• 한국가축번식학회지
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• 제22권3호
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• pp.277-286
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• 1998

The effect of MAP kinase activity on maturation of porcine oocytes was investigated. MAP kinase was detected by immunofluorescence staining in nucleus of oocytes just before entering GVBD (germinal vesicle breakdown)stage. In a Western blot with GV (germinal vesicle) from these oocytes (cultured for 25 hours), a shift of MAP kinase band a, pp.ared, suggesting an activated stage of the kinase. No activity was shown in the blot with GV isolated from, oocytes cultured for 0 hour. To confirm that activation of MAP kinase induce GVBD, we microinjected MAP kinase purified from matured oocytes starfish into the cytoplasm of oocytes in GV stage (cultured for 0 hour). The injected MAP kinase did not cause early a, pp.arance of GVBD. No oocytes showed GVBD state until 20 hours of culture. Activity of MAP kinase did not increase significantly after the injection. When the exogenous MAP kinase was injected into GV, GVBD was induced in about 20% of oocytes cultured for 5 to 10 hours. These results suggest that MAP kinase is traslocated to nucleus and function as a factor inducing GVBD.

• 한국동물학회지
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• 제30권1호
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• pp.89-98
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• 1987

생쥐 미성숙 난자를 재료로 하여 난자 성숙 억제제인 dbcAMP 존재하에서 GVBD 난자와의 융합에 따른 체외성숙 양상을 조사하였다. 기본 배양액 내에서 3시간이 경과하였을 때 GVBD 난자와 융합된 미성숙 난자 뿐만이 아니라 미성숙 난자 뿐만이 아니라 미성숙 난자끼리 융합된 것들도 모두 성숙을 재개하였다. 그러나 dbcAMP가 함유된 배양액 내에서 3시간 경과 하였을 때는, 미성숙 난자끼리 융합된 것들은 모두가 GV상태로 성숙이 억제된 채로 있었고 반면에 GVBD 난자와 융합된 미성숙 난자들은 비록 dbcAMP가 존재하더라도 보두 성숙을 재개하였다. dbcAMP가 함유된 배양액 내에서 20시간이 경과하였을 때 미성숙 난자끼리 융합된 것들은 여전히 성숙이 억제되어 있었으나 GVBD 난자와 융합된 미성숙 난자들은, 기본 배양액 내에서 배양된 융합 난자들과 마찬가지로 다수가 하나 혹은 두개의 극체를 형성하는 제2 감수분열 중기에 진입하였다. 두 개의 극체를 방출한 융합난자들 중에는 하나의 세포질 내에 감수분열 중기방추사가 두 곳에서 형성되는 것이 관찰되었다. 이 실험 결과로 보아 이미 성숙이 재개된 난자내에는 난자 성숙 억제제인 dbcAMP의 억제효과보다 더 영향이 큰 난자 성숙 유도 물질이 있으며, 이 물질이 융합하고 있는 미성숙 난자내로 이전하여 dbcAMP에 의해서 그 성숙이 억제된 미성숙 난자의 성숙을 유도한다는 것을 알 수 있다.

• Reproductive and Developmental Biology
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• 제28권4호
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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.

• 한국가축번식학회지
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• 제21권2호
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• pp.123-130
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• 1997

Normal maturation of the mammalian oocytes is prerequisite for the fertilization and the early embryonic development. We have been tested the effects of purine and its de novo synthetic inhibitor, azaserine(Aza) on the maturation of germinal vesicle(GV) and germinal vesicle breakdown(GVBD) mouse oocytes. Denude-immature oocytes were cultivated in the media containing adenosine, guanosine, and/or azaserine, and checked the matruation stage by monitoring the prominent morphological changes. In GV stage oocytes, GV was arrested temporarily by the adenosine(1.0%) and protractedly by the guanosine(65.9%, P<0.001). The regression was increased significantly at the adenosine(90%, P<0.001) but decreased at the guanosine(1.6%, P<0.05). Inhibiting the de novo synthesis of purine, nuclear maturation rate was increase(90.4% : 96.7%), but GV arrest was significantly increased by cotreatment with guanosine(P<0.001). Polar body extraction significantly was increased at the Aza(P<0.05), but not in others. In GVBD oocytes, adenosine itself did not affect GVBD arrest. Guanosine, on the other hand, elevated GVBD arrest rate(P<0.001), but co-treated with Aza, decreased GVBD arrest(P<0.001). Aza increased GVBD arrest rate(20.2%, P<0.05) compared with control. From those results, we know that guanosine shows more prominent effect on the inhibition of nuclear maturation at the GV stage, and of the 1st polar body extrusion at the GVBD stage. Adenosine showed the cytoplasmic toxicity at GV stage oocyte. Our data speculate that cytoplasmic cAMP level is auto-regulated by endogenous adenylate cyclase while GVBD is inhibited by guanosine, since purine toxicity is not observed in the GVBD stage. And it is showed that purine metabolism is concerned with nuclear maturation, that the amounts of purine metabolism is not even during the oocyte maturation.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2003년도 학술발표대회 발표논문초록집
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• pp.61-61
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• 2003

We have investigated the novel function of tissue transglutaminase (tTG) in the germinal vesicle breakdown (GVBD) of mouse oocyte. tTG was identified in ooplasm and germinal vesicle by immunostaining assay. Spontaneous maturation of the oocytes elevated in situ activity of tTG by over 2.5 fold at 3 hr, which was determined by a confocal microscopic assay. However, incubation with monodansylcadaverine (MDC), a tTG inhibitor, blocked the activation of tTG. The possible role of tTG in GVBD was investigated by the use of two tTG inhibitors, MDC and cystamine. MDC largely inhibited the GVBD by a concentration dependent manner. GV-stage oocytes were matured to the GVBD stage by 78% at 3 hr in BWW culture medium. However, in the oocytes incubated with MDC for 3 hr, the GVBD rates were 43 and 11% by 50 and 100 mM, respectively. MDC also blocked the entry of 70 kDa TRITC-dextran from the ooplasm to the compartment of germinal vesicle, indicating a possible inhibition of nuclear pore disassembly by MDC. The role of tTG in GVBD was further investigated by microinjection with cystamine. The control oocytes, injected with DPBS, showed about 80 % of GVBD at 3 hr. But the oocytes injected with cystamine showed 15% of GVBD at 3 hr and a little higher rate at 6 hr. In addition, the inhibition of GVBD maturation by MDC was reversible by washing. These results suggested that tTG was involved in the early event of mouse oocyte maturation

• 한국수산과학회지
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• 제34권6호
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• pp.584-587
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• 2001

성숙 시기의 넙치 (Paralichthys olivaceus) 어미를 대상으로 난 모세포의 GVBD와 배란유도과정에 TBT/TPhT와 Aroclor 1254의 저해효과를 조사한 결과, TBT (또는 TPhT)와 Aroclor 1254모두 GVBD 과정과 HCG로 전처리한 배란유도 과정에 저해효과를 보였으며, 난모세포의 반응은 Aroclor 1254보다는 TBT에 좀 더 민감한 것으로 나타났다. TBT의 처리 농도별 차이는 TBT 0.1과 1 ppm에서 가장 낮은 GVBD 유도율을 보였으나 $0.0001\~1\;ppm$ 사이에 유의한 차이는 관찰할 수 없었다 TBT의 배란저해 효과는 HCG 처리 구에 비해 HCG+TBT의 모든 실험구 (0.01, 0.1, 1 ppm)에서 뚜렷하게 나타났으며, TBT 처리구 중 가장 높은 농도인 HCG+TBT 1 ppm에서 가장 낮은 배란율을 보였다. 넙치의 GVBD와 배란과정을 유기주석화합물 (TBT, TPhT)이 방해함으로써 이 시기의 주요 호르몬인 progestogens 작용이 저해되는 것으로 생각되며, 앞으로 난소발달 단계별로 세분화하여 그 저해 작용 메커니즘에 대한 실험이 요구된다.

• Reproductive and Developmental Biology
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• 제31권2호
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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.

• 한국양식학회지
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• 제9권1호
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• pp.57-63
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• 1996

범가자미 난모세포의 최종성숙(GVBD=germinal vesicle breakdown) 유도를 위한 HCG (human chorionic gonadotropin)와 스테로이드 호르몬들의 효능 비교실험이 in vitro에서 이루어졌다. 실험에 사용한 스테로이드 호르몬, progestogens (progesterone, $17\alpha$-hydroxyprogesterone, $17\alpha$-hydroxy, $20\beta$-dihydroprogesterone)중에서는 $17\alpha$-hydroxy, $20\beta$-dihydroprogesterone ($17\alpha20{\beta}OHP$)이 매우 효과적으로 나타났으며, $17\alpha20{\beta}OHP$와 HCG와의 비교실험에서는 이들 모두 GVBD 유도에 효과적인 것으로 나타났다. 특히 $17\alpha20{\beta}OHP$는 저농도($10\~50$ ng/ml)에서도 효과적이며, HCG는 사용한 농도(50, 100, 500 IU/ml)사이에 유의한차를 보이지 않았다.

• 한국수산과학회지
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• 제50권1호
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• pp.41-47
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• 2017

Perhaps the most common type of reproductive dysfunction in captive fish is failure of females to undergo final oocyte maturation and thus to ovulate and spawn. The success of aquaculture could therefore be improved by developing techniques to enhance natural spawning, artificial maturation, and/or to induce ovulation in farmed fish. This study aimed to investigate the effects of prostaglandin $E_2$ ($PGE_2$) and prostaglandin $F_{2{\alpha}}$ ($PGF_{2{\alpha}}$) on in vitro oocyte maturation (germinal vesicle breakdown, GVBD) and ovulation in the marine fish Chasmichthys dolichognathus. Post-vitellogenic follicles (0.80-0.94 mm diameter oocytes) were incubated with $PGE_2$ or $PGF_{2{\alpha}}$ at concentrations of 5, 50, or 500 ng/mL for 24 hours. A significant increase in GVBD was seen in 0.84 mm and 0.94 mm oocytes incubated with 50 ng/mL $PGE_2$ compared with the control. There was no significant increase in GVBD in any of the other experimental conditions (5 or 500 ng/mL $PGE_2$ or 5, 50, or 500 ng/mL $PGF_{2{\alpha}}$). Neither of the prostaglandins induced ovulation at the concentrations tested.These results suggest that GVBD was induced by incubation with 50 ng/mL $PGE_2$.

• 한국수정란이식학회지
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• 제9권3호
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• pp.249-254
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• 1994

This study was undertaken to investigate effects of granulosa cells on mejotic maturation of porcine oocytes in vitro. The results obtained in this study were summarized as follows : The germinal vesicle breakdown(GVBD) rates were 91.5, 93.3 and 96.6%, respectively, when the cumulus oocy:e cornplexes(COC) in the TCM-199 medium with sodium bicarbonate, Na pyruvate, penicillin G, streptomycin sulfate and 10% FCS were cultured in the condition of FSH(0.02 Au/ml), LH(10 $\mu$g/ml) and FSH + LH added. And when the COC were co-cultured with granulosa cell (5$\times$ 106 cells /ml) in the condition of FSH, LH and FSH + LH added, GVBD rates were 94.3, 92.9 and 98.9%, respectively. However, when the COC were cultured in the condition of hormone free and co-cultured with granulosa cells in the condition of hormone free, the GVBD rates were 40.4 and 86.3%, respectively. The GVBD rates were 41.0, 62.7, 84.6, 88.1 and 93.6%, respectively, when the COC were co-cultured with granulosa cells that the concentrations are 0 cells /ml, 1 $\times$ 106 cells /ml, 5:: 106 cells /ml, 1$\times$ 107 cells /ml and 5$\times$ 107 cells /ml.