• Proceedings of the Ginseng society Conference
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• 1998.06a
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• pp.300-311
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• 1998

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), one of the most notorious toxic environmental pollutants, induces various toxic effects in many organs including testes and is regarded as an endocrine disruptor. Korean ginseng, on the other hand, has been well known for its preventive effects on lox- ins, diabetes melltus and hyperlipidemia. We investigated, histopathologically, the effect of Korean Red ginseng water extract (KR-WE) on guinea pig testes damaged by TCDD. Ninety guinea pigs were divided into 6 groups: normal control (NC) group received vehicle and saline; TCDD,1191kg b.w., was administered intraperitoneally to the single dose TCDD-treated (77) group; 100 mghg b.w.16 and 200mg1kg b.w./d KR-WE were injected intraperitoneally to the preventive groups (PIOO and P2OO, respectively) for 28 days from 1 week before TCDD injection, and to the therapeutic groups (CIOO and C2OO, respectively) for 14 days since 1 week after TCDD administration. Increment of body weight was retarded to a larger extent by TCDD. Moreover, body weight of the 77 group decreased significantly 7 days after TCDD exposure, while that of preventive groups kept increasing. Decrease in body weight was not observed in KR-WE-treated groups. Weight decrease in testes caused by TCDD was remarkably protected by KR-WE. Testicles in 77 group displayed decreased tubular size and maturation arrest at the primary or secondary spermatocyte stage. On the other hand, maturation arrest in germ cells by TCDD was improved in KR-WE treated groups. Almost complete protection of the testes was observed in PIOO and P2OO groups. In addition, the therapeutic effect was noticed in C 100 and C2OO groups. These results provided strong evidence that Korean Red ginseng might be a useful agent for the prevention and treatment of testicular damage induced by environmental pollutants.

• 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.

• Development and Reproduction
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• v.11 no.3
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• pp.263-272
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• 2007

Contrast to mouse where its in vitro maturation rates are high without specific supplements or presence of the cumulus cells, there are some species, such as porcine, where its in vitro oocyte maturation rates are still very low. This comparative study was conducted to investigate the role of malate dehydrogenase(Mor2) during oocyte maturation by RNAi in the mouse and porcine. The Mor2 double-stranded RNA(dsRNA) was prepared speciesspecifically and microinjected into the cytoplasm of denuded germinal vesicle(GV) oocytes. Oocytes were cultured for 48 h(porcine) and 16 h(mouse) in M199 with 10% porcine follicular fluid, pyruvate, p-FSH, EGF, cystein, and estradiol-$17{\beta}$. We measured changes in oocyte morphology, maturation rates and mRNA levels after Mor2 RNAi. We confirmed gene sequence-specific knock down of Mor2 mRNA in both species after Mor2 RNAi. In contrast to our previous finding that mMor2 RNAi resulted in GV arrest in the mouse, we found that pMor2 RNAi resulted in MI arrest in denuded porcine oocytes(58%), but developed to MII(84.4%) in COCs. To determine whether this difference between mouse and porcine RNAi is due to differences in culture media, we cultured mouse oocytes in the M199 media for 16 h after mMor2 RNAi. Mouse oocytes were developed to MII stage(62%) and there was no statistical difference compared to that of non-injected(76.8%) and buffer-injected(73.3%) control groups. Therefore, we concluded that the mouse and porcine oocytes are having different metabolic systems in relation to malate dehydrogenase for oocyte maturation. This could be a basis for differences in maturation rates in vitro in two species. Further scrutinized studies on the metabolic pathways would led us in finding better culture system to improve oocyte maturation rates in vitro, especially in more challenging species like the porcine.

• Clinical and Experimental Reproductive Medicine
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• v.34 no.4
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• pp.293-303
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• 2007

Objective: Previously, we identified differentially expressed genes between GV and MII stage mouse oocytes using ACP technology. When we study one of GV selective genes, Obox family, we found Obox4 mRNA expression in ovaries that has been reported as expressed exclusively in testis. Therefore, this study was conducted for characterization and functional analysis for Obox4. Methods: Expression of Obox4 mRNA was examined in gonads and oocytes by RT-PCR. To determine the role of Obox4 in oocyte maturation, Obox4 dsRNA was microinjected into the cytoplasm of GV oocytes followed by 16 h of incubation in the plain medium or by 24 h of incubation in the medium containing IBMX. After RNAi, phenotypes and maturation rates were observed, change in mRNA expression was evaluated, and chromosomal status was confirmed by orcein staining. Results: Obox4 has minimal expression in the ovary compared to that of the other family members. When oocytes were cultured for 16 h in M16 medium after RNAi, maturation rate was not changed significantly, compared with that of non-injected or buffer-injected control oocytes. Surprisingly, however, when oocytes were cultured for 24 h in M16 containing IBMX, in which oocytes were supposed to arrest at GV stage, Obox4 RNAi oocytes were advanced to MI and MII. Spindle structure was disappeared and the chromosomes were condensed in the oocytes after Obox4 RNAi. Conclusions: This is the first report on the expression of Obox4 in the ovary and oocytes. Results of the study suggest that Obox4 plays a crucial role in spindle formation and chromosome segregation during meiosis in oocytes. In addition, Obox4 may play an important role in cAMP-dependent signal cascades of GV-arrest in mouse oocytes.

• Clinical and Experimental Reproductive Medicine
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• v.31 no.3
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• pp.155-168
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• 2004

Objective: Melatonin, which is secreted by pineal gland play an important role in the regulation of ovarian function via seasonal rhythm and sleep in most mammals. It also has a role in the protection of cells by removing toxic oxygen free radicals brought about by metabolism. In the present study, effects of melatonin on the mouse oocyte maturation were examined using two different culture conditions provided with 5% or 21% oxygen concentration. Material and Method: Immature mouse oocytes were obtained from the ovarian follicles of $3{\sim}4$ weeks old ICR strain mice intraperitoneally injected with 5 I.U. PMSG 44 hour before. Under stereomicroscope, morphologically healthy oocytes with distinct germinal vesicle (GV) were liberated from the graafian follicles and collected using mouth-controlled micropipette. They were then cultured for 17 hour at $37^{circ}C$, 5% $CO_2$ and 21% $O_2$ (95% air) or 5% $CO_2$, 5% $O_2$ and 90% $N_2$. New modified Hank's balanced salt solution (New MHBS) was used as a culture medium throughout the experiments. Effects of melatonin were examined at a concentration of $0.0001{\mu}M$, $0.01{\mu}M$ or $1.0{\mu}M$. For the prevention of spontaneous maturation of immature oocytes during culture, dibutyryl cyclic AMP (dbcAMP) and/or hypoxanthine were included in the medium. Results: Under 21% oxygen condition, oocytes cultured in the presence of $0.01{\mu}M$ melatonin showed a significantly higher maturation rates, in terms of germinal vesicle breakdown (95.0% vs 89.0%) and polar body formation (88.1% vs 75.4%), compared to those cultured with $0.0001{\mu}M$ or $1.0{\mu}M$ melatonin. However, no difference was observed in oocytes cultured under 5% oxygen whether they were treated with melatonin or not. In the presence of $0.01{\mu}M$ melatonin, oocytes either cultured under 21% or 5% oxygen exhibited no difference in the polar body formation (85.6% vs 86.7%). However, in the absence of melatonin, oocytes cultured under 21% oxygen exhibited lower polar body formation (74.7%). When oocytes were cultured in the presence of dbcAMP alone or with varying concentrations of melatonin, those treated with both compounds always showed better maturation, i.e., germinal vesicle breakdown and polar body formation, compared to those cultured with dbcAMP alone. At the same concentration of melatonin, however, oocytes exposed to 21% oxygen showed poor maturation than those to 5% oxygen. Similar results were obtained from the experiments using hypoxanthine instead of dbcAMP. Conclusion: Based upon these results, it is suggested that melatonin could enhance the meiotic maturation of mouse oocytes under 21% oxygen concentration, and release oocytes from the meiotic arrest by dbcAMP or hypoxanthine regardless of the concentration of oxygen, probably via the removal of oxygen free radicals.

• Clinical and Experimental Reproductive Medicine
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• v.23 no.1
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• pp.87-94
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• 1996

For evaluating the suitability of human follicular fluid(HFF) as protein supplement in ART, this preliminary study was performed to examine the maturation promoting activity of HFF on mouse follicular oocytes in vitro. Mouse follicular oocytes were collected from ovaries of 21-28 day old ICR mice by puncturing the antral follicles with fine needle at 48 hours after PMSG injection. The oocytes were rinsed and cultured in modified Whittingham's $T_6$ medium containing purines or dbcAMP to maintain meiotic arrest, and different concentrations of HFF were added into the culture medium to examine the effect of HFF on releasing the oocytes from the suppressive influence of the meiotic inhibitors. As a control for HFF, the maturation promoting activity of human fetal cord serum(HFCS) was investigated and compared with the activity of HFF. While HFF was separated, by molecular weight(M.W), into high M.W. fraction(M.W>30,000) and low M.W. fraction(M.W<30,000) and the effects of the fractions on meiotic resumption were investigated in the presence of the meiotic inhibitors. Also hormone analysis was performed to compare the content of hormones in HFF with that in HFCS. Same concentrations of HFF and HFCS induced similar germinal vesicle break down(GVBD) rates of the oocytes meiotic arrested by purines(4mM hypoxanthine+0.75mM adenosine), but the extrusion rate of 1st polar body(PB) of the oocytes cultured in HFF(65.0%, P<0.05) was significantly higher than that(51.6%) in HFCS. While, in the presence of 200 M dbcAMP, the maturation promoting activity of HFF (GVBD: 70.5%, $p<10^{-6}$; 1st PB extrusion: 67.1%, $p<10^{-3}$) was significantly greater than that of HFCS(GVBD: 35.2%; 1st PB extrusion: 41.1%). The oocytes cultured in the fraction of HFF containing high M.W. components showed higher meiotic maturation rates than the oocytes cultured in the low M.W. fraction of HFF. Gonadotropins and $E_2$ were known to improve the completion of maturation changes, and the levels of these hormones were higher in HFF than in HFCS. Therefore, HFF was more effective than HFCS to use for promoting meiotic resumption of mouse oocytes in vitro.

• 대한생식의학회:학술대회논문집
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• 1997.11a
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• pp.50.2-51
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• 1997

• Clinical and Experimental Reproductive Medicine
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• v.35 no.4
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• pp.293-301
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• 2008

Objective: To evaluate outcomes of patients with non-obstructive azoospermia (NOA) undergoing the testicular sperm extraction (TESE) combined with intracytoplasmic sperm injection (ICSI) with different histopathologic subgroups. Method: A total of 122 embryo-transferred TESE/ICSI cycles were compared among NOA subgroups; Germ-cell aplasia (GA, 40 cycles), Maturation arrest (MA, 32 cycles) and severe hypospermatogenesis (S-HS, 50 cycles). Obstructive azoospermia (OA, 667 cycles) patients were served as a control. TESE/ICSI outcomes such as fertilization rate (FR), clinical pregnancy rate (CPR) and live birth rate (LBR) were evaluated. Results: The 2PN FR of embryo-transferred TESE/ICSI cycle was 58.1% in GA, 42.2% in MA and 48.0% in S-HS, which was significantly lower than that of OA (72.9 %, p<0.001). For ICSI-spermatozoa cycles, there were no significant differences in CPR (22.6%, 29.4% and 26.1%) and LBR (16.1%, 29.4% and 19.6%) among NOA subgroups. The CPR of ICSI-spermatid cycles was 0.0%, 9.1% and 0.0% without a live birth. For ICSI-spermatocyte cycles, no clinical pregnancies occurred in any group. Conclusion: There was no significant difference in the FR of embryo-transferred TESE/ICSI cycles among NOA subgroups. The FR among all NOA subgroups was significantly lower than that of OA. Testicular histopathology in NOA did not affect successful pregnancy if spermatozoa extraction from the testis is successful and embryo transfer is possible.

• Development and Reproduction
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• v.8 no.1
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• pp.35-42
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• 2004

Oocyte maturation refers to the process that prophase I arrested germinal vesicle(GV) drives the progression of meiosis to metaphase II(MII) to have the capacity for fertilization and embryo development. To better understand the molecular mechanism(s) involved in oocyte maturation, we identified differentially expressed genes(DEGs) between GV and MII mouse oocytes using a new innovative annealing control primer (ACP) technology. Using 20 ACPs, we successfully cloned 32 DEGs between GV and Mll oocytes, and 26 out of these 32 DEGs were functionally known genes. Four genes including Pscd2 were GV-specific, 10 genes including PKD2 and CSN3 were highly expressed in GV oocytes(GV-selective), and 12 genes including Diva were highly expressed in MII oocytes (MII-selective). Ail of the genes identified in this study were first reported in the oocyte expression using ACP system and especially, we could characterize the existence of PKD-CSW signaling pathwayin the mouse oocytes. Results of the present study would provide insight for studying molecular mechanisms regulating oocyte maturation.

• Korean Journal of Animal Reproduction
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• v.17 no.2
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• pp.75-83
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• 1993

Purine has been identified in the preparation of follicular fluid and shown an activity in maintaining oocyte meiotic arrest. Therefore this study was performed to examine the inhibitory effect of purine on germinal vesicle break down(GVBD) in the presence and absence of human fetal cord serum(HFCS) or human mature follicular fluid(HMFF), as a protein source, in vitro culture. Immature oocytes(GV stage) were collected from ovaries of 21∼28 days old ICR mice by puncturing the antral follicles with a fine needle, at 48 hrs after PMSG injection. Some of the oocytes were denuded by drawing the cumulus-enclosed(complex) oocytes in and out of a pasteur pipet. Complex oocytes and denuded oocytes were cultured 3 hrs. in T6 media containing 0.75mM adenosine or/and 4mM hypoxanthine, with HFCS or HMFF. Their GVBD rates were observed at every 1 hr. during the culture time. Both adenosine and hypoxanthine have shown a time-dependent inhibitory effect on GVBD in complex and denuded oocytes and the inhibitory effect was maximized in culture medium containing hypoxanthine and adenosine. HFCS and HMFF increased the GVBD rates in the presence of the purines, thus HFCS and HMFF may contain a factor that could reverse the inhibitory effect of purines. Also complex oocytes were more sensitive to not only the inhibitory effect of purines but the promoting action of HMFF on GVBD than denuded oocytes. Therefore it was reconfirmed that granulosa cells play an important part in meiotic arrest and resumption.