• Journal of Embryo Transfer
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• v.9 no.1
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• pp.117-125
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• 1994

In vitro development of parthenogenetic embryo was examined after ethanol treatment of follicular oocytes matured in vitro for 42, 48, 54 and 60h in the pig. The follicular oocytes were matured in TCM 199 containing 15% FCS and gonadotrophins in an atmosphere of 39 $^{\circ}C$ 5% $CO_2$. The cumulus-free oocytes were activated by 10% ethanol treatment in M2+4mg /ml BSA for 10 min. The ethanol-activated oocytes were washed and further cultured in TCM199+20%FCS containing granulosa cell monolayer. Maturation rates at 42, 48, 54 and 60h of IVM were 75.0, 86.5, 81.6 and 87.9%, respectively. Thus the oocytes maturated in vitro for longer periods did not improve nuclear maturation much. Pronuclear formation rates at 18h post-activation in ethanol-activated oocytes were 21.9, 25.0, 47.4 and 32.6%. The cytoplasmic maturation leading to pronuclear formation upon activation increased when the I VM period was extended from 42 to 54h. When the activated oocytes were cultured for 96~120h to analyse early development of the activated oocytes, the rates of embryonic development upto $\leq$ 5~8 cell were 5.3, 5.8, 12.0 and 11.7% among the cultured embryos. The result indicate that earlier development of activated porcine occyte is dependent on the duration of oocyte maturation, and that better development could be obtained from the oocyte matured for 54h.

• Journal of Embryo Transfer
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• v.22 no.4
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• pp.219-222
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• 2007

These study was carried out to investigate the effects of the collection time, culture time and activation of canine oocytes on in vitro maturation rates. The activated oocytes were cultured in 10% FCS+TCM-199 media containing hormonal supplements (10 IU/ml HCG, 10 IU/ml PMSG, 10 ug/ml gonadotropin) at 5% $CO_2$, 95% air, $38^{\circ}C$. 1. IVM rate of in vitro cultured cumulus-attached oocytes recovered from ovaries that collected at follicular and luteal stages of the reproductive cycles were 11.4% and 5.7%, respectively. IVM rate of oocytes recovered from ovaries that collected at follicular stages of the reproductive cycles was significantly higher than that of luteal stage (p<0.05). 2. When IVM was carried out at different periods of 40, 48, and 70 hrs, the IVM rates of oocytes matured in vitro were 2.9%, 8.6%, 5.7%, respectively. These results indicate that the IVM time between $48{\sim}70$ hrs gives the highest maturation rate for the oocytes matured at the different stages. 3. IVM rate of oocytes matured in vitro for 10 hrs after single and combined activation treatment by ET, IP and CH and Ca+DMAP, CH+DMAP, ET+CH were $11.5{\pm}1.2%,\;10.8{\pm}1.0%,\;9.6{\pm}1.2%\;and\;12.4{\pm}1.5%,\;11.8{\pm}1.5%,\;11.2{\pm}1.4%$ respectively. This was higher than that in both single and combined stimulated groups compared to control group ($6.2{\sim}7.2%$).

• Reproductive and Developmental Biology
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• v.28 no.1
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• pp.53-57
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• 2004

The study was carried out to investigate the effects of morphology, reproductive cycle, incubation time and activation of oocytes on in vitro maturation of cat oocytes and development of IVM/IVF embryos. The results were summarized as follows: 1. When recovered from ovaries collected at different stages of the reproductive cycle (inactive, follicular and luteal stage), the developmental rates of oocytes to GV and MI stage were 72.5% and 27.5%, 57.5% and 7.5%, 62.5% and 17.5%, respectively. 2. The developmental rates of oocytes with cumulus cells to GV and MI stage in different conditions of incubation (5% $CO_2$ , 95% $O_2$ and 10% $CO_2$, 90% $O_2$) were 70.0% and 27.5%, 52.5% and 20.0%, 55.0% and 12.5%, respectively. 3. The developmental rates to GV and MI oocytes when cultured at different time of incubation (17∼20, 21∼24, 25∼28 and 29∼32 h) were 67.5% and 20.0%, 67.5% and 30.0%, 62.5% and 22.5%, 65.0% and 15.0%, respectively. 4. The fertilization and cleavage rates of freshly collected oocytes with and without cumulus cells were 72.5% and 25.0%, 37.5% and 7.5%, respectively. The rates were greater in oocytes with cumulus cells than those without cumulus cells. 5. The fertilization and cleavage rates of oocytes recovered from ovaries collected at different stages of the reproductive cycle (inactive, follicular and luteal stage) were 75.0% and 25.0%, 40.0% and 7.5%, 50.0% and 15.0%, respectively.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.8-8
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• 2001

Mouse follicles require the addition of gonadotropins (Gns) to complete maturation and ovulation of oocyte and antrum formation of follicles in vitro. However, we tried examination of in vitro growth of mouse pre-antral follicles in medium without Gns and physiological factors. And also, pre-antral follicles were isolated from ovaries by mechanical method. Our present studies were conducted to evaluate on the growth of follicles and intra-follicular oocytes and antrum formation in vitro of mouse pre-antral follicles in two different media. Pre-antral follicles (91-120${\mu}{\textrm}{m}$) were isolated mechanically by fine 30G needles not using enzymes from ovary of 3-6 weeks old female ICR mice. Isolated pre-antral follicles were cultured in 20 ${mu}ell$ droplets of TCM (n=17; follicles: 107.8 $\pm$ 1.58 ${\mu}{\textrm}{m}$; oocytes: 59.9$\pm$1.2 ${\mu}{\textrm}{m}$) or MEM (n=12; follicles: 109.3$\pm$2.53 ${\mu}{\textrm}{m}$; oocytes: 55.4 $\pm$1.6${\mu}{\textrm}{m}$) under mineral oil on the 60mm culture dish. All experimental media was supplemented with 10% FBS but without Gns and/or physiological factors. Pre-antral follicles were individually cultured in drops for 8 days. Antrum formation and growth of pre-antral follicles and intra-follicular oocytes were evaluated using a precalibrated ocular micrometer at $\times$200 magnifications during in vitro culture. Results between different groups were analyzed using combination of Student's t-test and Chi-square, and considered statistically significant when P<0.05. Antrum formation of pre-antral follicles had started in two culture media on day-2. On day-8, antrum formation had occurred in 58.3%(7/12) of pre-antral follicles cultured in MEM, but only in 23.5% (4/17) of those cultured in TCM (P=0.0364). Growth of pre-antral follicles and intra-follicular oocytes were observed on day-4 and -8. On day-4, follicular diameters was similar (P=0.1338) in TCM (119.4$\pm$2.58 ${\mu}{\textrm}{m}$) and MEM (125.4$\pm$4.52 ${\mu}{\textrm}{m}$). However, on day-8, diameters of pre-antral follicles cultured in MEM (168.9$\pm$17.29 ${\mu}{\textrm}{m}$) was significantly (P=0.0248) bigger than that in TCM (126.7$\pm$4.28 ${\mu}{\textrm}{m}$). On day-4 and -8, diameters of intra-follicular oocytes were similar TCM (67.1$\pm$1.3 and 72.4$\pm$0.9${\mu}{\textrm}{m}$) and MEM (65.2$\pm$1.7 and 73.3$\pm$1.5 ${\mu}{\textrm}{m}$), respectively. We can conform that medium not supplemented with Gns and/or physiological factors can be used for in vitro antrum formation and growth of mouse pre-antral follicles and intra-follicular oocytes. In conclusion, MEM supplemented with FBS can be used for growth in vitro of mouse pre-antral follicles isolated mechanically.

• 대한생식의학회:학술대회논문집
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• 2002.05a
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• pp.76-76
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• 2002

• Development and Reproduction
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• v.16 no.3
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• pp.195-204
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• 2012

Recently, we reported growth arrest-specific gene 6 (Gas6) as a new maternal effect gene (MEG), that expressed in the oocytes but functioned principally during embryogenesis. Gas6 RNAi-treated oocytes developed to metaphase II (MII) stage but they have affected M-phase promoting factor (MPF) activity and incurred abnormal pronuclear (PN) formation during fertilization. Gas6 is a ligand of TAM family members (Tyro3, Axl and Mertk) of receptor tyrosine kinase (RTK). Purpose of the present study was to evaluate the expression of Tyro3, Axl and Mertk transcripts in oocytes and early embryos. Expression of Gas6 and Mertk mRNA was detectable in oocytes and follicular cells, while Tyro3 and Axl mRNA was expressed only in follicular cells. Expression of Mertk mRNA was relatively constant during oocytes maturation and embryogenesis, but the other receptors, Tyro3 and Axl, were not expressed in oocytes and PN stage of embryos at all. Knockdown of Mertk mRNA and protein by using sequence-specific Mertk double strand RNA (dsRNA) did not affect oocytes maturation. In this case, however, contrary to the ligand Gas6 RNA interference (RNAi), MPF activity had not been changed by Mertk RNAi. Therefore, we concluded that the Gas6-Mertk signaling is not directly related to the oocyte maturation. It is still required to study further regarding the function of Mertk as the receptor of Gas6 during preimplantational early embryogenesis.

• Reproductive and Developmental Biology
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• v.30 no.2
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• pp.135-141
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• 2006

The present study was conducted to investigate the effects of cumulus cells and porcine follicular fluid (pFF) on plasminogen activator (PA) activity and oocytes maturation in vitro in the pig. The cumulus-oocyte complexes (COCs) and denuded oocytes (DOs) were incubated in NCSU-23 medium with or without 10% pFF for 0, 24, or 48 hr. In the presence of cumulus cells, the proportions of oocytes matured to metaphase-II stage were significantly (P<0.05) higher in medium with pFF than without pFF (69.8 vs. 37.7%, respectively). When COCs and DOs were cultured in the presence of pFF, tissue-type PA (tPA), urokinase-type PA (uPA), and tPA-PA inhibitor (tPA-PAI) were observed in COCs, and PA activities were higher at 48 hr than 24 hr. When COCs and DOs were cultured in the absence of pFF, tPA and tPA-PAI were observed in COCs, and PA activities were increased as duration of culture increased. No PA activities were detected in DOs regardless of pFF supplementation. When porcine oocytes were cultured in the presence of pFF for 24 and 48 hrs, the activities of tPA-PAI, tPA, and uPA were observed in both COCs and DOs. In medium of absence of pFF, PA activities were observed in oocytes with cumulus cells only. On the other hand, three plasminogen-dependent lytic bands (tPA-PAI, tPA, and uPA) were observed in pFF cultures. Particularly uPA activity was higher than the other kinds of PA activity. When oocytes and cumulus cells were separated from porcine COCs at 0 hr of culture, tPA-PAI, tPA, and uPA were detected in cumulus cells at 48 hr of culture, but no PA activities were in DOs. The presence of pFF and cumulus cells in maturation medium stimulated not only nuclear and cytoplasmic maturation in porcine COCs, but also PA production by cumulus cells and COCs. It is possible that PAs produced by cumulus cells migrated through the gap junction between oocyte and cumulus cells. These results suggest that porcine oocytes have no ability to produce PA themselves.

• Journal of Embryo Transfer
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• v.9 no.3
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• pp.261-268
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• 1994

Immatured bovine follicular oocytes added with serum, hormones, granulosa cells and bovine oviduct epithelium cells were fertilized in vitro after in vitro maturation. In vitro maturation and early development capacity were examined and IVF-derived embryos were transferred and to recipients and effects of sperm treatment on in vitro capacitation were investigated. The rate of in vitro maturation was improved when they were co-culutred with granulosa cells in the TCM199 medium added with 10% FCS and hormones. The percentage of acrosome reaction was not differed between sperm treatments and sperm of above 25% under-went AR during 30 min preincubation with caffeine and heparin. The cleavage rate of oocytes in vitro fertilized in TCM199 medium added with 10% FCS and hormones, GC or BOEG higher than that in medium with 10% FCS and GC. But the rate was not significantly different between GC and BOEG The cleavage of rate oocytes cultured in medium containing serum, hormones and BOEG was 80.2% and more embryos were developed to Blastocyst (17.3%). The selected embryos were transferred to 9 recipients by surgical or nonsurgical method but did not result in pregnancy.

• Korean Journal of Clinical Laboratory Science
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• v.40 no.1
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• pp.31-40
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• 2008

Despite of the importance of the primordial follicle (PMF) recruitment, factors and mechanisms for process are poorly understood. To evaluate expression and role of the follicular transition from PMF to PMF/primary follicles (PMIF) in the present study, we evaluated expression of lats1, lats2, cyclin A1, and cyclin A2 mRNA and protein, and elucidated and role of lats1-cyclin A in the follicular transition from PMF to PRIF. To analysis of differential expression in PMF and PMIF, each stage follicles were collected by day1 and day5 of immuno-compromised rats (ICR) and analyzed by real-time PCR for the genes. For localization of mRNAs and proteins of the genes, in situ hybridization and immunohistochemistry were performed. We confirmed that the lats1, lats2, cyclin A1, and cyclin A2 mRNA were more expressed in PMF than PMIF. Localization of the four genes expression were observed in nuclei of oocytes from the arrested primordial, and in the surrounding granulosa cells of the growing follicles. The mRNA expressions were gradually decreased with follicular development. From immunohistochemistry studies, Cyclin A1 protein expression were observed in oocyte cytoplasmas of early stage follicles, while observed in granulose cells and oocyte nucleoli during growing follicles. This study suggested that the presence of lats gene family might perform negatively regulation of cell proliferation by modulation of the CDC2/Cyclin A complex activity. lats-cyclin A genes in oocytes of the early stage follicles might play a role in the meiotic cell cycle arrest of the primary oocytes at the primordial follicle stage as well as the follicular growth.

• Clinical and Experimental Reproductive Medicine
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• v.49 no.2
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• pp.135-141
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• 2022

Objective: This study investigated the impact of two stimulation protocols using highly purified human menopausal gonadotropin (HP-hMG) on the endocrine profile, follicular fluid soluble Fas levels, and outcomes of intracytoplasmic sperm injection (ICSI) cycles. Methods: This prospective clinical trial included 100 normal-responder women undergoing ovarian stimulation for ICSI; 55 patients received concomitant follicle-stimulating hormone (FSH) plus HP-hMG from the start of stimulation, while 45 patients received FSH followed by HP-hMG during mid/late follicular stimulation. The primary outcome was the number of top-quality embryos. The secondary outcomes were the number and percentage of metaphase II (MII) oocytes and the clinical pregnancy rate. Results: The number of MII oocytes was significantly higher in the concomitant protocol (median, 13.0; interquartile range [IQR], 8.5-18.0 vs. 9.0 [8.0-13.0] in the consecutive protocol; p=0.009); however, the percentage of MII oocytes and the fertilization rate were significantly higher in the consecutive protocol (median, 90.91; IQR, 80.0-100.0 vs. 83.33 [75.0-93.8]; p=0.034 and median, 86.67; IQR, 76.9-100.0 vs. 77.78 [66.7-89.9]; p=0.028, respectively). No significant between-group differences were found in top-quality embryos (p=0.693) or the clinical pregnancy rate (65.9% vs. 61.8% in the consecutive vs. concomitant protocol, respectively). The median follicular fluid soluble Fas antigen level was significantly higher in the concomitant protocol (9,731.0 pg/mL; IQR, 6,004.5-10,807.6 vs. 6,350.2 pg/mL; IQR, 4,382.4-9,418.4; p=0.021). Conclusion: Personalized controlled ovarian stimulation using HP-hMG during the late follicular phase led to a significantly lower response, but did not affect the quality of ICSI.