• Korean Journal of Fisheries and Aquatic Sciences
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• v.55 no.1
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• pp.67-72
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• 2022

We investigated the changes in the egg diameter of chub mackerel Scomber japonicus with the stages of egg development (and distinguished between hydrated oocyte and non-hydrated oocyte) for 1, 2, 3, 5, 10, 15 and 30 days. The chub mackerel oocytes were preserved in seven fixing solutions (70% ethyl alcohol, 99.9% ethyl alcohol, 5% formalin, 10% formalin, 5% neutral buffered formalin, 10% neutral buffered formalin and Gilson's solution). At 30 days, the chub mackerel hydrated oocytes preserved in 70% ethyl alcohol and 99.9% ethyl alcohol had shrunk by 5.2% and 7.9%, respectively. Similarly, the non-hydrated oocytes in the same solutions shrunk by 10.3% and 14.0%, respectively. Oocytes preserved in Gilson's solution had an average egg diameter decrease in both the hydrated oocyte (by 16.9%) and non-hydrated oocytes (by 15.6%). The diameter of the preserved hydrated oocytes did not significantly differ between the 5% formalin, 10% formalin, 5% neutral buffered formalin and 10% neutral buffered formalin, with shrinkage percentages of 0.6%, 0.1%, 1.9% and 3.4%, respectively (P>0.05). Similarly, the shrinkage percentages of the non-hydrated oocytes were 4.3% (5% formalin), 5.5% (10% formalin), 4.3% (5% neutral buffered formalin), and 4.1% (10% neutral buffered formalin).

• Journal of Embryo Transfer
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• v.16 no.3
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• pp.239-243
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• 2001

Selection of oocyte cryopreservation method is a prerequisite factor for developing an effective bank system. Compared with slow freezing method, the vitrification has various advantages such as avoiding intracellular ice crustal formation. In our previous, we attempted to employ a vitrification method using ethylene glycol and an electron microscope grid for cryopreservation of mouse oocytes. However, A high incidence of spindle and chromosome abnormalities was detected in thawed oocytes after vitrification. We examined whether the addition of a cystoskeleton stabilizer Taxol $^{TM}$, to the vitrification solution could promote the post-thawed survival and subsequent development of stored oocytes. More oocytes developed to the 4-cell (44.7% vs. 69.7%), 8-cell (31.8% vs. 64.2%), morula (24.7% vs. 54.3%), and blastocyst (20.3% vs. 49.2%) stages after the addition of Taxol$^{TM}$ to the cryoprotectant than after no addition. 21 and 26 mouse pups were born after transfer of blastocyst derived from oocytes vitrified without and with Taxol. The addition of Taxol to vitrification solution greatly promoted post-thaw preimplantation development of ICR morose oocytes.tes.

• Asian-Australasian Journal of Animal Sciences
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• v.17 no.5
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• pp.617-620
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• 2004

The present study compares development of rabbit embryos generated using different oocyte activation protocols and reconstructed with embryonic or cumulus cells as nuclear donor. In vivo matured oocytes were collected from New Zealand White rabbits at 16 h after ovulation treatment and were activated at18 h of post-ovulation treatment. The following schemes of oocytes activation were tested: 1) single electric pulse (EP, 3.2 kV/cm, 3${\times}$20 $\mu$s, 0.3 M mannitol)+5 min culture in the presence of 5 mM Ionomycin, 2) single electric pulse (EP, 3.2 kV/cm, (${\times}$20 $\mu$s, 0.3 M mannitol)+1 h culture in the presence of 2 mM 6-DMAP, and 3) three electric pulses 30 min apart. Cleavage rate, percentage of expanded and hatched blastocysts as well as total cell number of blastomeres of parthenogenetic embryos were significantly higher using either EP+6-DMAP or 3${\times}$EP schemes, comparing with EP+Ionomycin. Development rate up to hatched blastocyst stage of cloned rabbit embryos using the EP+6-DMAP for activation of nuclei were 19% for embryonic cell nuclei and 36% for cumulus cell nuclei. The best activation protocol optimalized in this study was the combined treatment "P+6-DMAP" which may be potentially used for nuclear transfer protocol.

• Asian-Australasian Journal of Animal Sciences
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• v.14 no.6
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• pp.754-758
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• 2001

Bovine oocytes with compact and complete cumulus cells were cultured for up to 24h in TCM199 buffered with 25mmol/l hepes and supplemented with 10% FBS (fetal bovine serum), 1mg/ml $17{\beta}$-estradiol, 20IU/ml hCG(human chorionic gonadotropin). All of the oocytes were divided into at 6 groups depending upon incubation times (control, 0 hour, 6 hours, 12 hours, 16 hours, 18 hours). To all experimental media, $200{\mu}g/ml$ puromycin was added at different incubation times mentioned above. Following these culture times, in vitro insemination was conducted with frozen-thawed bovine spermatozoa in medium BO (Brackett and Oliphant medium for in vitro insemination) with $10{\mu}g/ml$ BSA(bovine serum albumin) and 10 mg/ml heparin added. After 22h culture, the oocytes were fixed with acetic alcohol solution and stained with orcein acetic solution to evaluate sperm nuclear progression. Addition of puromycin after 0, 6 and 12 h of culture resulted in near of oocyte maturation at the M1 stage. Contrariwise, puromycin addition after 12 h of culture led to restoration of nuclear progression to M2 stage. On the one hand, puromycin affected the synthesis of Cyclin B protein that may be involved in the oocyte maturation and sperm capacitation for in vitro fertilization. The present study suggests the participation of protein synthesis, cyclin B, in the oocyte development from M1 to M2 stages in vitro.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.48 no.4
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• pp.483-488
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• 2015

We studied oocyte steroidogenesis in the blackfin flounder Glyptocephalus stelleri as a region-specific species, in the East Sea of Korea during the spawning season. Maturing oocytes (0.76, 0.82, 0.88, and 0.91 mm in oocyte diameter) were incubated in vitro in the presence of [$^3H$] $17{\alpha}$-hydroxyprogesterone ($[^3H]17{\alpha}$-OHP) as a precursor. Steroid metabolites were extracted from the incubated medium and oocytes, and the extracts were separated and identified by thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC) and gas chromatographymass spectrometry (GC/MS). The major metabolites produced from $[^3H]17{\alpha}$-OHP were androgens [androstenedione (A4) and testosterone (T)] and estrogens [$17{\beta}$-estradiol (E2) and estrone (E1)] and progestins [$17{\alpha},20{\alpha}$-dihydroxy-4-pregen-3-one ($17{\alpha}20{\alpha}P$) and $17{\alpha}20{\beta}$-dihydroxy-4-pregnen-3-one ($17{\alpha}20{\beta}P$)] in maturing oocytes. The metabolic rate of $17{\alpha}20{\beta}$ was elevated (29.04%) in oocytes measuring 0.88 mm (nucleus migration stage following the induction of germinal vesicle breakdown), but was very low in oocytes measuring 0.76, 0.82, and 0.91 mm (0.42, 0.67, and 2.62%, respectively). From these results, we suggest that $17{\alpha}20{\beta}P$ acts as a maturation-inducing steroid in the blackfin flounder.

• Clinical and Experimental Reproductive Medicine
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• v.42 no.4
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• pp.156-162
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• 2015

Objective: To investigate fertilization and embryo quality of dysmorphic mature oocytes with specific morphological abnormalities obtained from intracytoplasmic sperm injection (ICSI). Methods: The fertilization rate (FR) and embryo quality were compared among 58 dysmorphic and 42 normal form oocytes (control 1) obtained from 35 consecutive ICSI cycles, each of which yielded at least one dysmorphic mature oocyte, performed over a period of 5 years. The FR and embryo quality of 441 normal form oocytes from another 119 ICSI cycles that did not involve dysmorphic oocytes served as control 2. Dysmorphic oocytes were classified as having a dark cytoplasm, cytoplasmic granularity, cytoplasmic vacuoles, refractile bodies in the cytoplasm, smooth endoplasmic reticulum in the cytoplasm, an oval shape, an abnormal zona pellucida, a large perivitelline space, debris in the perivitelline space, or an abnormal polar body (PB). Results: The overall FR was significantly lower in dysmorphic oocytes than in normal form oocytes in both the control 1 and control 2 groups. However, embryo quality in the dysmorphic oocyte group and the normal form oocyte groups at day 3 was similar. The FR and embryo quality were similar in the oocyte groups with a single abnormality and multiple abnormalities. Specific abnormalities related with a higher percentage of top-quality embryos were dark cytoplasm (66.7%), abnormal PB (50%), and cytoplasmic vacuoles (25%). Conclusion: The fertilization potential of dysmorphic oocytes in our study was lower, but their subsequent embryonic development and embryo quality was relatively good. We were able to define several specific abnormalities related with good or poor embryo quality.

• Korean Journal of Ichthyology
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• v.29 no.4
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• pp.252-257
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• 2017

The oogenesis of the Microphysogobio yaluensis was investigated using light microscopy. Various developmental oocytes appeared in the ovary of the M. yaluensis. The oogenesis is largely divided into four stages: nuclear-chromatin stage, peri-nucleoli stage, vitellogenesis (yolk vesicle and yolk granule stages), and mature stage. The nuclear-chromatin is distributed in a large germinal vesicle as threads. The peri-nucleoli stage has many acidic nucleoli lining at the inner side of the nuclear membrane and an egg envelope just weakly starts. As the oogenesis gradually proceeds, they change to the vitellogenesis stage. The oocyte become to drastically increase and the marginal area of the ooplasm is covered with many vacuoles showing no negative reactions with hematoxylin and eosin staining, called the yolk vesicle stage. Many yolk vesicles-owned oocyte largely increase and as the development continues, its ooplasm is changed from the yolk vesicles to the yolk granules of eosinophilic. At the mature stage, lots of granules merged into a big yolk mass, acidophilic. Even at the mature stage, the egg envelope was still thin between the ooplasm and the follicular layer of the oocyte.

• Journal of Embryo Transfer
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• v.20 no.3
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• pp.201-206
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• 2005

This study was conducted to investigate the effects of oocyte activation after ICSI and of capacitation of insemination sperm before ICSI in Swine. There was no significant difference on cleavage rate and blastocyst developmental rate treated with ethanol, cycloheximide, or ethanol and cycloheximide jointly between treatment and control groups. However, significantly difference was found on cleavage rate and blastocyst developmental rate treated with caffeine and Ca-ionophore on capacitation of insemination sperm before ICSI (p<0.05). There was no significant difference on pronuclear formation rate and total oocyte activation rate treated with oocyte activation after ICSI between treatment and control groups, but was significant difference on pronuclear formation rate and total oocyte activation rate treated with capacitation treat of sperm (p<0.05).

• 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.

• Development and Reproduction
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• v.10 no.2
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• pp.115-125
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• 2006

The present experiment was performed to confirm the effects of selenium on maturation and viability of mouse oocyte. Maturation of oocytes was observed by microscope, Germinal vesicle breakdown(GVBD) and polar body formation(PB) were confirmed at 2.5, 13 hours after in vitro culture. Viability of oocytes was observed by microscope. Normal and abnormal oocytes were distinguished by morphological change in vitro culture for 72 hours. Glutathione(GSH) content of collected oocytes from individual stage also was measured by glutathione assay using spectrophotometer. The results obtained were as follows; The low concentration of selenium($0.005\;{\mu}g/mL{\sim}0.5\;{\mu}g/mL$) increased the maturation rate of germinal vesicle(GV) oocytes to GVBD and PB oocytes. The high concentration of selenium($5\;{\mu}g/mL$) decreased the maturation rate. The low concentration of selenium increased the viability rate of PB oocytes. The high concentration of selenium did not affect the viability rate. The low concentration of selenium increased the GSH content in PB oocytes. The high concentration of selenium decreased GSH content. GSH content in PB oocyte was much higher than that in GVBD oocyte. The results indicate that the low concentration of selenium increases the maturation rate by helping quality elevation of oocyte and minimizing damages of oxidative stress generated from metabolism process. The low concentration of selenium also increases the viability rate by increasing GSH content.