• Clinical and Experimental Reproductive Medicine
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• v.25 no.2
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• pp.207-216
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• 1998

Certain types of somatic cells, as well as follicular cumulus cells associating with mammalian oocytes, are known to produce beneficial effects on in vitro fertilization and pre implantation development of mammalian eggs when they are present in oocyte culture medium. To investigate the nature of the effects of somatic cells, the resistance of mouse oocytes against chymotrypsin treatment was examined after culture within various cell conditioned media. When mouse oocytes matured for 17-18 hr in the presence of cumulus cells were treated with 1 % chymotrypsin, half of them remained still alive even after 240 min $(t_{50}>240.0)$. In contrast half of mouse oocytes cultured without cumulus cells underwent degeneration within 65.0 min $(t_{50}=65.0{\pm}13.2min)$ of the same treatment. To see if the effects were duc to the secretory products of cumulus cells, mouse cumulus cells were cultured for 20 hr in medium containing 0.4% BSA and the supernatant of culture medium (conditioned medium) was taken. After maturation in the cumulus cell conditioned medium, mouse oocytes exhibited $t_{50}=190.0{\pm}10.8$ min upon chymotrypsin treatment whereas half of oocytes cultured without conditioned medium degenerated within 25.5 min. Human granulosa cell conditioned medium gave similar effects such that oocytes matured in conditioned medium exhibited $t_{50}=183.3{\pm}19.1$ min while $t_50$ of control group oocytes was $60.0{\pm}6.8$ min, Oocytes matured in vero cell conditioned medium exhibited $t_{50}=196.7{\pm}8.8$ min. On the other hand, amniotic cell conditioned medium resulted in the chymotrypsin resistance of $t_{50}=80.0{\pm}8.4$ min which was not statistically different from the control value of $t_{50}=48.0{\pm}13.2$ min. Based upon these results, it is suggested that certain somatic cell types including cumulus cells might change the biochemical properties of mouse oocyte membrane during meiotic maturation as revealed by the enhanced resistance against chymotrypsin treatment. Such effects of somatic cells appear to be mediated via the secretory products rather than direct communication between somatic cells and oocytes.

• Clinical and Experimental Reproductive Medicine
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• v.27 no.2
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• pp.165-172
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• 2000

Objective: In vitro fertilization (IVF) and a prolonging the time of culture may be helpful in establishing a viable pregnancy through a selection effect. Some embryos do not develop beyond the 4-cell stage and some may not develop to the blastocyst stage. We have evaluated the safety of SET and the outcomes of pregnancy. Methods: Sperms were treated with Ham's F-10 supplemented with 10% human follicular fluid (hFF). oocytes or fertilized oocytes were cultured in Dulbecco's Modified Eagle Medium (DMEM) with 10% or 20% hFF respectively. Up to five oocytes were inseminated with approximately 200,000 sperm cells/2 ml in each well. Fertilization was examined in the following morning and fertilized oocytes were co-cultured until embryo transfer. Vero cells for co-culture were prepared in Tissue Culture Medium - 199 (TCM-199) with 10% fetal bovine serum. At the two to four cell and blastocyst on day 2 and day 5, embryo and blstocyst grading were evaluated. Pregnancy rate was determined after transfer of human embryos at the two to four cell stage on day 2 (Group I) or subsequent transfer of embryos on day 2 and at the blastocyst stage on day 5 (Group II). For statistical analysis, Student's t-test and Chi-square (${\chi}^2$_test) were used. Results were considered statistically significant when p value was less than 0.05. Results: No differences was found in the fertilization between Group I (81.0%, 98/121) and Group II (81.8%, 180/220). In case of cleavage rate, no difference was found in Group I (95.9%, 94/98) and Group II (97.8%, 174/178). However, the rate of-clinical pregnancy was significantly higher (p=0.014) in Group II (66.7%, 12/18) than in Group I (26.3%, 5/19). Conclusion: The results of this study showed that SET is safe and effective, and significantly increases the pregnancy rate.

• Journal of Embryo Transfer
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• v.18 no.3
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• pp.237-242
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• 2003

These studies were carried out to establish an effective in vitro embryo transfer methods by analyzing several factors. The base media were TCM-199 solution for in vitro maturation(IVM) of bovine follicular oocytes and Fer-TALP solution for in vitro fertilization(IVF) and CRlaa medium for in vitro culture(IVC). IVC used the fertilized oocytes of 24-hr culture (day 1)after IVF. Embryos were cultured in drop-culture that contained 25 embryos per 10${mu}ell$. Blastocysts cultured for 7 to 9 in vitro were transferred to recipients. The results obtained were as follows: 1. The pregnancy rate according to different region of embryo transfer were 33.8％, 48.1％, 45.0％ and 35.3％ respectively. 2. The pregnancy rate according to the parity of recipient when embryos were transferred to nulliparous (42.9％) was higher than that of 1∼3nd parlous(36.9％), however there were not show significant difference each other. 3. According to the stage of blastocyst, the pregnancy rate when middle blastocysts (MB) (45.5％) were transferred to recipients were higher than that of late blastocysts (LB) (41.0％). 4. When IVF-derived blastocysts cultured for 7 to 9 day were transferred to recipients, the pregnancy rate was higher 7 day of blastocyst than that of 8 day or 9 day of blastocyst. The results of embryo transfer according to the regions, the parity of recipient and the development stage showed that blastocyst formed for 7day transferred to nulliparou were higher pregnancy rate than others.

• Journal of Embryo Transfer
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• v.30 no.3
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• pp.161-170
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• 2015

These studies were conducted to establish the practical Hanwoo (Korean native cattle) improvement system through the combining of embryo transfer technology and confirming individual Hanwoo oocyte culture system and to investigate that correlation of Hanwoo carcass classification (intramuscular marbling) and oocyte donor for blastocyst production in vitro. In case of Hanwoo, the carcass meat quality grades were divided to grade $1^{{+}{+}}$, $1^{+}$, 1, 2, and 3 depends on fat distribution of longest muscle cross-sectional surface. As results, the numbers of follicular oocytes collected from individual fundamentally-registered Hanwoo yielded $1^{{+}{+}}$, $1^{+}$, 1, 2 and 3 meat quality were 9.5, 9.4, 8.5, 8.8 and 8.8 per ovary, respectively. The numbers of retrieval oocyte from follicles were significantly higher in the cattle yield $1^{{+}{+}}$, $1^{+}$ meat quality than in the cattle yield 1, 2 and 3 meat quality (p<0.05). The rates of blastocyst formation were 18.2, 21.3, 29.4, 30.9, and 31.5% in the cattle yield $1^{{+}{+}}$, $1^{+}$, 1, 2 and 3 meat quality of after in vitro maturation, respectively. It was significantly lower in the cattle yield $^{{+}{+}}$ and $1^{+}$ meat quality than in the cattle yield 1, 2 and 3 meat quality (p<0.05). In order to evaluate embryos quality, TUNNEL assay was conducted for each meat quality grade using blastocyst stage embryo on day 8. The results showed that apoptosis cell number was higher tendency in the cattle yield $1^{{+}{+}}$and $1^{+}$ meat quality (81 and 79, respectively) than in the cattle yield 1, 2 and 3 meat quality (51, 48 and 50, respectively) but there was no statistical significance in each group. After embryo transfer, the conception rate of recipient was 53.5 (23 out of 43), 52.1 (38 out of 73) and 58.0% (58 out of 100) in the meat quality of 1, $1^{+}$ and $1^{{+}{+}}$, respectively. These results showed that the conception rate was significantly higher in the cattle yield 1 meat quality than in the cattle yield $1^{{+}{+}}$, $1^{+}$, 2, and 3 meat quality (p<0.05). In summary, these results indicate that the application of confirming Hanwoo individual oocyte culture system and embryo transfer technology can make good use of the genetic resources conservation and improvement of Hanwoo. Relevance of the meat quality grade and reproductive ability of carcasses of Hanwoo will be considered to be one of the effective means for the associated research with obesity and reproduction.

• Korean Journal of Animal Reproduction
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• v.26 no.2
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• pp.105-110
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• 2002

The objective of this study was to determine the developmental competence of in vitro matured oocytes after intracytoplasmic sperm injection(ICSI) with epididymal spermatozoa. The ovaries were obtained from slaughtered small species dogs. Oocytes matured in vitro for 24 hrs were fertilized by ICSI with epididymal spermatozoa. After ICSI, one group of oocytes was activated with 2.0 mM dimethylaminopurine or 7% ethanol for 5 min. and second group was not activated. The follicular oocytes were cultured in synthetic oviductal fluid(SOF) and TCM-199 medium containing hormones and 10% FCS for 24~48 hrs in a incubator with 5% $CO_2$ in air at 38.5$^{\circ}C$. 1. Results of IVM showed that the percentage of oocytes reaching MII after 24 h and 48 hrs of incubation were significantly higher(p<0.05) after culture with 48 hrs(9/30, 30.0%) than that after culture with 24hrs(a/30, 26.7%). 2. Results of IVM showed that the percentage of oocytes reaching MII after 48 hrs of incubation were significantly higher(p<0.05) after culture with SOF media(10/30, 30.3%) than TCM-199 media (7/30, 23.3%). 3. The rate of cleavaged embryos to blastocyst obtained by ICSI treated activation oocytes was significantly higher(p<0.05) than that of nonactivation oocytes(5/16, 25.0% vs 1/13, 5.0%). 4. The rates of development of cleavaged embryos to blastocyst obtained by ICSI treated sperm of fresh, epididymal and frozen-thawed epididymal were 8/18(44.43%), 5/16(31.3%), 2/14(14.3%), respectively. and these values of frozen-thawed epididymal sperm injection were lower than fresh sperm injection.

• Reproductive and Developmental Biology
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• v.28 no.2
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• pp.113-117
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• 2004

The present study was carried out to examine the effects of $O_2$ concentrations and culture media (North Carolina State University (NCSU)-23, porcine zygote medium(PZM)-3 or PZM-4) on in vitro development of porcine IVM/IVF embryos. Porcine oocyte-cumulus complexes were cultured in BSA-free NCSU-23 medium containing porcine follicular fluid (10%), cysteine (0.9 mM), $\beta$-mercaptoethanol (25 $\mu\textrm{g}$/$m\ell$), epidermal growth factor (10 ng/$m\ell$) and hormonal supplements (PMSG and hCG: 10 IU/$m\ell$) for 20∼22 h. They were then cultured in the same medium but without hormonal supplements for an additional 20∼22 h. After culture, cumulus-free oocyte were coincubated with liquid boar spermatozoa for 5∼6h. Putative zygotes were transferred to NCSU-23, PZM-3 and PZM-4 medium under the condition of 5% $O_2$ or 20% $O_2$ concentrations. At 48 h, no mean differences were found in cleavage rates. However, the rates of blastocyst formation at day 7 after in vitro fertilization were significantly higher in PZM-3 medium under the condition of 5% $O_2$ concentration than other treatments (19.9$\pm$2.4 vs. 11.1$\pm$2.0 to 16.0$\pm$2.5%, P<0.05). The total cell numbers of blastocysts were significantly higher in 5% $O_2$ than in 20% O2 (P<0.05). However, no differences was found among the culture media within each $O_2$ concentrations. In conclusion, the use of PZM-3 medium in 5% $O_2$ concentration was effective on in vitro development of porcine IVM/IVF embryos.

• Reproductive and Developmental Biology
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• v.36 no.4
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• pp.255-260
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• 2012

The purpose of this study was to assess follicular viability and competence through in vitro culture of preantral follicles isolated from vitrified mouse whole ovaries. Mouse preantral follicles were enzymatically isolated from vitrified- warmed and fresh ovaries and cultured for 10 days followed by in vitro oocyte maturation. In vitro matured oocytes were fertilized and cultured to the blastocyst stage. Five minutes pre-exposure to vitrification solution of whole ovaries had significantly higher (p<0.05) oocyte survival and maturation rates than between 10 min exposure groups. Oocyte diameter was significantly smaller (p<0.05) in the 5 and 10 min exposure groups ($69.4{\pm}2.8$ and $67.8{\pm}3.1$) when compared to that of control group ($71.7{\pm}2.1$). There was no statistical significant difference in blastocyst development rates between vitrification group (8.6%) and the fresh control group (12.0%). The mean number of cells per blastocyst was significantly lower (p<0.05) in the vitrification group ($41.9{\pm}20.2$) than in the fresh control group ($55.1{\pm}22.5$). The results show that mouse oocytes within preantral follicles isolated from the vitrified whole ovaries can achieve full maturation, normal fertilization and embryo development.

• 한국생물공학회:학술대회논문집
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• 2003.10a
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• pp.365-368
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• 2003

Hair follicles develop as a result of epithelial-mesenchymal interactions between epidermal keratinocytes and dermal cells. Moreover hair follicles constitute multiple cells that influence hair follicle development and cyclic activity. We isolated some cells using explantation and enzymatic digestion method from human scalp hair follicles. So we could culture some follicular cells, such as outer root sheath (ORS) cells, dermal papilla (DP) cells, dermal sheath (DS) cells, matrix cells and melanocyte.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.118-118
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• 2003

The aim of this study was to investigate the effect of glucose on embryonic development of mouse embryos. Two cell embryos were recovered from ICR female mice(3-4weeks) at 46~50 hrs after hCG 5 IU injection (mated just after hCG injection) and cultured in 50 $\mu m$ DMEM droplets supplemented with nothing (control: n=46), glucose 0.5mM (Group A; n=46) or glucose 3.15 mM(Group B; n=46) under mineral oil. All experimental media were supplemented with 20% human follicular fluid. Total blastocyst formation rates was lower (NS) in glucose groups (group A: 52.2% : B. 47.8%) than control group (60.9%). ZiB rates was the highest (P<0.05) in control (47.8%) than those in group A (21.7%) and B (28.3%). ZeB rates were the highest (NS) in group A (30.4%) than those in control (13.0%) and group B (19.6%). Blastocysts, cultured in group B (50.5), had the highest (NS) mean cell number compared with the others (control: 39.2 ; group A: (45.6). The ICM proportion (% ICM of total cells) in blastocysts cultured in group A (20.6%) was the highest (NS) than those of other tested groups (control: 15.2 ; group B: 13.9%). This study shows that a low dose of glucose added to culture medium increases the ICM proportion of blastocysts in mice.

• Journal of Animal Reproduction and Biotechnology
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• v.34 no.3
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• pp.240-246
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• 2019

The aim of this study was to investigate effect of heat stress on expression levels of plasminogen activators (PAs) related mRNAs and proteins, and changes of PAs activity in porcine endometrial explants. The endometrial explants (200 ± 50 mg) were isolated from middle part of uterine horn at follicular phase (Day 19-21) and were pre-incubated in serum-free culture medium at 38.5℃ in 5% CO₂ for 18 h. Then, the tissues were transferred into fresh medium and were cultured at different temperature (38.5, 39.5, 40.5 or 41.5℃) for 24 h. The expression level of urokinase-type PA (uPA), type-1 PA inhibitor (PAI-1), type-2 PAI (PAI-2), and heat shock protein-90 (HSP-90) mRNA were analysis by reverse-transcription PCR and proteins were measured by western blotting. The supernatant were used for measurement of PAs activity. In results, mRNA and protein levels of HSP-90 was higher in 41.5℃ treatment groups than other treatment groups (p < 0.05). The expression of uPA, PAI-1, and PAI-2 mRNA were slightly increased by heat stress, however, there were no significant difference. Heat stress condition suppressed expression of active uPA and PAI-2 proteins (p < 0.05), whereas PAI-1 protein was increased (p < 0.01). Although PAI-1 protein was increased and active uPA was decreased, PAs activity was greatly enhanced by exposure of heat stress (p < 0.05). These results suggest that heat stress condition could change intrauterine microenvironment through regulation of PAs activity and other factors regarding with activation of PAs might be regulate by heat stress. Therefore, more studies regarding with regulatory mechanism of PAs activation are needed.