• 한국동물학회지
• /
• 제36권1호
• /
• pp.109-115
• /
• 1993

본연구는 북방산개구리 여포난자의 자발적 성숙과 관련된 몇가지 요인들에 대해 조사하였다. 자발적 성숙을 일으키는 여포에서 여포조직을 제거하여 denuded 난자를 만든다음 배양했을 때에는 자발적 성숙을 일으키지 않았다. 이들 denuded 난자에 frog pituitary homogenate(FPH, 0.05 gland/ml)를 처리하여도 역시 성숙이 일어나지 않았으나 progesterone(P4, 0.5 Ig/ml)을 처리하였을 때에는 성숙이 일어났다. 동면기간에 여포들이 생성하는 P4와 testosterone(T)의 양을 측정해본 결과 동면초에는 비슷한 양의 P4와 T를 생성하였으나 후기에는 P4만 생성하고 T는 거의 생성하지 않았다. 배양액에 estradiol을 첨가하면 농도에 의존하여 FPH에 의해 촉진된 P4의 생성을 억제할 뿐 아니라 성숙도 비가역적으로 억제하였다. 배양액에 첨가한 hvpoxanthine은 CAMP와 항진적으로 난자의 성숙을 억제하였다. 이러한 결과들은 자발적 성숙과정에 여포세포와 P4/T 비율의 증가가 중요한 요인이 된다는 것과 일반적인 난자성숙억제제는 자발적 성숙을 역시 억제한다는 것을 보여준다.

• 한국발생생물학회지:발생과생식
• /
• 제17권1호
• /
• pp.63-72
• /
• 2013

A specific inhibitor of RNA polymerase II, ${\alpha}$-amanitin is broadly used to block transcriptional activities in cells. Previous studies showed that ${\alpha}$-amanitin affects in vitro maturation of cumulus-oocyte-complex (COC). In this study, we evaluated the target of ${\alpha}$-amanitin, and whether it affects oocytes or cumulus cells (CCs), or both. We treated ${\alpha}$-amanitin with different time period during in vitro culture of denuded oocytes (DOs) or COCs in comparison, and observed the changes in morphology and maturation status. Although DOs did not show any change in morphology and maturation rates with ${\alpha}$-amanitin treatment, oocytes from COCs were arrested at metaphase I (MI) stage and CCs were more scattered than control groups. To discover causes of meiotic arrest and scattering of CCs, we focused on changes of cumulus expansion, gap junctions, and cellular metabolism which to be the important factors for the successful in vitro maturation of COCs. Expression of genes for cumulus expansion markers (Ptx3, Has2, and Tnfaip6) and gap junctional proteins (Gja1, Gja4, and Gjc1) decreased in ${\alpha}$-amanitin-treated CCs. However, these changes were not observed in oocytes. In addition, expression of genes related to metabolism (Prps1, Rpe, Rpia, Taldo1, and Tkt) decreased in ${\alpha}$-amanitin-treated CCs but not in oocytes. Therefore, we concluded that the transcriptional activities of CCs for supporting suitable transcripts, especially for its metabolic activities and formation of gap junctions among CCs as well as with oocytes, are important for oocytes maturation in COCs.

• 한국수정란이식학회지
• /
• 제10권1호
• /
• pp.45-51
• /
• 1995

In order to determine the optimum condition and timing for in vitro maturation of oocytes to metaphase of meiosis II (M II), the immatured follicular oocytes were recovered by puncturing the large(1.0~1.5 mm in diameter) and small(<1.0 mm in diameter) follicles in the ovaries of rabbits treated intramuscularly with a single dose of 100 TU PMSG 68 hours previously. The follicular oocytes were classified into three grades by the attachment of cumulus cells. The Grade I and II follicular oocytes from large follicles were cultured in BO-DM medium with 10% FCS, 35 $\mu$g /nl of FSH, 10 $\mu$g /ml of LH and 1 $\mu$g /ml of estradiol-17$\beta$ at 39t in a 5% $CO_2$ incubator for 11 to 23 hours. In 3 hours interval during the culture period, the oocytes were harvested and their cumulus cells were removed with hyaluronidase. The denuded oocytes were stained with Hoechst 33342 dye and their meiotic status and extrusion of the first polar body (PB) were examined under a fluorescence microscope. Also the fragmentation of the first PB and the distance between the first PB and nucleus were examined. The results obtained were as follows: 1. The mean recovery rate of follicular oocytes from the large and small follicles was 59. 9 and 31.3%, respectively. The mean number of oocytes recovered per rabbit and the Grade I percentage were 14.6 and 94.4% in large follicles, but 2.1 and 61.1% in small follicles, respectively. All the parameters examined were different significantly (p<0.05) between both the folliclular size. 2. Most of the follicular oocytes(86.8%) were matured in vitro to M II phase in 14 hours in Grade I oocytes, but the significantly(p<0.05) less oocytes(45.5%) were matured in Grade II oocytes. 3. The first PB was extruded in most of the oocytes(94.7%) in 14 hours of culture with the fragmentation rate of 29.6%, but the fragmentation rate of the first PB increased significantly (p<0.05) as the culture period for maturation was longer to 20 hours(63.5%). 4. The distance between the first PB and nucleus was increased linearly (p<0.05) as the maturation time passed from 14(7.1$\mu$rn) to 23 hours(58.4$\mu$m). 5. From the above results it was concluded that the optimum time for in vitro maturation culture might be 14 hours in the follicular oocytes from rabbit primed with PMSG for 68 hours, expecially when these follicular oocytes were used for recipient cytoplasms in embryo cloning.

• 한국동물번식학회:학술대회논문집
• /
• 한국동물번식학회 2001년도 춘계학술발표대회
• /
• pp.66-66
• /
• 2001

Since the first birth of pig derived from embryonic cells by nuclear transfer, many researches to produce cloned pig have been carried out. Recently, two reports about the birth of somatic cell cloned pigs using in vivo oocytes and also Betthauser et al. (2000) reported the birth of somatic cell cloned pigs using in vitro oocytes. So here we investigated the effect of activation method and culture medium on in vitro development of porcine nuclear transfer embryo using fetal fibroblast. Oocytes derived from slaughter house obtained ovaries were matured for 42 to 44 h in TCM 199. Matured oocytes were denuded using 0.1% hyaluronidase and then Oocytes with the first polar body were used for enucleation by aspirating the first polar body and adjacent cytoplasm in TCM 199 supplemented with 7.5 $\mu\textrm{g}$ cytochalasin B. Petal fibroblast cells were prepared from 35 days old fetus. To be used as donor cells, fetal fibroblast cells were serum starved for 3 to 5 days and then isolated into single co:1 by trypsinization. Nuclear transfer embryos were fused using 2 times 1.25㎸ for 30$mutextrm{s}$. Fused NT embryos were activated with calcium ionophore (CI) and 6-dimethyl-aminopurine (6-DMAP). Activated oocytes were cultured in NCSU 23 or BECM 3 for 6 days. There was no significant difference between chemical activation and no chemical activation for blastocyst development rate(11.6 vs. 14.8%). However, cell number was significantly higher when NT embryos were activated with CI and 6-DMAP (31.2 vs. 22.6). When NT embryos were cultured in NCSU 23 or BECM 3, blastocyst development rate was 16.4 and 13.2%, respectively, and cell number was 31.5 and 24.1, respectively. These results suggest that chemical activation after fusion and culture in NCSU 23 could increase cell number of porcine NT embryos.

• Asian-Australasian Journal of Animal Sciences
• /
• 제14권10호
• /
• pp.1353-1359
• /
• 2001

The present study was undertaken to assess the influence of dimethyl-sulfoxide plus sucrose solution as a cryoprotectant on the adenosine triphosphate (ATP) content, the ultrastructure and the embryonic development of bovine oocytes matured in vitro. We measured the amount of ATP in cumulus cells enclosed oocytes (CO) or denuded oocytes (DO) equilibrated with or removed from the cryoprotectant (1.5 M DMSO + 0.25 M sucrose + 20% fetal bovine serum in physiological saline). As a result, the ATP contents in both CO and DO, equilibrated with the cryoprotectant, were significantly lower (p<0.05) than that of the each control group. However, ATP content of DO was recovered to the level of the control group ailer removal of the cryoprotectant, but failed to restore for CO. In the observation of the ultrastructure by a transmission electron microscope, all of the mitochondria in the ooplasm of CO and DO equilibrated with the cryoprotectant were swollen with peripherally located cristae following decreased electron density. However, a large proportion of these swollen mitochondria were restored to the normal shape which can be observed usually in the control group after removal from the cryoprotectant. To the contrary, the morphology of many mitochondria of the cumulus cells in CO were not recovered to that of the control group after removal of the cryoprotectant. CO with removed cryoprotectant had significantly lower embryonic development up to the blastocysts stage (p<0.05) after in vitro fertilization compared with that in the control group. These results suggest that the addition and removal of a cryoprotectant has a negative effect for the ATP content of cumulus enclosed oocytes. One of the factor(s) causing the lower embryonic development after removal of cryoprotectant, may be associated with ATP metabolism.

• 한국수정란이식학회지
• /
• 제11권3호
• /
• pp.249-258
• /
• 1996

To improve the efficiency of in vitro production of embryos with follicular oocytes in Korean Native cows, the recovery rates, in vitro maturation, fertilization and development, and the time required for collecting and processing oocytes by aspiration with or without slicing were evaluated comparatively. The ovaries were obtained from a local abattoir and placed in physiological saline at 25~28$^{\circ}C$ and brought to the laboratory within 3 hrs. The oocytes were collected by aspiration of follicles(2~6mm) with or without slicing ovaries after aspiration, and classified into Grade I, Grade II, Denuded, Expanded oocytes by the morphology of cumulus cells attached and the homogeneity of cytoplasmic granules. Also the time required for each step of collecting and processing oocytes were measured. The cumulus cells were removed in some Grade I oocytes to measure their size and nuclear configuration before and after in vitro maturation. The Grade I oocytes were matured in vitro(IVM) for 24 hrs. in TGM-199 supplemented with 35$\mu$g /ml FSH, 10$\mu$g /ml LH, 1 $\mu$g /ml at 39$^{\circ}C$ under 5% C02 in air. They were fertilized in vitro(IVF) by epididymal spermatozoa treated with heparin for 24hrs. and then the zygotes were cocultured in vitro (IVC) with bovine oviductal epithelial cells for 10 days. The results obtained were as follows: The number of oocytes recovered per ovary was averaged 6.6 by aspiration and 11.2 by slicing post aspiration, which summed to 17.8. The number of Grade I oocytes recovered per ovary was averaged 3.1 by aspiration and 3.6 by slicing, which summed to 6.7. The percentage of Grade I to total oocytes recovered was significantly(P<0.05) higher as 48.0 % in aspiration than 31.6% in slicing post aspiration. The time requlred for recovering a Grade I oocyte by aspiration and slicing was 1.1 and 2.5 min, respectively. The mean diameter of Grade I oocytes by aspiration and slicing was similar as 148.7 and 151.5$\mu$m, respectively. The percentage of Metaphase II stage oocytes after IVM for 24 hours was significantly (P

• Clinical and Experimental Reproductive Medicine
• /
• 제27권1호
• /
• pp.23-29
• /
• 2000

Objective: Zona pellucida (ZP) has been thought to be the barrier of egg to sperm penetration before and after fertilization. The phenomenon of ZP hardening has been considered as a post-fertilization event until now, and it is generally accepted that it is caused by the secretory products of cortical granules released during the cortical reaction. Hardening of ZP could occur "spontaneously" in mammalian oocytes in standard culture conditions, and that it is probably not a consequence of cortical reaction. The purpose of our study was to investigate the effect of human amniotic fluid (HAF) on nuclear maturation (NM) and fertilization ability of mouse immature oocytes. Methods: HAF was obtained from patients undergoing amniocentesis at $16{\sim}20$ weeks of gestation. HAF from five to ten patients was centrifuged and the supernatants was pooled. Cumulusenclosed mouse immature oocytes were incubated in the medium containing HAF, and examined to confirm NM and fertilization. Female ICR mice (about 3 weeks old) were stimulated with 7.5 IU PMSG. Immature oocytes were isolated at $48{\sim}52$ hrs post PMSG injection and cultured in TCM-199 supplemented with 20% HAF for 18 hrs. FBS was used as a control for the examination. Matured oocytes (MII) were fertilized with sperms collected from the epididymis of male mice (over 10 weeks old). Fertilization was in conducted T6 medium containing 15 mg/ml BSA, and confirmed at 6 hrs post-insemination. Fertilization rate was assessed in zona-intact or zona-free oocytes (denuded by trypsin). Evaluation of NM and fertilization was carried out by rapid staining method. ZP hardening was evaluated by incubating cumulus cell-free mature oocytes in 0.001% chymotrypsin at $37^{\circ}C$ for 10 min. Results: There was no significant difference between the effects of HAF (86.6%) and FBS (87.7%) supplements on NM of immature oocytes. When maturation medium was supplemented with HAF, total fertilization rates (7%) were significantly lower (p<0.01) than that of FBS (85.1%). In HAF group, fertilization rate was increased (p<0.01) in zona-free oocytes (7% versus 100%). The resistance of mouse oocyte ZP to digestion by chymotrypsin after maturation in vitro was significantly higher (p<0.01) in HAF group (86.7%) than in FBS (6.7%). To culture oocytes in FBS were very effective in preventing ZP hardening. However cultured oocytes in HAF showed high rate of ZP hardening (p<0.01). Conclusions: These results suggest that HAF can be used as a supplement for the NM of mouse immature oocytes in vitro. However, HAF induces spontaneous hardening of ZP of mouse immaure oocytes during maturation in vitro.

• 한국수정란이식학회지
• /
• 제18권2호
• /
• pp.157-161
• /
• 2003

본 연구는 소형 고양이의 불임 해결과 체외수정란을 생산하기 위한 방안으로서 난자의 형태, 번식주기, 배양시간 및 활성화 처리가 난포란의 체외수정 및 체외발생에 미치는 영향을 조사하였다. 1. 신선 및 salt에 보존한 난소로부터 회수한 난구세포부착 및 나화 난자를 각각 배양했을때 체외수정율 및 분할율은 65.7%와 17.1%, 28.6%와 8.6% 및 57.1%와 13.3%, 23.3%와 3.3%로서 난구세포 부착 신선난자가 나화 난자에 비해 높은 체외발생률을 나타냈다. 2. 휴지기, 발정기 및 황체기 단계로 구분하여 채취한 난포란을 성숙배양 후 수정시켰을 때 체외수정율은 각각 68.9%, 44.4%, 48.9%였으며, 분할율은 각각 17.8%, 8.9%, 12.8%로 나타났다 3. 24, 36 및 48시간 각각 배양한 난포란을 성숙 배양 후 수정시켰을 때 체외수정율은 각각 66.7%, 46.7%, 48.9%였으며, 분할율은 각각 17.8%, 11.1%, 8.5%로 나타났다. 4. 난자를 활성화처리 및 비활성화처리 후 각각 체외수정시켰을 때 체외수정율은 각각 57.4%와 31.4%였고, 체외분할율은 22.9%와 11.4%로서 활성화 처리를 한 난자가 높은 체외발생율을 나타냈다.

• 한국수정란이식학회지
• /
• 제27권1호
• /
• pp.57-62
• /
• 2012

Efficient oocyte activation is a key step for the success of nuclear transfer in cloning. Ionomycin sequentially combined with 6-DMAP is now widely used to activate normal oocytes for analytical studies of oocyte activation and to activate reconstructed oocytes after nuclear transfer. The present study investigated sources of oocytes, duration of ionomycin and 6-DMAP, laser and electric stimulation in goat oocyte activation in order to optimize the protocols. Goat ovaries were collected in individual abattoirs during the breeding season and were delivered to the laboratory within 6 h in saline with 100 IU/ml streptomycin and 0.05 mg/ml penicillin. The oocytes were denuded from the cumulus cell by pipetting with 0.2% hyaluronidase in PBS at 20~22 hr post maturation. Oocytes with the polar body were selected and assigned to four groups for parthenogenetic activation. To examine the effect of duration of ionomycin treatment, oocytes after 20~22 hr of maturation were treated with 2.5 uM ionomycin for 1 or 5 min times and then cultured in 2 mM 6-DMAP for 2 or 4 hr. The activated oocytes were cultured in mSOF at $38.5^{\circ}C$ in $CO_2$ 5%, $O_2$ 5% and $N_2$ 90% multi incubator. Cleavage and blastocyst development was observed at 48 hr and day 8 of culture $in$ $vitro$, respectively. Activation rates of oocytes exposed to ionomycin for 1 min(86.4%) were significantly higher than those treated for 5 min(74.3%) duration. This indicated that 1 min ionomycin treatment was most suitable for activation of goat oocytes. The duration of 6-DMAP treat duration was in 2 mM 6-DMAP for 2 hr after 1 min exposure to 2.5 uM ionomycin. The activation rate of oocytes incubated in 6-DMAP for 2 hour(82.5%) was significantly higher than those in oocytes treated with 4 hr(75.5%).

• 한국수정란이식학회지
• /
• 제18권1호
• /
• pp.27-33
• /
• 2003

본 연구는 소형 개의 불임 해결과 체외수정란을 생산하기 위한 방안의 하나로써 난자의 형태, 번식주기, 배양시간 및 활성화 처리가 난포란의 체외성숙 및 체외발생에 미치는 영향을 조사하였다. 1. 신선, salt 및 4$^{\circ}C$에 보존한 난소로부터 채취한 난구세포부착 난자와 나화 난자로 각각 체외수정시켰을 때 16세포기로의 발생율은 14.3%, 5.0% 및 7.5%, 2.8%, 5.7% 및 0.0%로써 난구세포 부착난자군의 체외발생율이 나화 난자군에 비해 높게 나타났다. 2. 발정주기를 inactive, follicular, luteal 단계로 구분하여 채취한 난포란을 각각 체외배양시켰을 때 GV 및 MII로의 발생율은 11.3%와 9.4%, 50.7%와 26.7%, 16.9%와 13.8%였고, 16세포기로의 체외발생율은 0.0%, 10.7%, 1.5%였다. 3 신선한 난구세포 부착 난자를 각각 24, 32, 48시간 성숙배양 후 체외수정시켰을 때 분할율은 8.6%, 15.8%, 23.5%였으며, 16세포기로의 체외발생율은 각각 0.0%, 5.3%, 11.8%로써 48시간 배양군이 가장 높은 발생율을 나타냈다. 4. 활성화 처리 및 비활성화 처리 난자를 각각 체외수정시켰을 때 분할율은 각각 42.5%, 22.2%였고 16세포기로의 체외발생율은 각각 15.0%, 6.7%로써 활성화 처리 난자군이 비활성처리 난자군에 비해 높은 발생율을 나타냈다.