• Asian-Australasian Journal of Animal Sciences
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• v.21 no.4
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• pp.532-537
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• 2008

In order to investigate the factors affecting the culture of mouse preantral follicles in vitro, we examined the effect of culture media, protein supplements, and culture period on their growth. The oocyte diameter (initial size: $55.6{\pm}2.5{\mu}m$) was progressively increased during culture, and the maximum size ($72.0{\pm}2.4{\mu}m$) was reached on day 10 of the in vitro culture. The chromatin configuration in the germinal vesicle (GV) oocyte progressively shifted from a non-surrounded nucleolus (NSN) to a surrounded nucleolus (SN). On day 10 of the culture, most of the oocytes progressed to the SN pattern. The survival and metaphase II rates of the oocytes in alpha-minimal essential medium (alpha-MEM) were significantly higher (p<0.05) than those in Waymouth and tissue culture medium (TCM)-199. As a protein source, fetal bovine serum (FBS) was more suitable for the culture of mouse preantral follicles as compared to human follicular fluid (hFF) and bovine serum albumin (BSA); the optimal concentration of FBS was 5%. These results suggest that in a culture of mouse preantral follicles, alpha-MEM and 5% FBS are an optimal medium and a protein source, respectively; further, the 10 days of culture is required for the complete growth of oocytes in this culture system.

• 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.

• Journal of Embryo Transfer
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• v.26 no.4
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• pp.251-256
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• 2011

Presently, the effect of 0.5 mM dibutyryl cAMP (dbcAMP)-supplemented maturation medium during different incubation time on meiotic arrest (germinal vesicle) and resumption (metaphase II) of porcine oocytes and embryonic development of porcine oocytes following in vitro fertilization (IVF) or parthenogenetic activation (PA) was determined. Porcine cumulus oocyte complexes (COCs) were cultured in 0.5 mM dbcAMP for 17, 22, 27, or 42 h, and an additional 22 h without 0.5 mM dbcAMP. The nuclear status was examined at each time point. Oocytes cultured from 39~49 h displayed more than 80% meiotic resumption. More than 85 % of meiotic arrest was presented at 17~22 h. Oocytes were cultured for 22 h with 0.5 mM dbcAMP and additional 22 h without dbcAMP to assess developmental potential following IVF or PA. There were no significant differences in blastocyst rates among the dbcAMPIVF, IVF, dbcAMP-PA, and PA groups, although cleavage rate of IVF group was significantly higher than those of dbcAMP-PA, and PA groups. In conclusion, 0.5 mM dbcAMP influenced meiotic maturation of porcine oocytes depending on incubation time of oocyte, although embryonic development was not improved in both IVF and PA.

• Journal of Embryo Transfer
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• v.9 no.3
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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.

• Development and Reproduction
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• v.1 no.1
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• pp.1-8
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• 1997

본 연구는 단순배양체계내 소 난포란의 성숙에 요구된는 탄수화물 (glucose, lactate와 pyruvate)을 검토하기 위해 수행하였다. GV(germinal vesicle) 단계의 난구세포로 둘러싸인 소 난자를 단백질, 아미노산 및 호르몬이 첨가되지 않은 modified Tyrodes (mT)에서 24시간, 5% $CO_{2}$ 배양기를 이용하여 성숙배양하였다. Glucose 무첨가군 (0-61%)에 비해 5.6mM의 glucose 첨가군 (71-74%)이 유의적으로 높은 (P<0.05) M-II단계로의 난자 발육을 나타내었다. Glucose를 함유한 배지내에서는 lactate(10mM0와 pyruvate(0.5mM)의 첨가에 따르는 M-II 단계로의 발육에 있어 차이를 보이지 않았다. 그러나 glucose 무첨가 배지에서는 pyruvate와 lactate를 첨가하는 것이 첨가하지 않은 것에 비해 condensed GV(76%vs. 0-2%), M-II, (43-61%vs. 0%)단계에 이른 난자수가 유의적으로 높았다. Glucose 함유 mT배지에 lactate와 pyruvate를 첨가하여 난자를 배양하였을 때, 동결융해 정자와 24시간 정치한 후 난자중 87-93%가 정자침투되었고 39-44%가 전핵단계로 발육하였다. 침투난자의 26-30%는 다정자수정이었다. 결론적으로, GV 단계의 소 난포란은 에너지원으로 lactate, glucose와 Pyruvate를 이용하지만, 감수분열 성숙을 유지하는데 glucose가 가장 효과적이다. 이 결과들로 보아 단순배양배지는 소 난포란의 체외성숙에 영향을 미치는 다양한 물질들을 연구하는데 잠재적으로 유용하다.

• 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.

• Reproductive and Developmental Biology
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• v.32 no.2
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• pp.71-79
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• 2008

Surface-enhanced laser desorption and ionization time-of-flight mass spectrometry (SELDI-TOF MS) is one of the recently developed proteomic technologies which is based on capturing proteins and peptides by chemically modified surfaces and highly sensitive for the analysis of complex biological samples. In the present study, to gain insights into oocyte maturation and early embryo development, SELDI-TOF-MS was used to find the protein candidates that are specifically or prominently expressed in porcine oocytes at the in vitro matured metaphase II (MIIl) and germinal vesicle (GV) stages. By selected CM10 chip, 16 candidates were found to be up-regulated in GV stage oocytes compared with in MII stage oocytes, their molecular weights were 8,180 (2 candidates), 10,226 (5 candidates), 15,767 (5 candidates) and 16,770 (4 candidates) Da respectively. And the expression of 29 candidates were higher in MII than in GV stage oocytes, their molecular weight were 10,832 (3 candidates), 17,743 (8 candidates), 20,122 (3 candidates), 22,131 (3 candidates), 24,857 (7 candidates) and 33,507 (5 candidates) Da, respectively. The expression of selected 13 candidates (0.2 and 1.0 % error tolerances) were analyzed using real time RT-PCR. The proteins that differentially regulated during oocyte in vitro maturation in the pigs may be potential biomarkers of oocyte maturation and quality.

• The Korean Journal of Malacology
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• v.32 no.2
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• pp.67-71
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• 2016

In order to obtain a large number of fertilized eggs for seedling production, experiment was carried out examine effects of serotonin on spawning of the Pacific oyster Crassostrea gigas. The shorter response time to initial spawning in case of serotonin injection showed, the higher serotonin injected with 7.6-27 min. The response time to initial sperm releasing showed the same tendency with female. The highest response rates and eggs amount spawned were showed in the highest concentration. The serotonin injection had no effect on frequency of germinal vesicle breakdown (GVBD), fertilization and hatching rate.

• Asian-Australasian Journal of Animal Sciences
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• v.17 no.8
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• pp.1069-1075
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• 2004

In an attempt to evaluate the function of MAP kinase in porcine oocytes and to develop a method of the assessment of its activity, myelin basic protein (MBP) was used as a substrate to detect the MAP kinase activity of porcine oocytes which had undergone maturation in vitro. The existence of MAP kinase and MAP kinase kinase (MAPKK) was verified in immature porcine germinal vesicle (GV) oocytes at 0 h culture via Western blotting. Porcine oocytes exhibited a low level of MAP kinase activity during the first 20 h of culture, which increased at 25 h, during which time a breakdown in the nuclear membrane occurred. Significantly higher increases (p<0.05) of MAP kinase activity were detected at 30 h of culture. Using the gel phosphorylation method, MBP was phosphorylated at two positions corresponding to mammalian MAP kinase-extracellular signal-regulated kinase (ERK 1) (44 kDa) and ERK 2 (42 kDa). The absolute levels of those proteins did not increase during 40 h of culture, suggesting that the detected increase in MAP kinase activity was the result of phosphorylation rather than changes in the total amount of protein. MAPKK and MAP kinase were dephosphorylated in first-stage (MI) meiotic oocytes by the addition of cycloheximide, a protein synthesis inhibitor. These results of this study indicate that the MAP kinase cascade does exists in porcine oocytes and that its activation leads to oocyte maturation.

• Animal cells and systems
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• v.3 no.4
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• pp.375-383
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• 1999

Gonadal development, gametogenesis, reproductive cycle, and first sexual maturity of Reishia clavigera were investigated monthly from July 1998 to June 1999 through cytological and histological observations. R. clavigera had separate sexes, and was an internal fertilizer. The ma1e penis was located near the two tentacles. The ovary and testis were composed of a great number of oogenic lobules and spermatogenic tubules, respectively. The size of ripe oocyte ranged from 130 to 140 ${\mu}$m in diameter. The peripheral cytoplasm of the germinal vesicle of the ripe oocyte in many cases were surrounded by smaller yolk granules, while the eccentric cytoplasm was occupied with larger ones. The reproductive cycle of R. clavigera could be classified into five successive stages: early active, late active, ripe, spawning, and recovery. Spawning of females occurred from early July to August when the seawater reached above 24.8$^{\circ}C$. Spawning of males occurred from early June to August in the water above 22.8$^{\circ}C$. Minimum size for sexual maturity of both sexes was above 10.0 mm in shell height. Each egg capsule was a cylinder or spindle in shape, 4-6 mm in length and 1-2 mm in width. Colors of newly spawned egg capsules showed yellowish white or pale yellow, while those with veliger larvae showed pale black, and released larvae or dead egg capsules showed black violet. The fecundity in an egg capsule ranged from 70 to 91 eggs (mean＝80.28 eggs).