• The Korean Journal of Zoology
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• v.36 no.2
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• pp.277-284
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• 1993

A possible involvement of maturation promoting factor (nfPF) in the two-cell block phenomenon was studied by fusion experiments. Germinal vesicle (GlF) ooeyte was fused with a blastomore from late or blocked 2-cell mouse embryos. and germinal vesicle breakdoum (GVBD) of fused GV oocvtes in the presence of dbcAMP (100$\mu$g/ml) was scored as an index of MPF aniviD. GnD was induced approximately 30% by fusion of a blastomere derived from late 2-cell embryos, but not from blocked 2-cell embryos. The rate of GVBD was changed when GV oocyte was fused with a blastomere from late 2-cell embryos which were treated with u-amanitin, puromvcin or colcemid before and after hsion: Treatment of late 2-cell embryos with puromycin (50 Is/mll but not with u-amanitin (100 Is/ml) clearly inhibited GVBD, indicating that do novo protein synthesis maw be required for the appearance of MPF activity in late 2-cell embryos. Treatment of late 2-cell embryos w기h colcemid (0.1 Is/mll doubled GVBD, presumably due to the maintenance of metaphase or mitotic phase. SDS-PAGE and twoiimensional electrophoresis revealed that there was no difference in protein synthetic pattern in late and blocked 2-cell embryos, but three phosphoproteins with 27, 35 and 46 M)a, presumsblv M-phase components were phosphorylated in late 2-cell embryos but not in blocked 2-cell embryos. It seems then that MPF activity is closely related to phosphorylstion of M-phase components in late 2-cell embryos.

• 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.

• Clinical and Experimental Reproductive Medicine
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• v.21 no.1
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• pp.99-110
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• 1994

One consequence of fertilization in mammals is an increased resistance of the zona pellucida (ZP) to proteases and various chemical reagents. This phenomenon has been called 'zona pellucida hardening' (ZPH), and it is generally accepted that it is caused by the secretory products of cortical granules released by the egg at fertilization. ZP of mouse oocytes maturing in vitro in a chemically defined medium becomes progressively more resistant to solubilization by chymotrypsin ("Spontaneous" ZP hardening). In the present study, it was aimed to find the specificity of spontaneous ZPH in relation to its possible relevance to the cortical reaction and the physiological block to polyspermy. When a maturation inhibitors, cAMP analog(dbcAMP) and phosphodiesterase inhibitor (IBMX) was added to culture medium, it prevent spontaneous ZPH of mouse oocyte during in vitro culture. Thus spontaneous ZPH requires GVBD, since it is prevented by those agents, which inhibit GVBD in vitro. However, culture for 3 hours in the presence of PMA(lOng/ml), a protein kinase C activator, resulted in ZPH without GVBD, thus suggesting that ZPH may be regulated independently apart from the event of GVBD. Pretreatment of mouse oocyte with FBS result in partially inhibitory effect on subsequent spontaneous ZPH. Induction of GVBD in vivo had a inhibitory effect on the spontaneous ZPH, but subsequent spontaneous ZPH. Induction of GVBD in vivo had a inhinbitory effect on the spontaneous ZPII, but had no inhibitory effect on PMA-induced ZPH. Treatment with a microfilament formation blocker(cytochalasin-B) at 1${\mu}g$/ml concentration, resulted in the excellent inhibitory effect on spontaneous ZPH. However cytochalasin-B did not inhibit PMA-induced ZPH. Thus this suggesting that spontaneuse ZPH had a different mechanism from PMA-induced ZPH.

• Clinical and Experimental Reproductive Medicine
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• v.23 no.3
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• pp.357-366
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• 1996

The present experiments aimed to investigate the metabolism of calcium during oocyte maturation in rat. The concentration of free calcium and calmodulin in oocytes was measured respectively by using of fluo-3/AM and FITC with microscope fluorescence spectrometer. The ultrastructural localization of calcium precipitates in oocytes was observed with the transmission electron microscope. Cumulus-free immature oocytes(GV-oocyte) were cultured in vitro through 15 hours. The free calcium concentration in GV oocyte was $55.9{\pm}3.5nM$. In calcium-containing medium, the free calcium concentration was increased in germinal vesicle breakdown(GVBD) oocyte($64.2{\pm}7.3nM$). In normal medium after calcium chelator treatment ($10{\mu}M$ BAPTA/AM), the free calcium contents were slightly lower than those in control group. In calcium-free medium, the free calcium content was drastically increased in GVBD($72.7{\pm}3.4nM$) and metaphase I - anaphase I ($88.0{\pm}3.4nM$) oocyte. In maturation rate of oocytes, GVBD rate was high in control group($82.9{\pm}6.55%$) and calcium chelator treatment group($91.2{\pm}4.4%$), but in calcium-free medium group, it was low and then the oocyte was degenerated without polar body formation. Relative content of calmodulin in oocyte was significantly(P<0.001) increased in metaphase I - anaphase I than in GV and GVBD oocyte. The calcium precipitates were observed in mitochondria and cytoplasm of GV oocyte but that were not observed in mitochondria of GVBD and metaphase I - anaphase I oocyte. And then the calcium precipitates reappeared in mitochondria of metaphase II oocyte. The above results indicate that changes in free calcium and calmodulin concentration of oocyte occur according to the maturational stages and the extracellular calcium is required during oocyte maturation. Also change of calcium localization in oocyte occurs according to the maturational stages.

• Clinical and Experimental Reproductive Medicine
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• v.23 no.1
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• pp.33-39
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• 1996

This objective of this experiment was to test the effect of EGF on GVBD and MII of nuclear maturation of pig immature oocytes in vitro. Experiment 1 examined to the effect of EGF on nuclear maturation of pig immature oocytes according to different maturational times. The percentage of GVBD of EGF 10mg/ml treated groups were significantly higher than untreated groups after 24hr (p < 0.001). Experiment 2 examined to the effect of duration of exposure of oocytes to EGF supplement in maturation medium. Nuclear maturation rates (M II) of EGF treated groups (during 0-24: 72.8% and 0-42hr: 84.8%) were significantly higher than 53.5 and 26.1% of EGF treated group (during 24-42hr) and untreated group (p < 0.001). Also, experiment 3 examined to the effect of EGF on nuclear maturation of CEOs or CFOs. Nuclear maturation rate (M II) 84.6% of EGF treated group of CEOs was significantly higher than 53.0, 27.6, and 44.2% of EGF treated group of CFOs and untreated groups of CEOs and CFOs (p < O.001). These results conclude that EGF alone can stimulate GVBD and M II of nuclear maturation in pig immature oocytes.

• Reproductive and Developmental Biology
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• v.28 no.3
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• pp.187-190
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• 2004

Protein kinase C exists as a family of serine/threonine kinases which are broadly classified into three groups as cPKC nPKC and aPKC depending on their cofactor requirements. Previous studies have shown that the role of PKC in the process of mouse oocyte maturation. For example, phorbol 12-myristate 13-acetate which is known as an activator of cPKC and nPKC inhibits germinal vesicle break down and 1st polar body extrusion in maturing oocytes. In this study, the effect of thymeleatoxin, a specific activator of cPKC not nPKC, was tested comparing with PMA to address the roles of cPKC and nPKC during mouse oocyte maturation. Cumulus-oocyte complex were cultured in M16 medium for 6 or 12 hr with each of these PKC activators to investigate the effect of germinal vesicle breakdown (GVBD) or the extrusion of 1st polar body. IC/sup 50/ of GVBD were at concentrations of 50nM in PMA and 400nM in thymeleatoxin and of 1st polar body extrusion were 20nM in PMA and 200nM in thy- meleatoxin. The results suggest that activation of nPKC is more closely related to the inhibition of GVBD and 1st polar body extrusion than activation of cPKC. Additionally, we found that the oocytes inhibited 1st polar body extrusion with PMA or thymeleatoxin were arrested in metaphase I of first meiosis.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.30 no.2
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• pp.203-210
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• 1997

The aim of this study was to determine the effect of steroids and human chorionic gonadotropin (HCG) on in vitro maturation and ovulation of oocyte in Pseudobagrus fulvidraco. Oocytes were incubated in the media Leibovitz L15 supplemented with the various concentration of $17\alpha,\;20\beta-dihydroxy-4-pregnen-3-one(17\alpha20{\beta}OHP),\;17\alpha-hydroxyprogesterone(17{\alpha}OHP),\;progesterone(P_4),\;estradiol-17\beta(E_2)and\;HCG$. After 60 hours incubation, the maturation ability of oocyte was assessed by the appearance of germinal vesicle breakdown (GVBD). GVBD was significantly enhanced by the addition of $17\alpha20{\beta}OHP,\;17{\alpha}OHP,\;P_4\;and\;HCD(P<0.05)$. The highest CVBD was observed when $17\alpha20{\beta}OHP$ and HCG were supplemented to media. When oocytes were cultured for 16 hours in media containing $10\~1,000\;ng/ml\;17\alpha20{\beta}OHP,\;17{\alpha}OHP\;and\;P_4$, the rate of GVBD in oocytes cultured in the medium supplemented with 100 ng/ml $17\alpha20{\beta}OHP(65\%)$ was significantly higher than that with $17{\alpha}OHP\;(40\%)\;and\;P_4(35\%)$. The efforts of $17\alpha20{\beta}OHP$ and HCG on GVBD were assessed by various concentration of these hormones. When oocytes were cultured for 60 hours in various media containing $1\~1,000\;ng/ml\;17{\alpha}20{\beta}OHP\;or\;5\~1,000\;IU/ml$ HCG, the GVBD of oocytes was significantly increased in the medium with $10\~100\;ng/ml\;17\alpha20{\beta}OHP$ and 500 IU/ml HCT. When oocytes were cultured in the various media supplemented with $1\~1,000\;ng/ml\;17\alpha20{\beta}OHP\;or\;5\~1,000\;IU/ml$ HCG for 60 hours, the media with $1\~100\;ng/ml\;17\alpha20{\beta}OHP\;or\;50\~1,000IU/ml$ HCG significantly increased in the rate of ovulation. However supplementation with $1,000\;ng/ml\;17\alpha20{\beta}OHP$or 5 IU/ml HCG did not improve the rate of ovulation compared to controls. This results indicate that supplementation of steroid and HCG except $E_2$ can improve the in vitro maturation and ovulation of oocyte in P. fulvidrac; HCG and $17\alpha20{\beta}OHP$ may be more effective than other steroids on oocyte maturation and ovulation in P. fulvidraco.

• Clinical and Experimental Reproductive Medicine
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• v.36 no.3
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• pp.175-186
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• 2009

Objective: We examined the effect of different culture media on oocyte maturation. Methods: Four groups of media, (1) 0.3% BSA mBASAL-XI-HTF, (2) 0.3% BSA mBASAL-XI-HTF with FSH, (3) 10% FBS mBASAL-XI-HTF and (4) 10% FBS mBASAL-XI-HTF with FSH were prepared. Mouse cumulus enclosed oocytes (CEOs) were incubated in each group of medium. Hypoxanthine (Hx) was mixed to each group of medium in increasing concentrations of 1 mM, 2 mM and 4 mM. CEOs were incubated and assessed for GVBD and MII development at 3, 6, 18 hours. Results: CEOs maturation to GVBD was seen in all four groups during 3 hours of culture, however MII stage of oocytes was seen after 6 hours. Complete arrest of GV stage in 4 mM Hx media without FSH and partial arrest in 2 mM Hx media without FSH were seen during 18 hours of culture but development was not suppressed in 1 mM Hx media without FSH. More prominent GVBD suppression was noted at early 3, 6 hours culture in 1 mM, 2 mM Hx media with FSH compared to media without FSH. But the suppression was recovered at 18 hours. This result suggests that low concentrated Hx and FSH supplemented media can suppress CEOs maturation during early culture period but recovery is resumed or even stimulated at late period. 1 mM, 2 mM Hx 10% FBS medium with FSH had significantly higher rates of MII development (71.7%, 66.7%) at 18 hours compared to other media. Conclusion: Our results show that low concentrated Hx and FSH supplemented 10% FBS media may stimulate MII development after an initial inhibitory effect.

• Journal of Aquaculture
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• v.11 no.1
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• pp.119-124
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• 1998

The relative effectiveness of C21-steroids and human chorionic gonadotropin(HCG) on maturation and ovulatin was investigated in vitro using the isolated oocytes or ovarian fragments from the sea bass, Lateolabrax japonicus. ${\alpha}$-hydroxy, 20${\beta}$-dihydroprogesterone(17${\alpha}$20${\beta}$OHP : 5, 50, 500, 1000ng/ml), 17${\alpha}$-hydroxy, 20${\alpha}$-dihydroprogesterone(17${\alpha}$20${\alpha}$OHP : 5, 50, 500, 1000ng/ml) and HCG (5, 50, 500IU/ml) were effective in inducing oocyte maturation, GVM (germinal vesicle migration) or GVBD(germinal vesicle breakdown), compared to control except 17${\alpha}$20${\beta}$OHP and 17${\alpha}$20${\alpha}$OHP at 5ng/ml. 17${\alpha}$20${\beta}$OHP showed the greatest effect on oocyte maturation at 50ng/ml. A combination of 17${\alpha}$20${\beta}$OHP(50ng/ml) and HCG(500IU/ml) led to a significant increase (p<0.05) in GVBD when compared with 17${\alpha}$20${\beta}$OHP or HCG alone. These findings suggest that the two in combination acts synergistically to induce GVBD. 17${\alpha}$20${\beta}$OHP (1~1000ng/ml) and HCG(1~500IU/ml) also induced ovulation in ovarian fragments at all concentrations used ; more effective at lower concentrations(1~50ng/ml or IU/ml). It was shown that HCG was more potent in inducting ovulatin than 17${\alpha}$20${\beta}$OHP.

• Development and Reproduction
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• v.15 no.3
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• pp.223-230
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• 2011

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants derived from incomplete combustion of carbons and crude oil. In this study, we investigated the effects of benzo[a]pyrene (B[a]P), a representative PAHs on in vitro sex steroid hormone production and germinal vesicle breakdown (GVBD) using isolated oocytes of longchin goby (Chasmichthys dolichognathus) and chameleon goby (Tridentiger trigonocephalus). Oocytes in diameters of 0.8-0.9 (end vitellogenic stage) and 0.9-1.0 mm (germinal vesicle migratory stage) from longchin goby and 0.5 mm (fully vitellogenic stage) from chameleon goby were used. In GVBD assay, B[a]P at 10 nM stimulated GVBD in the oocytes of 0.8-0.9 mm from longchin goby. B[a]P at 1 nM stimulated GVBD in the oocytes with diameter 0.5 mm from chameleon goby. In steroid production from oocytes of longchin goby, B[a]P at 100 nM decreased testosterone (T) production, B[a]P at 1,000 nM increased estraiol-17 (J (E2) production and 10 and 100 nM increased $17,20{\beta}$-dihydroxy-4-pregnen-3-one ($17{\alpha}20{\beta}P$) production in the oocytes with diameter 0.8-0.9 mm. B[a]P at 1,000 nM increased E2 production, 100 and 1,000 nM increased $17{\alpha}20{\beta}P$ production in the oocytes with diameter 0.9-1.0 mm. In steroid production of oocytes from chameleon goby, B[a]P at 1,000 nM increased $E_2$ production. B[a]P at 10 nM increased $17{\alpha}20{\beta}P$ production. In the ratio of $E_2$ to T ($E_2$/T), B[a]P at 100 and 1,000 nM increased $E_2$/T in the oocytes of longchin goby. B[a]P at 100 nM also increased $E_2$/T in the oocytes of chameleon goby. Taken together, these results suggest that B[a]P have not only weak estrogenic effects but progestogenic effects on oocyte maturation.