• The Korean Journal of Zoology
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• v.31 no.4
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• pp.265-272
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• 1988

세포융합방법을 사용하여 성장증인 포유동물의 난자에 들어있는 성숙억제요인(maturation inhibiting activity, 1연Al에 대해 조사하였다. 성장중인 생쥐난자와 성장한 미성숙난자를 1:1로 융합하여 배양했을 서 (14-17시간)에는 거의 모두 핵붕괴를 일으키었으나(90oyo), 2:1로 융합했을 때는 대부분(약 64%) 3개의 핵을 모두 간직하고 있었다. 돼지난자의 경우는 성장중인 것깎 성장한 것을 1:1로 융합하여 배양했을 때에도 융합체들은 모두 핵을 간직하고 있었으며 돼지의 성장중인 난자와 생쥐의 성장한 난자를 융합했을 때에도 모두 핵을 보존하고 있었다. 이에 반하여 돼지와 생쥐 모두에서 성장한 난자끼리 융합했을 때에는 예외없이 핵붕괴가 일어났다. 이러한 결과는 성장중인 생쥐나 돼지의 난자에 각IA가 존재한다는 열과 이종간에도 효과가 있다는 것을 보여주고 있다. 또한 이는 MIA와 성숙촉진요인(maturation promoting factor, MPH의 상대적인 양의 변화가 난자의 성숙조절에 증요한 9f할을 한다는 것을 시사해주고 있다.In an attempt to elucidate the nature of maturation inhibiting activity (MIA) in growing mamma-lian oocvtes, growing mouse and pig oocytes incompetent to resume meiosis were fused with fully grown immature oocvtes in various combinations and cultured for 14-17 hours. In slant cells composed of two mouse growing ooh임es and one large immature oocyte (2:기, their GVs remained well conserved (about 64%) after culture, but not in the ceils composed of one by one pairs. In giant cells of pig composed of one growing and onto large immature oocytes, both GVs remained conserved. In the cells composed of one pig growing and one mouse large oocytes, both GVs were also conserved. In contrast to this, pairs of large mouse oocvtes or those of large pig oocvtes had no CVs after culture. Thus, we could acertain the existEnce of MIA and none-pecificty of it in the mouse and pig growing oocvtes. The results also suggest that the relative amount of substances showlns MfA or MPF activity may be important in the regulation of oocyte amount of substances showing MIA or MPF activity may be important in the regulation of oocyte maturation.

• Clinical and Experimental Reproductive Medicine
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• v.38 no.4
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• pp.193-202
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• 2011

Objective: We found previously that $interferon$ $regulatory$ factor ($Irf$)-1 is a germinal vesicle (GV)-selective gene that highly expressed in GV as compared to metaphase II oocytes. To our knowledge, the function of $Irf-1$ in oocytes has yet to be examined. The present study was conducted to determine the relationship between retinoic acid (RA) and RA-mediated expression of $Irf-1$ and the mouse oocyte maturation. Methods: Immature cumulus-oocyte-complexes (COCs) were collected from 17-day-old female mice and cultured $in$ $vitro$ for 16 hours in the presence of varying concentrations of RA (0-10 ${\mu}M$). Rate of oocyte maturation and activation was measured. Gene expression was measured by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) and cytokine secretion in the medium was measured by Bio-Plex analysis. Apoptosis was analyzed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Results: The rates of oocyte maturation to metaphase II and oocyte activation increased significantly with RA treatment (10 nM-1 ${\mu}M$). With 100 nM RA treatment, lowest level of $Irf-1$ mRNA and cumulus cell's apoptosis was found. Among 23 cytokines measured by Bio-Plex system, the substantial changes in secretion of tumor necrosis factor-${\alpha}$, macrophage inflammatory protein-$1{\beta}$, eotaxin and interleukin-12 (p40) from COCs in response to RA were detected. Conclusion: We concluded that the maturation of oocytes and $Irf-1$ expression are negatively correlated, and RA enhances the developmental competence of mouse immature oocytes $in$ $vitro$ by suppressing apoptosis of cumulus cells. Using a mouse model, results of the present study provide insights into improved culture conditions for $in$ $vitro$ oocyte maturation and relevant cytokine production and secretion in assisted reproductive technology.

• Clinical and Experimental Reproductive Medicine
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• v.25 no.1
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• pp.9-16
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• 1998

This study was performed to investigate the stimulating effect on oocyte maturation and cumulus cell expansion in TC199 media by human cord serum (HCS) supplementation. Immature mouse oocyte cumulus complexes (OCCs) were cultured in TC199 media supplemented with bovine serum albumin (BSA), HCS and human chorionic gonadotropin (hCG) instead of luteinizing hormone (LH) respectively, and the expression of cumulus expansion and oocyte maturation were observed. After 4hr and 24hr culture with or without OCCs, media containing 0.4% BSA, 10% HCS and 10 IV hCG respectively were collected and analyzed for changing concentrations of estradiol $(E_2)$, progesterone $(P_4)$, testosterone (T), and $PGF_{2\alpha}$. There were no elevation of $E_2$, T, and $PGF_{2\alpha}$ by OCCs culture, but minute elevation of $P_4$ level by 24hr OCCs culture in hCG supplementation (p=0.048). The stimulating pattern of cumulus expansion of OCCs by HCS and hCG supplementation was similar to our previously report using Ham's F-10 media, however oocyte maturation rates after 24hr OCCs culture in all media were increased by $20\sim30%$ compared to Ham's F-10 media. These results suggest that LH in HCS induce cumulus expansion probably by $P_4$ secretion of OCCs, and TC199 is efficient media for immature mouse oocyte maturation.

• Clinical and Experimental Reproductive Medicine
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• v.27 no.1
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• pp.23-29
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• 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.

• Development and Reproduction
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• v.1 no.1
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• pp.45-56
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• 1997

Befor fertilization, mammalian oocytes undergo meiotic maturation, which consists of nuclear and cytoplasmic differentiation. In this study, changes of $Ca^{2+}$ stores in mouse oocytes were examined during meiotic maturation and the role of $Ca^{2+}$ in the regulation of the maturation was investigated by using monoclonal antibodies against smooth endoplasmic reticulum $Ca^{2+}$-ATPase(SERCA-ATPase) and calreticulin. Observations were made under epifluorescence microscope and/or confocal laser scanning microscope. In immature oocytes which did not resume meiotic maturation, SERCA-ATPases were mostly localized in the vicinity of the germinal vesicle and calreticulins were distributed evenly throughout the cytoplasm. In mature oocytes, SERCA-ATPases were observed throughout the cytoplasm, butwere absent from the nuclear region. In contrast, calreticulins were localized mostl in the cortex of the oocyte and were absent from the cytoplasm. However, bright fluoresence stainings were wbserved in the perimeiotic spindle region of mature oocyte when labeled with antibodies against calreticulin. These results indicate that mouse oocytes undergo distinct rearrangement of the localization of $Ca^{2+}$-ATPases and calreticulins during meiotic maturation. Thus it can be suggested that redistribution of the $Ca^{2+}$ stores, as revealed by differential fluorescence stainings, is deeply involved in the regulatory mechanism of mammalian oocyte maturation.

• Clinical and Experimental Reproductive Medicine
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• v.20 no.2
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• pp.157-160
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• 1993

The human follicular f1uids(F.F.) may be considered to contribute the maturation of the oocytes on the in vitro culture. To investigate the effects of epidermal growth factor (E.G.F.), which is present in mature and immature follicular fluids, we had experiments of mouse oocytes maturation in vitro. The endpoints assayed were rated as percentage of oocytes undergoing germinal vesicle breakdown(G.V.B.D.) and polar body(P.B.) formation at 12 hours after in vitro culture. The rates of G.B.B.D. were 87% in mature F.F. 68% in immature F.F. and 78% in Ham's F-10 medium respectively. And overall the mature F.F. seem to stimulate on in vitro oocyte maturation compared with either immature F.F. or Ham's F-10 medium. As the concentration of addition of E.G.F. in immature F.F., the rates of G.V.B.D. and P.B. formation were 82 %, 23% in addition with 2 ng/ml while 84%, 32% in addition with 4 ng/ml respectivly. And at the concentration of addition of E.G.F. in Ham's F-10 media as well, the rates of G.V.B.D. and P.B. formation were 84%, 40% and 82%, 44% in addition with each 2ng, 4ng. AccordinglY there was no influence on the oocytes maturation at the addition of E.G.F. to each immature F.F. and Ham's F-10 medium. In conclusion, the E.G.F. is not able to induce oocyte maturation independent of it's effects in immature F.F. and Ham's F-10 media.

• Reproductive and Developmental Biology
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• v.28 no.4
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• pp.267-273
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• 2004

Maturation of oocytes is maintained by complex procedures along with follicular genesis and is a critical step for embryonic development. Purine known as an oocyte maturation regulator is present in follicular fluid. In this study, the roles of guanosine as a strong inhibitor of GVBD and a modulator of cyclic GMP concentration in ooyctes were revealed. Denuded immature oocytes were treated with guanosine, and the maturation rates and cGMP concentration of oocytes were measured. GVBD was blocked in a concentration dependent manner by guanosine, but this effect was reversible. However, GVBD was lagged yet not significant by adenosine. Both guanosine and adenosine modified cGMP concentration in oocytes. The characteristic of the guanosine-treated oocyte was significantly higher cGMP compared with the adenosine-treated oocyes at initial time of the maturation. Based these results, guanosine may be a strong and reversible GVBD inhibitor. Although the precise mechanism of guanosine presently is unclear, the results suggest that guanosine may lead the accumulation of cGMP in oocyte cytoplasm, which in turn suppresses GVBD.

• Reproductive and Developmental Biology
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• v.31 no.2
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• pp.71-76
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• 2007

Previous studies have shown that BMI-1026 is a potent inhibitor of the cyclin-dependent kinases (cdk). In cell culture, the compound also arrests G2/M strongly and G1/S and S weakly. Two key kinases, cdk1 (p34cdc2 kinase) and mitogen-activated protein (MAP) kinase (erk1 and 2), perform crucial roles during oocyte maturation and, later, metaphase II (MII) arrest. In mammalian oocytes, both kinases are activated gradually around the time of germinal vesicle breakdown (GVBD) and maintain high activity in eggs arrested at metaphase II. In this study, we examined the effects of BMI-1026 on GVBD and MII arrest in mouse oocytes. BMI-1026 inhibited GVBD of immature oocytes and activated MII-arrested oocytes in a concentration-dependent manner, with more than 90% of oocytes exhibiting GVBD inhibition and MII activation at 100 nM This is approximately 500$\sim$1,000 times more potent than the activity reported for the cdk inhibitors roscovitine (${\sim}50{\mu}M$) and butyrolactone (${\sim}100{\mu}M$). Based on the results of previous in vitro kinase assays, we expected BMI-1026 to inhibit only cdk1 activation in oocytes and eggs, not MAP kinase. However, in our cell-based system, it inhibited the activity of both kinases. We also found that the effect of BMI-1026 is reversible. Our results suggest that BMI-1026 inhibits GVBD and activates MII-arrested oocytes efficiently and reversibly and that it also inhibits both cdk1/histone HI kinase and MAP kinase in mouse oocytes.

• Development and Reproduction
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• v.13 no.3
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• pp.145-153
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• 2009

Previously, we obtained the list of genes differentially expressed between GV and MII oocytes. Out of the list, we focused on functional analysis of Zap70 in the present study, because it has been known to be expressed only in immune cells. This is the first report about the expression and its function of Zap70 in the oocytes. Synthetic 475 bp Zap70 dsRNA was microinjected into the GV oocytes, and the oocytes were cultured in vitro. In addition to maturation rates, meiotic spindle and chromosome rearrangements, and changes in expression levels of transcripts of three kinases, Erk1/2, JNK, and p38, were determined. Zap70 is highly expressed in immature GV oocytes, and gradually decreased as oocyte matured. When dsRNA of Zap70 was injected into the GV oocytes, Zap70 mRNA specifically and completely decreased by 2 hr and its protein expression also decreased significantly. Absence of Zap70 resulted in maturation inhibition at meiosis I (57%) with abnormalities in meiotic spindle formation and chromosome rearrangement. Concurrently, mRNA expression of Erk2, JNK, and p38, were affected by Zap70 RNAi. Therefore, we concluded that Zap70 is involved in MI-MII transition by affecting expression of MAP kinases.

• 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.