• 한국가축번식학회지
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• 제8권1호
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• pp.29-35
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• 1984

These experiments were carried out to obtain basic information necessary for in vitro culture of aggregated mouse embryos. Inbred ICR mice were used to obtain embryos. The zona pellucida was removed by placing the embryos in Whittingham's medium containing 0.5% protease for about 5-10minutes at 37$^{\circ}C$. Total 263 pairs of 2-, 4- and 8-cell zona free mouse embryos were subjected to aggregation by physical pressure and cultured in Whittingham's medium under the gas phase of 5% CO2 in air at 37$^{\circ}C$ for 24 to 60 hours. The results obtained in these experiments were summarized as follows: 1. Time needed for fusion of 2-, 4- and 8-cell embryos were 0-3, 0-3 and 0-3 hours, respectively and average time needed for in vitro development of 2-, 4- and 8-cell embryos after aggregation to morula and blastocyst were 42, 30 and 13.5 hours, and 51, 39 and 27 hours, respectively. 2. Of total 263 pairs of naked embryos, 227 were firmly aggregated together and the rats of aggregation in 2-, 4- and 8-cell embryos were 71.8, 88.3 and 97.0%, respectively. 3. The rates of aggregated pairs which obtained from 2-, 4- and 8-cell embryos developed to morula were 96.7, 95.6 and 96.9%, respectively, and embryos developed to blastocysts were 88.5, 89.7 and 90.8%, respectively. 4. Conspicuous differences in size of volume and inner cell masses between single and double blastocysts were observed. Although a single blastocolic cavity was formed in most double blastocysts, several formed two distinct cavities from the very beginning.

• 한국가축번식학회지
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• 제18권2호
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• pp.141-150
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• 1994

전자현미경에 의해 자궁부착 전후의 돼지 수정란의 형태형성 및 분화에 따른 배발생 과정을 검토하였다. 돼지 초기배는 자궁이주후 균일하게 자궁에 배분되기전 약 2~3일간은 자궁각의 proximal portion에 존재하며, 임신 4일째에 할구와 할구의 경계를 상실하는 tight한 gap junction을 가진 상실배로 발달한다. 배반포를 형성하는 시기에 estradiol 17$\beta$는 compact한 상실배를 cavitated blastocyst로 발달을 촉진시키면서, steroid hormone이 이후의 배발생을 지배한다. Hatching의 시기는 교배후 6~7일경 zona pellucida을 둘러사고 있는 glycoprotein의 thinning과 lysis에 의해 이루워지는데, hatching 과정은 embryo의 세포수와 무관하였으며, 이때의 embryo의 직경은 0.5~1.0mm인 것을 본 실험에서 확인하였다. 12일경부터는 embryo는 prostaglandins, IGF-binding protein, retinol binding protein, plasminogen activator등의 단백질이 풍부해 이들 인자가 elongation 개시 후보로 고려될 수 있었다. 또한 이 시기의 embryo는 embryonic disc로 발달시 progesterone과 estrogen을 estradiol 17$\beta$로 전활할 수 있으며, 이러한 변화와 함께 spherical stage로부터 tubular 혹은 filamentous form으로 변형되었다. Estrogen이 임신을 통해 prostagladins의 분비를 uterine lumen에 지시하는지는 알 수 없으나 13일 경을 전후해 conceptus estrogen이 uterine arterial blood flow, uterine vasular permeability을 증가시키는 것으로 나타났으며, 자궁에서 protein과 calcium, PGF2$\alpha$, plasminogen inhibitor를 증가시키는 것으로 나타났다. 이 시기의 자궁 변화와 함께 embryo의 attachment는 trophoblast와 uterine membrane사이의 느슨한 결합에 의해 개시되었으며, 18일경 uterine과 trophoblastic microvili의 interdigitation에 의해 완성된다. 이 시기에 conceptus attachment를 위해 필요한 uterine microvili에서의 glycocalyx의 형성과 endometrial epithelium의 erosion을 야기하기 위해 plasminogen activator을 분비하였으며, 반면 자궁에서 plasminogen 역할을 하는 것은 estrogen이며, blastocyst cell 표면의 lectin binding이 attachment에 중요한 역할을 한다. 이상과 같은 일련의 과정을 거친 초기배는 성공적인 임신으로 유도된다고 본다. 따라서, 본 연구는 이상과 같이 착상을 전후한 시기의 배를 전자현미경에 의해 형태형성의 변화를 특히 착상을 전후해 배 취사율이 높은 시기를 대상으로 분석하였다. 이 분석 시기중 성공적인 착상성공율은 56%(71/126)였다.

• Clinical and Experimental Reproductive Medicine
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• 제45권1호
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• pp.25-30
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• 2018

Objective: This study conducted a preliminary examination of the effects of three-area laser-assisted zona thinning (LAZT) during the cleavage stage of embryo development on the hatching process in human in vitro fertilization-embryo transfer (IVF-ET) with subjects of advanced female age or frozen-thawed (FT) embryos. Methods: Eight-cell stage embryos were treated with LAZT in three areas of the zona pellucida at $120^{\circ}$ intervals. The control group was embryos without LAZT. Of the 72 consecutive fresh cycles and the 28 FT embryo transfer cycles, the patients in 55 fresh cycles and 17 FT cycles declined LAZT, and those cycles were defined as the control group. Results: In the fresh cycles, the pregnancy rates were similar in the LAZT and control groups. However, in the FT cycles, the pregnancy rate was significantly higher in the LAZT group than in the control group (45.5% in the LAZT group vs. 23.5% in the control group, p< 0.05). Conclusion: These results show that multi-area LAZT resulted in significantly improved pregnancy outcomes in human 8-cell embryos compared to controls.

• 한국가축번식학회지
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• 제25권4호
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• pp.327-337
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• 2001

본 연구는 superoxide dismutase (SOD)가 동결-응해 돼지정자의 lipid peroxidation과 체외수정능력에 미치는 영향을 검토하였다. 그 결과 동결-응해 정자는 10 units/$m\ell$의 SOD가 첨가된 배양액에 의해 처리했을 때 가장 높은 정자생존율을 나타냈으나 서로 다른 농도에 의한 차이는 인정되지 않았다. 그러나 SOD농도에 관계없이 정자처리 직후의 생존율은 120분간 배양 후에 비해 유의적(P＜0.05)으로 높은 생존율을 나타냈다. 또한 정자처리 후 배양시간이 0, 60 및 120분으로 길어짐에 따라 정자의 첨체반응 유기율이 증가하였지만 SOD 첨가 또는 무첨가구 사이에서 유의적인 차이는 인정되지 않았다. 한편, 체외수정시 1 unit/$m\ell$의 SOD를 첨가한 경우 0, 10 및 100 units/$m\ell$ 첨가시 보다 유의적(P＜0.05)으로 높은 정자침입율을 나타냈으며, 10과 100 units/$m\ell$의 SOD 첨가시 낮은 다정자 침입율을 나타냈다 (P＜0.05). 정자의 peroxidation은 malondialdehyde의 생성에 기초를 두고 평가하였는데 SOD 무첨가에 비해 첨가농도가 높아질수록 malondialdehyde의 생성이 낮아졌지만 유의적인 차이는 인정되지 않았다. 또한 동결-융해된 정자는 sulfhydrl(-SH) group의 용량을 측정한 결과 SOD무첨가시 보다 첨가시 높은 용량이 측정되었지만 유의적인 차이는 인정되지 않았다. 한편, 동결-융해된 정자가 체외에서 성숙시킨 난자의 투명대에 접착하는 정도를 평가한 결과 SOD 무첨가 보다는 첨가농도가 높아짐에 따라 접착정자수가 증가하였으며 100 units/$m\ell$ 첨가시 유의적(P＜0.05)으로 높은 접착정자 수를 나타냈다 본 연구의 결과로부터 SOD는 돼지 동결-융해정자에 있어서 난자의 투명대 접착능력의 증가와 함께 체외수정능력 향상에 영향을 미치는 것으로 나타났다.

• 한국가축번식학회지
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• 제19권2호
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• pp.129-134
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• 1995

This study was carried out to investigate on the survival rate and in vitro developmental rate of bisected porcine embryos and immature oocytes by manipulator and micropipette. Bisected embryos and immature oocytes cultured for 1∼5 days in TCM 199 medium with 20% FCS. Survival rate was defined as development rate on in vitro culture or FDA-test. The results are summarized as follows ; 1. The survival rate of bisected porcine embryos and oocytes were 26.1%, 22.7%, respectively. The survival rate of bisected embryos and oocytes was significantly lower than that of non-bisection embryos(62.5%). 2. The survival rate of bisected porcine embryos in cultured for 12, 24, 48, 72 hrs with 20% FCS＋TCM-199 medium were 26.9%, 19.2%, 19.2% and 11.5%, respectively. 3. The in-vitro developmental rate with and without zona pellucida of bisected porcine embryos by micromanipulator were 30.8%, 25.0%, respectively.

• 한국수정란이식학회지
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• 제7권2호
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• pp.81-88
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• 1992

Cryopreservation of mouse embryos biopsied at 4-cell stage was investigated by ultrarapid freezing. Four-cell embryos were obtained from ICR mice on 55h after hCG injection. Zona pellucida of the embryos were partially dissected with a cutting pipet, and then single blastomeres were biopsied from the embryos followed by incubation in $Ca^2$+ and $Mg^2$+-free M16 medium for 30min. Biopsied embryos cultured for lh or 15h were frozen by ultrarapid freezing method using 3M DMSO or 5M glycerol as a cryoprotectant, respectively. The developmental rate of biopsied embryos after ultrarapid freezing and thawing to blastocysts was 81 % in the group of biopsied embryos cultured for lb and 98% in the group of biopsied embryos cultured for 15h, respectively. When biopsied embryos after ultrarapid freezing and thawing were transferred to the uteri of pseudopregnant recipients, normal live young were born. These results suggest that this freezing method can efficiently cryopreserve biopsied mouse embryos.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2002년도 춘계학술발표대회 발표논문초록집
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• pp.48-48
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• 2002

Chimerism has become an important tool for investigating fundamental aspects of early embryonic development and differentiation in mammals for producing transgenic animals. The objective of this study was to evaluate the developmental capacity of chimeric embryos reconstructed with parthenotes and IVF bovine embryos into empty zona pellucida. The MII oocytes were activated by two treatment groups [Group 1, 5 μM inomycin, 5min, ＋ 10 ㎍/㎖ cycloheximide (CHX)/5 ㎍/㎖ cytochalasin B (CCB), 3 h; Group 2, 5 μM ionomycin, 5 min ＋ 1.9 mM 6-dimetylaminopurine (6-DMAP), 3 h]. (omitted)

• 한국가축번식학회지
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• 제20권2호
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• pp.179-190
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• 1996

Sexing and developing from splitted embryos which were fertilized in vitro implicate a possibility of production of the superior and sex controlled individuals. This study was carried out to investigate the production of transferable late blastocysts from in vitro fertilized embryos and to analyze sex by chromosome analysis from same embryos. In results, the ratio of cleavage and fertility of bovine follicular oocytes matured in vitro was 90% in co-cultured with granulosa cells. The competence of embryonic development from in vitro matured and fertilized bovine oocytes was 38% in co-cultured with bovine oviductal epithelial cells. To produce a lot of transferable embryos, therefore, the best conditon of culture system was co-cultured with granulosa cells for immature bovine oocytes and then co-cultured with bovine oviductal eptithelial cells for matured and fertilized oocytes. In chromosome analysis, 93% of in vitro fertilized embryos were very important aspect in chromosome preparation from bovine embryos such as duration of colcemid treatment, weakening of zona pellucida, methods of hypotonic treatment and fixation treatment.

• BMB Reports
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• 제35권6호
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• pp.604-608
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• 2002

The effects of progesterone on the acrosome reaction, as well as the effects of RU486 on the progesterone-induced acrosome reaction in capacitated boar spermatozoa, were investigated. Progesterone, a major steroid that is secreted by the cumulus cells of oocyte, clearly induced the acrosome reaction in a dose-dependent manner in capacitated boar spermatozoa, even though it failed to show similar effects in non-capacitated spermatozoa. RU486, a potent antiprogestin, significantly reduced the effects of progesterone on the progesterone-induced acrosome reaction; however, when treated alone, it showed no inhibitory effects on the acrosome reaction. The inhibitory effects of RU486 were also shown to be dose-dependent. These results imply that in addition to the well-known inducer of the acrosome reaction, zona pellucida, progesterone can also induce the acrosome reaction through its specific receptors on spermatozoa after the spermatozoa undergo capacitation.

• Reproductive and Developmental Biology
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• 제35권4호
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• pp.491-498
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• 2011

During mammalian fertilization, germ cell-specific hyaluronidases, such as sperm adhesion molecule 1 (SPAM1) and hyaluronoglucosaminidase 5 (Hyal5), are important for the dispersal of the cumulus mass. In this study, we demonstrated that bull Hyal5 is a single copy gene on chromosome 4 that is expressed specifically in the testis. In addition, we expressed recombinant bull SPAM1 and Hyal5 in human embryonic kidney 293T cells and showed that these enzymes possessed hyaluronidase activity. We also demonstrated that a polyclonal antibody against bull sperm hyaluronidase inhibits sperm-egg interactions in an in vitro fertilization (IVF) assay. Our results suggested that bull Hyal5 may have a critical role in bull fertilization.