DNA and loads histones onto it. We examined how the histone H3-lysine 9 methylation system is established on the remodeled sperm chromatin in mice. We found that the paternal pronucleus was not stained for dimethylated H3-K9 (H3-$m_2K9$) during pronucleus development, while the maternal genome stained intensively. Such H3-$m_2K9$ asymmetry between the parental pronuclei was independent of $HP1{\beta}$ localization and, much like DNA methylation, was preserved to the two-cell stage when the nucleus appeared to be compartmentalized for H3-$m_2K9$. A conspicuous increase in H3-$m_2K9$ level was observed at the four-cell stage, and then the level was maintained without a visible change up to the blastocyst stage. The behavior of H3-$m_2K9$ was very similar, but not identical, to that of 5-methylcytosine during preimplantation development, suggesting that there is some connection between methylation of histone and of DNA in early mouse development.

• Clinical and Experimental Reproductive Medicine
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• 제24권3호
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• pp.311-318
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• 1997

Bypassing acrosome reaction and fusion process in intracytoplasmic sperm injection(ICSI), most of injected spermatozoa still contain intact acrosome contents and plasma membrane. It Is not known yet what acrosome contents and plasma membrane of spermatozoa have effect on the development of embryo. For further understanding of fertilization process after ICSI, we studied the time of pronucleus formation, disappearance and first cleavage in human zygote, and pregnancy rate in relation to acrosome reaction rate of spermatozoa after ICSI. Seventy cycles undergoing ICSI program were randomly selected. Sperm suspension from 38 cycles were treated 50% human follicular fluid(hFF) for 3 hours in order to induce acrosome reaction, others were not treated as control. Acrosome reaction in hFF treated and non-treated group was assessed by fluorescein isothiocyanate(FITC)-conjugated Arachis hypogea(PNA) and Pisum sativum agglutinin(PSA). Oocytes were classified into 'good' and 'poor' according to their morphology. After ICSI, fertilization of oocytes were assessed by detection of two pronuclei at 16 hours. The pronuclei disappearance and first cleavage of zygotes were observed at 24 hours, and then embryos were transferred to uterus after culture for 72 hours. The rate of acrosome reaction of spermatozoa in hFF treated group was significantly higher than that in control(p<0.01). Fertilization rates of good oocytes were not different both control and hFF treated group(81.3%(174/206) vs. 72.1%(102/130)). But, in poor oocytes, the fertilization rates in hFF treated group(72.1%(149/183)) were increased compared than those of control group (63.6%(98/140), p<0.01). In either good or poor oocytes, the rates of pronuclei disappearance in hFF treated-spermatozoa injected oocytes were higher than control (59.1%(103/174), 56.4%(84/149) vs. 32.4%(33/102), 37.8%(37/98), p<0.01). Also, the rates of thirst cleavage were increased in hFF treated group (31%(54/174), 24.1%(36/149)) compared than those of control group (10.8%(11/102), 13.2%(13/98), p<0.01). The pregnancy rates of hFF treated group (42.1%(16/38)) were slightly higher than control group (28.1%(9/32), p>0.05). But, the pregnancy rate of group which possessed more than one cleavaged zygote at 24 hours was higher than group which did not (45.2%(19/42) vs. 21.4%(6/28), p<0.05). From these results, the development of zygotes were faster in higher acrosome reacted sperm group than lower acrosome reacted sperm group after ICSI. Our results may be explained that acrosomal membrane and plasma membrane are easily detached from spermatozoa in acrosome reacted spermatozoa compared with acrosome intact sperm in the cytoplasm of oocyte during pronuclear formation. We conclude that the injection of acrosome reacted spermatozoa will increase the pregnancy rate as they can induce fast embryonic development in ICSI.

• 한국수정란이식학회지
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• 제25권2호
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• pp.97-101
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• 2010

Intracytoplasmic sperm injection (ICSI) is one of the artificial fertilization methods when only a few sperm are available for insemination, and an important tool for the preservation of genetic materials of endangered animal species, especially the male is infertile. Different from other species such as mice and pigs, the conventional ICSI method which uses spiked pipette for injection (Spike-ICSI) is exhibited low success rates in cattle because the bovinesperm head membrane is hard to break during injection procedure. We chose piezo-assisted ICSI (Piezo-ICSI) for the improvement of the injection procedure including sperm head membrane rupture and efficient puncture of the plasma membrane of the oocytes. In this experiment, we compared the efficacy of the bovine ICSI embryo production between the Piezo-ICSI and Spike-ICSI. The second polar body extrusion, pronuclear formation, cleavage and blastocyst formation were evaluated after implementation of two different ICSI techniques. The Piezo-ICSI tended to show comparably higher rates of the second polar body extrusion (41.7%), the pronuclei formation (42.9%) and the two-cell cleavage (41.4%) than Spike-ICSI does (33.3%, 28.6% and 23.5%, respectively) although there is no statistic significance between two groups. In addition, the blastocysts were only obtained from the Piezo-ICSI group (10.3%). Our finding shows that the Piezo-ICSI may be used as an artificial fertilization method in cattle when in vitro fertilization is not applicable.

• Clinical and Experimental Reproductive Medicine
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• 제42권1호
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• pp.14-21
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• 2015

Objective: It has previously been suggested that embryos developing from intracytoplasmic sperm-injected (ICSI) zygotes with three pronuclei (3PN) are endowed with a mechanism for self-correction of triploidy to diploidy. 3PN are also observed in zygotes after conventional in vitro fertilization (IVF). The parental origin, however, differs between the two fertilization methods. Whereas the vast majority of 3PN IVF zygotes are of dispermic origin and thus more likely to have two centrioles, the 3PN ICSI zygotes are digynic in origin and therefore, more likely to have one centriole. In the present study, we examine whether the parental origin of 3PN embryos correlates with the karyotype. Methods: The karyotype of each nucleus was estimated using four sequential fluorescence in situ hybridizations-each with two probes-resulting in quantitative information of 8 different chromosomes. The karyotypes were then compared and correlated to the parental origin. Results: 3PN ICSI embryos displayed a significantly larger and more coordinated reduction from the assumed initial 3 sets of chromosomes than 3PN IVF embryos. Conclusion: The differences in the parental origin-and hence the number of centrioles-between the 3PN IVF and the 3PN ICSI zygotes are likely to be the cause of the differences in karyotypes.

• Clinical and Experimental Reproductive Medicine
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• 제28권1호
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• pp.65-72
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• 2001

Objectives: This study was to investigate the fertilization rate after intracytoplasmic sperm injection (ICSI) or partial zona dissection (PZD) of human and hamster sperm into hamster oocyte in in vitro fertilization (IVF). In addition, the possibility of clinical application was evaluated by the comparison of usefulness and difference of these method. Materials and Methods: Hamster immature oocytes were obtained from oviducts superovulated by PMSG and hCG, and hamster sperms were obtained from epididymis. The freezed human sperms were thawed before use. Fertilization were confirmed by two pronuclei, one pronucleus, swollen sperm head or/and two polar bodies at $7{\sim}8$ hour after ICSI or PZD. Results: The fertilization rates after ICSI and PZD of human sperm to hamster oocyte were 3.6%, 64.2%,73.6%, and 55.6% for negative control, positive control, ICSI, and PZD respectively, suggesting that ICSI only showed improved fertilization rate (p<0.01). The fertilization rates after ICSI and PZD of hamster sperm to hamster oocyte were 11.1%, 51.2%, 39.6%, and 72.7% for negative control, positive control, ICSI, and PZD respectively, suggesting that PZD only showed improved fertilization rate (p<0.01). PZD showed significantly higher fertilization rate than ICSI (p<0.05). Conclusions: As for the fertilization rate by ICSI and PZD using hamster oocyte in IVF, ICSI technique was considered to be more useful for human sperm and PZD technique for hamster sperm. Therefore, ICSI technique was considered more appropriate for experimental application using human sperm and hamster oocyte.

• 한국수정란이식학회지
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• 제15권2호
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• pp.157-165
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• 2000

The efficiency of transgenic livestock production could be improved by early screening of transgene-integration and sexing of embryos at preimplantational stages before trasferring them into recipients. We examined the effciency of multiplex PCR analysis for the simultaneous confirmation of the trasgene and sex during the preimplantational development of bovine embryos and the possibility of green fluorescent protein(GFP) gene as a non-invasive marker for the early screening of transgenic embryos. The GFP gene was microinjected into the male pronuclei of bovine zygotes produced in vitro. The injected zygotes were co-cultured in TCM-199 containing 10% FCS with boving oviductal epithelial cells in a 5% CO2 incubator. Seventeen(13.0%) out of 136 gene-injected bovine zygotes developed by multiplex PCR analysis and the expression of GFP was detected by observing green fluorescence in embryos under a fluorescent microscope. Eight(67%) of 12 embryos at 2-cell to blastocyst stage were positive in the PCR analysis, but only two(11.8%) of 17 blastocysts expressed the GFP gene. Their sex was determined as 7 female and 5 male embryos by the PCR analysis. The results indicate that the screening of GFP gene and sex in bovine embryos by PCR analysis and fluorescence detection could be a promisible method for the preselection of transgenic embryos.

• Clinical and Experimental Reproductive Medicine
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• 제45권1호
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• pp.52-55
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• 2018

This study retrospectively assessed whether time-lapse data relating to developmental timing and morphology were associated with clinical outcomes, with the eventual goal of using morphokinetic variables to select embryos prospectively for cryopreservation. In this study, we examined the clinical outcomes of single vitrified-warmed blastocyst transfer cycles that were cultured in a time-lapse incubation system. The morphokinetic variables included uneven pronuclei, an uneven blastomere, multinucleation, and direct, rapid, and irregular division. A total of 164 single vitrified-warmed blastocyst transfer cycles were analyzed (102 cycles of regularly developed blastocysts and 62 cycles of blastocysts with morphokinetic variables). No significant differences in the age of females or the standard blastocyst morphology were found between these two groups. The regularly developed blastocysts showed significantly higher implantation and clinical pregnancy rates than the blastocysts exhibiting morphokinetic variables (30.4% vs. 9.7% and 37.3% vs. 14.5%, respectively; p< 0.01). The blastocysts that exhibited morphokinetic variables showed different mean development times compared with the regularly developed blastocysts. Although morphokinetic variables are known to have fatal impacts on embryonic development, a considerable number of embryos developed to the blastocyst stage. Morphokinetic variables had negative effects on the implantation and clinical pregnancy rates in vitrified-warmed blastocyst transfer cycles. These findings suggest that blastocysts cultured in a time-lapse incubation system should be considered for selective cryopreservation according to morphokinetic variables.

• Clinical and Experimental Reproductive Medicine
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• 제25권3호
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• pp.331-340
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• 1998

본 연구에서는 돼지 난자내에 돼지정자, 여러 가지 처리된 정자두부 (1% Triton, 0.1% Trypsin, 100mM NaOH)및 이종정자 (소, 쥐, 사람)를 미세 주입한 후 난 활성과 웅성 전핵형성, 전핵의 이동 및 배발달을 관찰하였다. 전자현미경으로 관찰한 결과 Triton X-100을 처리하였을 때 첨체가 제거되었으나 핵 주변 물질은 제거되지 않았고 Trypsin 또는 NaOH를 처리 할 경우 핵주변 물질 (perinuclear material)이 제거됨을 볼 수 있었다. 돼지 난자는 정자, 정자두부 및 Triton X-100을 처리한 정자두부의 주입을 통해 난 활성이 유도되었으며 쥐, 소, 사람의 정자를 주입하였을 때 난 활성이 유도되고 정상적인 전핵형성이 이루어졌다. 그러나 정자꼬리나 Trypsin 또는 NaOH에 의해 정자 핵주변 물질(perinuclear material)이 제거된 정자두부를 주입하였을때는 난 활성은 야기되지 않았다. 유사분열 및 2-세포기까지 정상적인 수정은 동종의 정자 및 정자두부를 주입한 난자에서 관찰할 수 있었으나 이 종정자를 주입한 난자에서는 관찰되지 않았다. 또한 상실배 및 배 반포까지 정상적인 수정은 동종의 정자 및 정자두부를 주입한 난자에서 관찰할 수 있었다. 이러한 결과는 돼지에서 정자 및 정자두부의 미세주입에 의해 수정이 이루어지는 것을 시사하며 수정시 정자유래의 난할성인자는 정자 핵주변 물질(porinuclear material)에 존재하며 종특이적이지 않다는 것을 보여주는 것이다.