• Reproductive and Developmental Biology
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• 제36권3호
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• pp.141-145
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• 2012

The present study was conducted to develop a simple method for porcine oocyte maturation without $CO_2$ regulation. In experiment 1, we evaluated that the effect of $CO_2$ non-supplement on porcine oocyte maturation. Cumulusoocyte complexes (COCs) were collected from 2~6 mm follicles and divided into three groups (Control, tube-$CO_2$, and tube-non-$CO_2$). For control, COCs were cultured in 4-well multidish in a $CO_2$ incubator. For tube-$CO_2$, COCs were cultured in a round-bottom tube in a $CO_2$ incubator, and for tube-non-$CO_2$, COCs were cultured in a round-bottom tube sealed tightly without $CO_2$ supplement in a dry incubator. The proportion of oocytes reached to metaphase II (M-II) was not significantly different among three groups (87.9% to 91.4%). In experiment 2, we evaluated the effect of $CO_2$ non-supplement during oocyte maturation on development of embryos. Oocytes with a polar body were divided into two groups (Control and tube-non-$CO_2$) and applied 1.1 kV/cm or 1.2 kV/cm voltages for parthenogenetic activation. After activation, embryos were cultured for 6 days and examined the development. The proportion of embryos cleaved was not significantly different among treatment (86.3% to 91.5%). The proportion of embryo reached to blastocyst stage was not significantly different among treatment (13.9% to 25.2%). The cell number of blastocysts was not significantly different among treatment (29.0 to 32.4). In conclusion, oocytes cultured in a dry incubator without $CO_2$ supplement have enough competence to development after parthenogenetic activation. These results would be useful for transporting oocytes or embryos a long distance.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2003년도 학술발표대회 발표논문초록집
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• pp.26-26
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• 2003

Hyaluronic acid (HA)-binding sites have been shown the diagnostic potential fur assessment of sperm maturity, which is related to male fertility. This study was designed to evaluate chromosomal patterns in porcine embryos produced by in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) with non- or HA-binding sperm (HABS). For binding of sperm with HA, sperm incubated in 10 ${mu}ell$ drop containing HA (0.8 mg/ml)-agarose (0.8%) mixture for 15 min. IVF and ICSI with non- or HA-bound sperm examined with matured oocytes at 44 hr after in vitro maturation. Embryos were cultured in 50 ${mu}ell$ of NCSU 23 containing 0.5% BSA for 5 days and then in 50 ${mu}ell$ of NCSU 23 containing 10% FBS for 2 days. For the evaluation of chromosomal aneuploidies, chromosome 1 sub-metacentric specific probe was used in sperm and embryos by fluorescence in situ hybridization (FISH). The frequency of aneuploidy sperm for chromosome 1 was 6.25%. The significant differences following IVF and ICSI with non- or HA-bound sperm were not observed in blastocyst formation rates (18.6, 23.5, and 23.8%) and cell number (61.8 $\pm$ 12.5, 55.5 $\pm$ 7.3, and 59.3 $\pm$ 9.6). Moreover, the percentage of diploidy in 4-cell stage embryos was 57.1% (IVF), 68.8% (ICSI), and 76.3% (ICSI-HABS). These results suggest that HA-binding sites may be a material for selection of normal sperm for ICSI. Therefore HA selection of normal sperm may be reduce the loss to embryonic mortality prior to embryo transfer in pig.

• Journal of Animal Science and Technology
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• 제66권5호
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• pp.936-948
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• 2024

The Conical 9 well dish (C9 well dish) is characterized by a decreasing cross-sectional area towards the base. This design was hypothesized to enhance embryonic development by emulating the in vivo physical environment through density modulation. Comparative analyses revealed no significant difference in nuclear maturation rates between the C9 well dish and the 5-well dish. Reactive oxygen species (ROS) generation was lower in the C9 well dish compared to the 5-well dish; however, this difference was not statistically significant. On the second day of in vitro culture, the cleavage rate in the C9 well dish was 4.66% higher, although not statistically significant, and the rates of blastocyst development were similar across both dishes. No significant differences were observed in the intracellular levels of glutathione (GSH) and ROS, as well as in the total cell number within the blastocysts between the dish types. The expression of mitogen-related factors, TGFα and IGF-1, in the blastocysts was consistent between the dishes. However, PDGFβ expression was significantly lower in the C9 well dish compared to the 35 mm petri dish. Similarly, the expression of the apoptosis factor Bax/Bcl2l2 showed no significant differences between the two dishes. Despite the marked difference in PDGFβ expression, its impact on blastocyst formation appeared negligible. The study also confirmed the feasibility of culturing a small number of oocytes per donor, collected via Ovum Pick-Up (OPU), with reduced volumes of culture medium and mineral oil, thus offering economic advantages. In conclusion, the present study indicates that the C9 well dish is effective for in vitro development of a small quantity of oocytes and embryos, presenting it as a viable alternative to traditional cell culture dishes.

• Reproductive and Developmental Biology
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• 제28권2호
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• pp.113-117
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• 2004

본 연구는 돼지 난포란을 이용하여 생산된 1-세포기의 체외수정란을 NCSU-23, PZM-3 및 PZM-4의 세 가지 배양액과 서로 다른 산소농도를 부여하여 돼지 체외수정란의 적합한 체외배양조건을 구명하고자 실시하였다. 돼지 난포란의 체외성숙은 BSA가 미첨가 된 NCSU-23 배앙액에 10% pFF, 0.9 mM crysteine, 25 $\mu/ml \beta$-mercaptoethanol 및 10 ng/$\mu/ml$ epidermal growth factor와 호르몬(10 IU/$ml$ PMSG, hCG)을 첨가하여 20∼22시간과 추가로 호르몬을 제거한 배양액에 20∼22시간을 배양하여 성숙을 유기하였고, 5∼6시간 동안 돼지 액상정액과 공배양함으로써 체외수정을 유기하였다. 체외수정 5∼6시간후 각각 5% 및 20%의 산소조건하의 NCSU-23, PZM-3 및 PZM4 배양액에서 배발달을 유기하였다. 돼지체외수정란을 체외배양하였을 때, 배발달 48시간에 처리구간 난할율에는 차이가 없었으나, 배양 7일째 배반포형성률은 5% 산소조건의 PZM-3 배양액에서 가장 높게 나타났다(19.9 $\pm$ 2.4 vs. 11.1$\pm$2.0 to 16.0$\pm$2.5%, P<0.05). 그리고, 총세포수에 있어서 5%의 산소조건 하에서 배양하는 것이 20% 산소조건보다 유의적(P<0.05)으로 높았으나, 배양액간 차이는 인정되지 않았다. 따라서, 체외생산된 돼지초기수정란의 체외배발달은 5% 산소조건하의 PZM-3 배양액에서 배양하는 것이 좋은 것으로 나타났다.

• Clinical and Experimental Reproductive Medicine
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• 제21권1호
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• pp.77-81
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• 1994

To improve in vitro embryonic cell development, this study was desigend to culture in vitro fertilized early embryos of mouse in two different systems; conditioned medium alone and ampullary cells co-culture. Thirty two of 83 embryos(38.6%) were blocked in the 2 cell stage by co-culture, as compared to forty of 42 embryos(95.2%) in control group for 24hours culture. And all the embryonic cells cultured for conditioned medium alone were blocked for 48 hours culture. Twenty seven of 46 embryos (58.7 %) which overcome culture block in 2 cell stage by cocultured were developed morular and expanded blastocyst, and ninteen of 46 embryos(26.1 %) underwent hatching for 96 hours culture. The cellular fragmented rates for embryo were 26.2% in medium alone; 10 fragmented blastomere were graded mild status and 1 fragmented blastomere in severe status. On the other hand, the fragmented rate for 48 hours co-cultured were 15.7%03/83); 8 fragmented embryos were graded mild status, moderate status in 3 fragmented embryos and severe in 2 fragmented embryos respectively. In conclusion, the co-culture of embryos with ampullary cells is good to improve quality of embryos and overcome of culture block as well as development of cell cleavage.

• Molecules and Cells
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• 제40권8호
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• pp.558-566
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• 2017

Regular monitoring on experimental animal management found the fluctuation of ART outcome, which showed a necessity to explore whether superovulation treatment is responsible for such unexpected outcome. This study was subsequently conducted to examine whether superovulation treatment can preserve ultrastructural integrity and developmental competence of oocytes following oocyte activation and embryo culture. A randomized study using mouse model was designed and in vitro development (experiment 1), ultrastructural morphology (experiment 2) and functional integrity of the oocytes (experiment 3) retrieved after PMSG/hCG injection (superovulation group) or not (natural ovulation; control group) were evaluated. In experiment 1, more oocytes were retrieved following superovulation than following natural ovulation, but natural ovulation yielded higher (p < 0.0563) maturation rate than superovulation. The capacity of mature oocytes to form pronucleus and to develop into blastocysts in vitro was similar. In experiment 2, a notable (p < 0.0186) increase in mitochondrial deformity, characterized by the formation of vacuolated mitochondria, was detected in the superovulation group. Multivesicular body formation was also increased, whereas early endosome formation was significantly decreased. No obvious changes in other microorganelles, however, were detected, which included the formation and distribution of mitochondria, cortical granules, microvilli, and smooth and rough endoplasmic reticulum. In experiment 3, significant decreases in mitochondrial activity, ATP production and dextran uptake were detected in the superovulation group. In conclusion, superovulation treatment may change both maturational status and functional and ultrastuctural integrity of oocytes. Superovulation effect on preimplantation development can be discussed.

• 한국동물생명공학회지
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• 제39권2호
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• pp.131-137
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• 2024

Background: Porcine embryonic development is widely utilized in the medical industry. However, the blastocyst development rate in vitro is lower compared to in vivo. To address this issue, various supplements are employed. Extracellular vesicles (EVs) play the role of communicators that carry many bioactive cargoes. Additionally, the contents of EVs can vary on the estrous cycle. Methods: We compared the effects of adding EVs derived from porcine uterine fluid (UF), categorized as non-EV (G1), EVs in estrus (G2) and EVs in diestrus (G3). After in vitro culture (IVC) was performed in three different groups, cleavage rate and blastocyst development rate were examined. In addition, glutathione (GSH) and reactive oxygen species (ROS) levels were measured 2 days after activation to assess oxidative stress. Results: Using NTA and cryo-TEM, we confirmed the presence of EVs with sizes ranging from 30 nm to 200 nm, that the particles were suitable for analysis for analysis. In IVC data, the highest cleavage rate was observed in G2, which was significantly different from G1 but not significantly different from the next highest, G3. Similarly, the highest blastocyst development rate was observed in G2, which was significantly different from G1 but not significantly different from the next highest, G3. Conclusions: These results indicate that estrus derived EVs contain biofactors beneficial for early blastocyst development, including GSH which protects the blastocyst from oxidative stress. Additionally, although diestrus-derived EVs are expected to have some effect on blastocyst development, it appeared to be less effective than estrus-derived EVs.

• Journal of Microbiology and Biotechnology
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• 제29권8호
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• pp.1193-1203
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• 2019

We investigated the protective effects of 3-bromo-4,5-dihydroxybenzaldehyde (BDB) from Polysiphonia morrowii Harvey against hydrogen peroxide ($H_2O_2$)-induced apoptosis in Vero cells. BDB exhibited scavenging activity for DPPH, hydroxyl, and alkyl radicals. BDB also inhibited $H_2O_2$-induced lipid peroxidation, cell death, and apoptosis in Vero cells by inhibiting the production of ROS. To evaluate the molecular mechanisms of apoptosis inhibition, the expression of Bax/Bcl-xL and $NF-{\kappa}B$ was assessed by western blot assay. BDB significantly suppressed the cleavage of caspase-9 and PARP and reduced Bax levels in $H_2O_2$-induced Vero cells. Besides, BDB suppressed the phosphorylation of $NF-{\kappa}$B and the translocation of p65 in $H_2O_2$-induced cells. Furthermore, we evaluated the effect of BDB on ROS production, cell death, and lipid peroxidation in an $H_2O_2$-stimulated zebrafish embryo model. Taken together, these results indicated that ROS generation and cell death were significantly inhibited by BDB in zebrafish embryos, thereby proving that BDB exerts excellent antioxidant activity in vitro and in vivo.

• Journal of Veterinary Science
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• 제25권3호
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• pp.40.1-40.19
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• 2024

Importance: The creation of robust maternal-embryonic interactions and implantation models is important for comprehending the early stages of embryonic development and reproductive disorders. Traditional two-dimensional (2D) cell culture systems often fail to accurately mimic the highly complex in vivo conditions. The employment of three-dimensional (3D) organoids has emerged as a promising strategy to overcome these limitations in recent years. The advancements in the field of organoid technology have opened new avenues for studying the physiology and diseases affecting female reproductive tract. Observations: This review summarizes the current strategies and advancements in the field of 3D organoids to establish maternal-embryonic interaction and implantation models for use in research and personalized medicine in assisted reproductive technology. The concepts of endometrial organoids, menstrual blood flow organoids, placental trophoblast organoids, stem cell-derived blastoids, and in vitro-generated embryo models are discussed in detail. We show the incorportaion of organoid systems and microfluidic technology to enhance tissue performance and precise management of the cellular surroundings. Conclusions and Relevance: This review provides insights into the future direction of modeling maternal-embryonic interaction research and its combination with other powerful technologies to interfere with this dialogue either by promoting or hindering it for improving fertility or methods for contraception, respectively. The merging of organoid systems with microfluidics facilitates the creation of sophisticated and functional organoid models, enhancing insights into organ development, disease mechanisms, and personalized medical investigations.

• Clinical and Experimental Reproductive Medicine
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• 제24권3호
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• pp.347-353
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• 1997

본 실험은 초자화 동결된 생쥐 배반포기배의 융해 후 배양조건 및 이식방법이 난자의 생존에 미치는 효과를 조사하고자 실시하였다. 체외수정후, M16배양액에서 4일동안 배양하여 얻어진 생쥐 배반포기배는 EFS40 (40% ethylene glycol, 18% Ficoll, 0.5 M sucrose가 함유된 PBS)으로 초자화동결하였다. 실험 I에서는 융해 후 배양조건에 따른 난자들의 체외/체내 생존율을 조사하였다. 융해된 난자가 M16과 4 mg/ml 소혈청알부민과 20 가지 아미노산이 함유된 m-CR1 (2% BME 아미노산 용액, 1% MEM 아미노산 용액) 및 단층배양이 유도된 난구세포 (10% FBS가 함유된 m-CR1배양액)에서 각각 배양되었을 때, 융해 후 24시간째 체외 생존율은 배양조건에 따라 차이가 없었다(75.6, 83.1, 82.4%). 그러나 체내 발달율에 있어서 임신 15일째 생존 산자율은 39.0, 49.0, 38.1%로서 유사한 성적을 나타냈으나, 전체 착상율에 있어서는 m-CR1 (80.4%)에 배양되었을 때, M16 (51.2%), 난구세포와 공배양시 (57.1%)보다 유의하게 높은 생존율을 보였다(p<0.05). 실험 II에서는 수정란 이식 방법에 따른 체내 발달율을 조사하였다. 배반포기배를 융해 후 체외배양없이 곧바로 가임신 2, 3일째 대리모에 이식을 실시하였을 때, 가임신 2일째 대리모에서는 임신징후를 얻지 못하였고, 가임신 3일째 대리모에서는 50.0%의 착상율과 15.4%의 정상산자율을 얻었다. 그러나, 이러한 결과는 융해 후, 16시간 배양하여 가임신 3일째 대리모에 이식 (73.5, 57.1%)하는 경우보다 유의하게 낮은 결과였다(p<0.05). 실험 III에서는 초자화 동결된 배반포기배의 융해 후 배양시 발달이 늦어진 수정란의 이용효율을 극대화시키기 위해 융해한 4일째 초기, 5일째 초기, 5일째 팽창 배반포기배의 체외/체내 생존율을 조사하였다. 가장 높은 체외 생존율은 5일째 팽창 배반포기배 (78.3%)에서 얻었으나, 체내 발달율 (산자율, 착상율)에 있어서는 4일째 초기 배반포기배 (33.3, 66.7%)의 경우가, 5일째 팽창 배반포기배(29.0, 38.7%)의 경우보다 높았다(p<0.05). 따라서 본 연구의 결과는 배양조건과 수정란 이식방법에 따라 초자화 동결된 배아의 체외/체내 발달율을 높일 수 있으며, 발달이 늦은 배반포기배의 체내 발달율은 체외 배양시간이 길어질수록 낮아짐으로, 5일째 팽창 배반포기배보다 4일째 초기 배반포기배를 동결하는 것이 더 유용하다는 것을 알 수 있었다.