• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.42-42
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• 2001

The objective of this study was to assess the development of porcine follicular oocytes fertilized by ICSI. Cumulus-oocyte-complexes (COCs) were collected by aspiration from follicles of 2-7 mm in diameter from a local slaughterhouse ovaries. Oocytes matured for 40-44 h were centrifuged at 12,000g for 6 min and then injected with sperm prepared by swim-up procedure in the presence or absence of 5 mM dithiothreitol (DTT). Injected oocytes were cultured in NCSU 23 medium during 6 to 8 days. IVF controls were compared to those of resulting embryos. The results obtained were as. follow: 1, The rates of cleavage and development rates into blastocyst by ICSI were not significantly (P<0.05) different between with (53.0% and 19.7%) or without (48.3% and 23.8%) centrifugation, respectively. 2. The cleavage and developmental rates to blastocyst after ICSI with or without 5mM DTT treated-sperm were not significantly (P<0.05) different (60.4% vs 16.4% and 48.5% vs 22.2%, respectively). 3. The cleavage and the developmental rates to blastocyst were not significantly (P<0.05) different between the zygotes obtained by IVF (51.8% vs. 22.4%) and ICSI (51.4% vs. 21.6%). 4. The number of blastomere in blastocyst stages after IVF or ICSI was not significantly different (46.7 $\pm$2.9 and 41.9$\pm$4.6).

• Reproductive and Developmental Biology
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• v.34 no.1
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• pp.55-62
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• 2010

Matrix metalloproteinases (MMP) play important roles in extracellular matrix (ECM) remodeling during ovarian follicular development, oocytes development and ovulation. In an attempt to investigate the effect of MMP activation in development cumulus-oocytes complexes, we examined the localization and expression of MMP, and monitored MMP expression profile. Cumulus-oocytes complexes were collected and matured in vitro for 24 hr, 36 hr and 48 hr. A mRNA expression of MMP-2, MMP-9, TIMP-2 and TIMP-3 was detected in all culture medium regardless of CC, DC and CDCs. Activity of MMP-2 in the DC progressively was increased from 24 hr to 48 hr. But MMP-9 was not detected in all culture medium. The localization of MMP-2 was also measured by immunohistochemistry analysis. The MMP-2 and TIMP-2 was detected in cumulus cell and oocyte zone pellucida. Expression of MMP-2 protein in the COCs was progressively increased from 24 hr to 48 hr. However, MMP-9 protein was progressively decreased from 24 hr to 48 hr. And TIMP-2 protein was most highly expressed in the CDCs 36 hr. Expression of TIMP-3 protein in the CDCs was progressively increased from 24 hr to 48 hr. In conclusion, these results suggest that MMP-2 plays a role in maintaining normal maturation and development by controlling the ECM inhibitor concentration on cumulus cell and oocytes.

• Animal cells and systems
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• v.3 no.4
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• pp.385-391
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• 1999

Changes in the levels of prostaglandian F$_{2a}$ (PGF$_{2a}$) and E$_2$ (PGE$_2$) in culture medium during in vitro ovulation of Rana dybowskii follicles were examined. The ovulation was induced by frog pituitary homogenate (FPH) or TPA (12-O-tetradecanoylphorbol-13-acetate, a protein kinase activator) and the levels of PGs were measured by radioimmunoassay. When the ovarian follicles were cultured, only a few oocytes were ovulated by 12 h, but half of them were ovulated by 24 h in response to FPH, whereas around 30% of oocytes were ovulated by 12 h and maximum ovulation (around 50%) occurred by 24 h in response to TPA. Without any stimulation (control), no ovulation occurred. TPA elevated the level of PGF$_{2a}$ to high levels when compared to control (basal levels), but the increase by FPH was less evident. Likewise, the levels of PGE$_2$ increased markedly in response to TPA, but rather decreased by FPH treatment. Interestingly, PGF$_{2a}$ induced ovulation but PGE$_2$ suppressed FPH- or PGF$_{2a}$-induced oocyte ovulation. Basal levels of PGs Increased steadily during culture. When theca/epithelium (THEP) layer and granulosa cell-enclosed oocytes (GCEOs) were separated by microdissection and cultured independently, higher levels of both PGs were secreted by THEP than by GCEOS. Synthesis of PGs by follicle or follicular components was strongly suppressed by exogenous cAMP or indomethacin. These results suggest that: 1) PGF$_{2a}$ plays an important role in Rana ovulation, 2) protein kinase C is involved in PGs production, and 3) thecal epithelium layer is responsible for the PGs production in Rana.

• Korean Journal of Animal Reproduction
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• v.17 no.1
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• pp.57-68
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• 1993

This stduy was carried out to find a reliable method for the production of in vitro fertilized embryos having more excellent development capacity and freezability in the rabbit and cattle. The greatest number of rabbit oocytes was recovered 6hrs after HCG injection(P<0.05). The maturation rate in vitro was slightly higher in the oocytes(6-h-oocytes) from 6h than those (8-h-oocytes)from 8 hrs after HCG injection and the beneficial effect of FSH during oocyte maturation was significantly great in the oocytes from large follicles. The cleavage rate into 2-to-6-cell stage was not differ between the 6-h-oocytes and 8h-oocytes, but the cleavage of these oocytes was greatly promoted by FSH addition to maturation medium and the cleavge of 8-h-oocytes matured without FSH was significantly low. The embryo development into 16-cell to morula was not promoted by the co-culture with rabbit oviduct epithelial cells. The freezability by embryo stages was ovidusly high at 4-cell and morula stage in 6-h-oocytes and the viability of 16-cell embryos from 8h-oocytes was similar to that of morula stage. The implantation sites after surgical tranfer of fresh rabbit embryos were not implanted. In bovine experiment, the in vitro development into 16-cell and morula after in vitro maturation and fertilization in the follicular oocytes was slightly improved by the co-culture with granulosa cells compared to that with oviduct epithelial cells and the frozen-thawed viability rate of these embryos ranged from 14 to 40%. The excellent fresh embryos were transferred nonsurgically to 6 recipients, but were not pregnant.

• Reproductive and Developmental Biology
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• v.34 no.4
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• pp.275-281
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• 2010

The aim of this study was to investigate what protein(s) of porcine epididymal fluid (pEF) are able to enhance the nuclear maturation of porcine germinal vesicle (GV) oocytes in vitro. Proteins of pEF were fractionated by affinity, ion exchange, and gel filtration chromatography. Porcine cumulus-oocytes complexes (COC) from follicles were cultured in tissue culture medium (TCM 199) containing various fractions obtained by chromatography. Porcine COCs were also cultured in TCM 199 containing various meiosis inhibitors and pEF. After 24 or 48 h culture, oocytes were examined for evidence of GV breakdown, metaphase I, anaphase-telophase I, and metaphase II. When porcine COCs were cultured in the medium with meiosis inhibitor such as, dibutyryl cAMP (dbcAMP) and forskolin (Fo), more than 80% of oocytes were unable to resume meiosis. However, porcine COCs supplemented with pEF were able to overcome the inhibitory effect of dbcAMP and Fo. Maturation rate of oocytes was significantly (p<0.05) increased in the media supplemented with cationic protein(s) during in vitro maturation than in those with anionic protein(s) (44.1% vs 20.0%). When oocytes were cultured in the TCM 199 with fractions obtained by gel filtration, the maturation rate of oocytes was significantly (p<0.05) higher in fraction 11 containing 18 kDa than other fractions. The present study suggests that 1) dbcAMP and Fo prevent the spontaneous maturation of oocyte after isolation from follicles, and that pEF contain a substance(s) that improves meiosis resumption in vitro of porcine COCs, 2) cationic 18 kDa protein(s) are responsible for promotion of Mil stage.

• Korean Journal of Clinical Laboratory Science
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• v.38 no.1
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• pp.72-81
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• 2006

Wee1 is a kinase regulator of the M-phase promoting factor (MPF; a complex of cdc2 and cyclin B1). The present study was undertaken to determine the role(s) of wee1 in the early stages of mouse ovarian follicles. The expression of wee1 and the correlated cell-cycle components, namely cdc2, cyclin B1, and cdc25C, were evaluated by immunohistochemistry. In addition, the expression of Tyr15-phosphorylated cdc2 (cdc2-p) was also examined to determine whether wee1 kinase phosphorylates cdc2 existed. Each component except cdc25C was found cytoplasmic in the oocytes at all stages of follicles, while cdc25C was not detected in primordial follicles. It was found primarily in ovarian somatic cells and to a small extent in granulosa cells of the growing follicles. To further confirm the expression of cell-cycle components in the primordial follicular oocytes, day1 ovaries were enzymatically and mechanically dissociated, then oocytes were isolated from somatic including pre-granulosa cells, and we confirmed that cdc2-p was expressed in oocytes of primordial follicles. From the results of the present study, we concluded wee1, without the counteracting cdc25C, would cause meiotic arrest of oocytes by the inhibitory phosphorylation of cdc2. The expression of all these proteins in the granulosa cells of growing follicles may regulate their mitosis concurrently with the growth of oocytes and follicles.

• Ocean and Polar Research
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• v.38 no.1
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• pp.21-33
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• 2016

Gametogenesis of the comb pen shell, Atrina pectinata (Linnaeus, 1767) (Bivalvia: Pinnidae) on the southern coast of Ulleungdo Island, Korea was assessed monthly (November 2013 to October 2014) using histology. Gametogenesis commenced in January when the surface water temperature was $12.6^{\circ}C$ and pen shells evidenced an early development phase with small oogonia from January to April, although few females exhibited ripe eggs in their follicular epithelium. In April, the oocyte diameter increased rapidly, and fully mature eggs appeared in May. First spawning males and females were observed in June as the surface water temperature reached $19.3^{\circ}C$ and July ($23.2^{\circ}C$) respectively. The spawning activity continued until the end of September. Histology indicated that the spawning peak of the females in Ulleungdo Island was July to August. During October to January, most of the pen shells were in spent and resting stages. Our data suggested that A. pectinata is a summer spawner, and their annual gametogenesis is closely associated with the seasonal variation in the surface water temperature. The present study is the first provided fundamental information on the life history of A. pectinata in Ulleungdo Island, and this can be put to good use in the management of this pen shell in the study area.

• Clinical and Experimental Reproductive Medicine
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• v.27 no.1
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• pp.75-81
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• 2000

Objective: The objective of this study is to influence of sequential embryo transfers in an invitro fertilization was examined. Method: After in vitro fertilization, a maximum of 6 fertilized oocytes was enrolled in this study. At day 3 after an oocytes retrieval, embryos with good quality were transferred (mean 4.9), remaining embryos (mean 2.0/cycle) were cryopreserved at blastocyst stage (Group 1). At day 5 after oocytes collection, second a embryo transfer (mean 1.2/cycle) was performed, if one of these embryos had reached the blastocyst stage (Group 2) using P1 supplemented with 10 SSS and 30% Follicular fluid. No statistical difference in the pregnancy rate could be seen between the group without a second embryo transfer (n=21; 28.6%) and the group with a second transfer (n=52; 28.8%). Results: The incidence of multiple pregnancy rate per embryo transfer was not statistically different between both group and no high-rank multiple pregnancy (greater than triplete) were observed (0.9%, 15.4%, respectively, p=0.74, ${\chi}^2$). Out of 114 cycles (506 embryos) cultured embryos in group 2, 52 cycles (159 embryos, 29.8%) reached the blastocyst stage. Conclusion: The second transfer did not have a significant effect on the pregnancy rate. The most important factor for the pregnancy seems to be the quality of the embryos transferred on day 3 following oocyte retrieval. We recommend embryo transfer is performed only one, day $2{\sim}3$ or D5.

• Journal of Embryo Transfer
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• v.14 no.3
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• pp.185-193
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• 1999

To improve the efficiency of in vitro production of embryos with follicular oocytes in pig, the recovery rates, in vitro fertilization and development. The results obtained were as fellows: The number of oocytes recovered 37 ovary was 1,365 by aspiration, 1,884 by slicing and 3,830 aspiration post slicing, per ovary was averaged 103.5 aspiration post slicing than 30.7 by aspiration and 50.8 by slicing (P<0.05). The percentage of grade I and II oocytes recovered was 0.05∼0.2% and 1.7∼2.3% respectively(p<0.05). The fertilization rates of ejaculate and epididymis sperm was 83.0 and 83.1%. And cleavaged rate was 60.8 and 69.0% respectively(P<0.05). However, there were no significant differences between sperm sources. The clevage rates of fertilized oocyte was significantly(P<0.05) higher as B.O(92.8%) than TALP (90.1%) or mTBM (80.1%). And in vitro developed to blastocyst rates of mTBM media used for fertilization was significantly (P<0.05) higher as 12.4%, compared with the results using the media of TALP(1.6%) or B.O (0.0%). The embryos developed 2-cell stage after in vitro fertilization were co-cultured with or without POEC and BOEC in NCSU-23 and TCM-199 media. In vitro developed to blastocyst rates was NCSU-23 with POEC(2.3%) or BOEC(1.2%), but in vitro cultured in TCM-199 medium with POEC or BOEC was not developed to blastocyst. The percentage of embryos that developed to morula stage in 0, 50, 100, 200 and 250uM was 16.6, 22.0, 13.5, 19.0 and 22.0%, respectively.

• Korean Journal of Veterinary Research
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• v.59 no.2
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• pp.81-88
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• 2019

This study was performed to examine the effects of various macromolecules in in vitro growth (IVG) media on the growth, maturation, and parthenogenesis (PA) of pig oocytes derived from small antral follicles (SAF). Immature oocytes were cultured for two days in IVG medium supplemented with 10% (v/v) fetal bovine serum (FBS), 10% (v/v) pig follicular fluid (PFF), 0.4% (w/v) bovine serum albumin (BSA), or 0.1% (w/v) polyvinyl alcohol (PVA) and then maintained for 44 h for maturation. After IVG, the mean diameters of the SAF treated with FBS, PVA, and no IVG-MAF ($113.0-114.8{\mu}m$) were significantly larger than that of no IVG-SAF ($111.8{\mu}m$). The proportion of metaphase II oocytes was higher in PFF (73.6%) than in BSA (43.5%) and PVA (53.7%) but similar to that in the FBS treatment (61.5%). FBS and PFF increased cumulus expansion significantly compared to PVA and BSA while the intraoocyte glutathione content was not influenced by the macromolecules. Blastocyst formation of PA oocytes treated with FBS (51.8%), PFF (50.4%), and PVA (45.2%) was significantly higher than that of the BSA-treated oocytes (20.6%). These results show that the PFF and FBS treatments during IVG improved the growth, maturation, and embryonic development of SAF.