• Development and Reproduction
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• v.2 no.1
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• pp.9-20
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• 1998

It has been wel known that phospholipase C(PLC) plays an important role in the intracellular signaling in a variety of cell types. However, involvement of PLC in mouse oocyte maturation and preimplantation embryo development remains unknown. The present study examined the expression patterns of the mouse PLC \beta 1 and \gamma 1 during oocyte maturatio and preimplantation embryo development study examined the expression patterns of the mouse PLC \beta 1 and \gamma 1 during oocyte maturation and preimplantation embryo development by the competitive reverse transcription-polymerase chain reaction (RT-PCR method). PLC \gamma 1 mRNA (0.1 fg) was readily detected in germinal vesicle (GV)-stage oocyte and its level was reduced as meiotic resumption proceeded. PLC-\beta 1 mRNA (<0.1 fg) as detected at low level at GV-stage oocytes and scarcely detected at germinal vescle breakdown (GVBD)-stage oocytes. After fertilization, both PLC \beta 1 and \gamma 1 mRNA levels began to increase at morula-stage embryos (0.2 fg) and were more prominent in blastocyst-stage embryos(1 fg). to elucidate the possible involvement of PLC via protein kinase C(PKC) pathway during oocyte maturation and preimplantation embryo development , the effects of sphingosine (PKC inhibitor), sn-$diC_{8}$(PKC activator) anc U73122 (PLC ingibitor) were examined. Treatment of GV-stage oocytes with sphingosine (20 \mu M) facilitated the meiotic resuption by 10-20 over the control within 1 h as judged by GVBD, whereas U73122 failed to show any significant effect. U73122 (10 \mu M) effectively blocked the compaction of morula, while sn-$diC_{8}$(50 \mu M). In summary, the present study shows that the mouse PLC \beta 1 and \gamma 1 are expressed in a developmental stage-specific manner and PLC-PKC pathway may be involved in early preimplantation embryo development.

• Journal of Embryo Transfer
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• v.19 no.2
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• pp.165-172
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• 2004

This study was carried out to examine the effects of maturation media on in vitro maturation (IVM) of porcine immature oocytes, and on subsequent in vitro fertilization (IVF) and development (IVD). Cumulus-oocyte complexes (COCs) were collected from antral follicles of porcine ovaries collected from abattoir, and were matured in vitro in modified NCSU-37 (mNCSU-37), modified NCSU-23 (mNCSU-23), or TCM-199 supplemented with 10% porcine follicular fluid (pFF). Oocytes matured in vitro, were fertilized in vitro in modified Tris-buffered medium(mTBM) with the final motile sperm concentration of 1${\times}$105 sperm/mL, and subsequently putative embryos were developed in vitro in NCSU-23. The results are as follows. 1. In the result of IVM, the rate of germinal vesicle breakdown (GVBD) and of nuclear maturation were not significantly different among the media, though numeric value of them were slightly lower in TCM-199 than in mNCSU-37 or in mNCSU-23. 2. In the result of IVF, though the rate of sperm penetration was not significantly different among the maturation media, the percentage of oocytes with male pronucleus (MPN) of ones matured in mNCSU-37 (88.0%) was significantly higher than in TCM-199 (71.1%) (p<0.05). 3. In the result of IVD, the percentage of cleaved oocytes of ones matured in mNCSU-37 (52.3%) or in mNCSU-23 (53.7%) was significantly higher than in TCM-199 (43.1%) (p<0.05), but the rate of blastocysts at day 6 was not significantly different among the maturation media, though putative embryos from oocytes matured in mNCSU-37 or in mNCSU-23 were developed more than in TCM-199. These results suggested that mNCSU-37 or mNCSU-23 was more appropriate than TCM-199 as IVM medium for porcine immature oocytes.

• Journal of Embryo Transfer
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• v.19 no.2
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• pp.173-183
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• 2004

This study was carried out to examine the effects of epidermal growth factor (EGF) and the number of cumulus-oocyte complexes (COCs) on in vitro maturation (IVM) of porcine immature oocytes, and on subsequent in vitro fertilization (IVF) and development (IVD). COCs were collected from antral follicles of porcine ovaries collected from abattoir, and were maturated in modified NCSU-23 (mNCSU-23) with 10% pFF, 0.6 mM cysteine, 50 ${\mu}mM{\beta}-mercaptoethanol$, 1 mM dbcAMP, 10 IU/mL PMSG and 10 IU/mL hCG, which was supplemented with or without 10 ng/mL EGF and into which 50 or 15 COCs per droplet was put. Oocytes matured in vitro, were fertilized in vitro in modified Tris-buffered medium (mTBM) with the final motile sperm concentration of 1${\times}$105 sperm/mL, and subsequently putative embryos were developed in vitro in NCSU- 23. The results are as follows. 1.In the result of IVM, 10 ng/mL EGF supplement duplicated the percentage of C4 group of COCs(41% vs 81%). But the rate of germinal vesicle breakdown (GVBD) and of nuclear maturation were not significantly different between control and EGF supplemented, or between the number of COCs per culture droplet, and there was not a significant interaction between the two factors, either. 2. In the result of IVF, there was not significantly different between control and EGF supplemented, or between the number of COCs per culture droplet, or was not a significant interaction between the two factors, in the rate of sperm penetration, in the percentage of oocytes with male pronucleus (MPN), and in the rate of polyspermy. 3. In the result of IVD, there was not significantly different between control and EGF supplemented, or between the number of COCs per culture droplet in the percentage of cleaved oocytes. There was not significantly different between the number of COCs per culture droplet, but between control and EGF supplemented (p<0.01) in the percentage of blastocysts, the number of inner cell mass (ICM), trophectoderm (TC) and total cells. There was no significant interaction between the two factors anywhere. These results suggested that 10 ng/mL EGF supplement into mNCSU-23 for IVM was effective in the production of more as well as better blastocysts during IVD through increasing the number of cells in those.

• Korean Journal of Animal Reproduction
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• v.22 no.4
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• pp.385-393
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• 1998

This study was conducted to investigate the effect of cysteine (CySH) and glutathione (GSH) on in vitro maturation of porcine follicular oocytes. The results obtained were summarized as follows: 1. When the immature oocytes were cultured at 0, 0.04, 0.14, 0.6 and 1.2 mM of cysteine (CySH) for 36h, the germinal vesicle breakdown (GVBD) rates were 90.8, 89.9, 90.5, 92.0 and 91.3%, respectively, and the maturation rates of the oocytes with metaphase-II were 56.1, 50.7, 41.9, 49.0 and 61.5%, respectively. Especially, the maturation rates of 0.14 and 0.6 mM treated groups were significantly lower than those of control (non-treated) group (P＜0.05). After 44h of culture in the same treatments of CySH, the GVBD rates of porcine immature oocytes were 90.0, 91.8, 89.8, 90.5 and 89.6%, respectively, and the maturation rates were 80.2, 76.3, 69.4, 66.7 and 72.6%, respectively. Especially, the maturation rates of 0.14 and 0.6 mM treated groups were significantly lower than those of control group (P＜0.05). 2. When the immature oocytes were cultured at 0, 0.05, 0.1, 0.5 and 1.0 mM of glutathione (GSH) for 36h, the GVBD rates of porcine immature oocytes were 91.0, 90.9, 89.5, 92.0 and 91.1%, respectively, and the maturation rates were 59.0, 48.5, 47.8, 38.6 and 37.5%, respectively. All treated groups of GSH showed lower maturation rates than the control group (P＜0.05). After 44h of culture in the same treatments of GSH, the GVBD rates of porcine immature oocytes were 91.8, 94.1, 89.1, 91.3 and 91.1%, respectively, and the maturation rates were 84.6, 57.1, 69.6, 71.3 and 64.3% respectively. All treated groups of GSH showed lower maturation rates than the control group (P＜0.05).

• Korean Journal of Animal Reproduction
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• v.25 no.1
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• pp.19-28
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• 2001

The present study was undertaken to investigate the effects of cooling rate and equilibration time on the survival, in vitro maturation and development to embryos of frozen-thawed bovine immature oocytes(Germinal Vesicle Stage). The cryoprotectants are used 10% ethylene glycol(EG) as permeating cryoprotectant and 0.05M soc.ose(S) or trehalose(T) as low molecular weight nonpermeating cryoprotectants and 5% ficoll(F) or polyvinylpyrrolidone(PVP) as high molecular weight nonpermeating cryoprotectants. Four freezing solution were uysed in this experiment(EFT: 10% EG + 5% F + 0.05M T, EFS: 10% EG + 5% F + 0.05M S, EPT: 10% EG + 5% P + 0.05M T, EPS: 10% EG + 5% P + 0.05M S). The best equilibration time and freezing solution was 15 min in EPT(83% survival rate of frozen-thawed bovine immature oocytes). When frozen-thawed bovine oocytes were cultured following IVM and IVF, there was no significant difference in cleavage and development rates among the EFT, EFS, EPT and EPS solutions. When 9 blastocysts derived from frozen bovine oocytes were transferred to 6 recipients, two recipients were pregnant. And one was aborted at 45 days of pregnancy and the other had a stillbirth.

• Clinical and Experimental Reproductive Medicine
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• v.33 no.3
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• pp.189-198
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• 2006

Objective: We previously described that Diva is highly expressed in matured metaphase II (MII) oocytes compared to immature germinal vesicle (GV) oocytes in mouse. We report here that the expression of Diva transcript as well as protein is oocyte-specific. To elucidate its physiological role in oocyte, the binding partner(s) of Diva has been identified by using immunoprecipitation (IP) followed by Mass Spectrometry. Methods: NIH/3T3 cells were transiently transfected for 24 h with either empty vector for control or FLAG-tagged mouse Diva construct, and IP was performed with anti-FLAG antibody. The immuno-isolated complexes were resolved by SDS-PAGE on a 12% gel followed by Coomassie Blue staining. For in-gel digestion, 15 bands of interest were excised manually and digested with trypsin. All mass spectra were acquired at a positive reflector mode by a 4700 Proteomics Analyzer (Applied Biosystems, Framingham, MA). Proteins were identified by searching the NCBI nonredundant database using MASCOT Peptide Mass Fingerprint software (Matrixscience, London). Results: Diva-associated complexes were formed in FLAG-tagged mouse Diva-overexpressed NIH/3T3 cells via IP using anti-FLAG-conjugated beads. Among the excised 15 bands, actin and actin-binding proteins such as tropomyosin, tropomodulin 3, and ${\alpha}$-actinin were identified. Binding between Diva and actin or tropomyosin was confirmed by IP followed by Western blot analysis. Both bindings were also detected endogenously in mouse ovaries, indicating that Diva works with actin and tropomyosin. Conclusions: This is the first report that immuno-isolated Diva-associated complexes are related to actin filament of the cytoskeletal system. When we consider the association of Diva with actin and tropomyosin, oocyte-specific Diva may play a role in modulating the cytoskeletal system during oocyte maturation.

• Clinical and Experimental Reproductive Medicine
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• v.35 no.4
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• pp.285-291
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• 2008

Objective: This study was performed to investigate whether cumulus morphology and oocyte diameter influence on in vitro maturation (IVM) of human germinal vesicle (GV) stage oocytes obtained from stimulated in vitro fertilization (IVF) cycles. Methods: Forty-one GV stage oocytes were obtained from 21 patients who received ovarian hyperstimulation and IVF. According to cumulus morphology before denudation, GV oocytes were classified into oocytes with dispersed cumulus cells (CCs) or compacted CCs. The diameters of denuded oocytes, both including and excluding the zona pellucida, were measured. All oocytes were cultured in commercial IVM medium. Maturation was defined as extrusion of the first polar body and the matured oocytes were inseminated by ICSI method. Results: Overall maturation and fertilization rate were 56.1% and 73.9%. Matured oocytes had significantly higher proportion of oocytes with dispersed CCs compared to oocytes failed to mature (91.3% vs. 55.6%, p=0.023). There were no significant differences of oocytes outer ($155.7{\mu}m$ vs. $152.4{\mu}m$, NS), inner ($114.3{\mu}m$ vs. $113.4{\mu}m$, NS) diameters and zona thicknesses ($41.3{\mu}m$ vs. $39.1{\mu}m$, NS) between matured and not-matured oocytes. In-vitro maturation rate of oocytes with dispersed CCs was significantly higher than which of oocytes with compacted CCs (67.7% vs. 20.0%, p=0.044). Oocyte diameters (outer and inner) and thicknesses were not related with maturational competence. Conclusion: Our results suggest that in vitro maturational competence of GV stage oocytes obtained from stimulated IVF cycles is closely associated with the cumulus morphology but not oocyte diameter.

• Development and Reproduction
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• v.11 no.3
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• pp.263-272
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• 2007

Contrast to mouse where its in vitro maturation rates are high without specific supplements or presence of the cumulus cells, there are some species, such as porcine, where its in vitro oocyte maturation rates are still very low. This comparative study was conducted to investigate the role of malate dehydrogenase(Mor2) during oocyte maturation by RNAi in the mouse and porcine. The Mor2 double-stranded RNA(dsRNA) was prepared speciesspecifically and microinjected into the cytoplasm of denuded germinal vesicle(GV) oocytes. Oocytes were cultured for 48 h(porcine) and 16 h(mouse) in M199 with 10% porcine follicular fluid, pyruvate, p-FSH, EGF, cystein, and estradiol-$17{\beta}$. We measured changes in oocyte morphology, maturation rates and mRNA levels after Mor2 RNAi. We confirmed gene sequence-specific knock down of Mor2 mRNA in both species after Mor2 RNAi. In contrast to our previous finding that mMor2 RNAi resulted in GV arrest in the mouse, we found that pMor2 RNAi resulted in MI arrest in denuded porcine oocytes(58%), but developed to MII(84.4%) in COCs. To determine whether this difference between mouse and porcine RNAi is due to differences in culture media, we cultured mouse oocytes in the M199 media for 16 h after mMor2 RNAi. Mouse oocytes were developed to MII stage(62%) and there was no statistical difference compared to that of non-injected(76.8%) and buffer-injected(73.3%) control groups. Therefore, we concluded that the mouse and porcine oocytes are having different metabolic systems in relation to malate dehydrogenase for oocyte maturation. This could be a basis for differences in maturation rates in vitro in two species. Further scrutinized studies on the metabolic pathways would led us in finding better culture system to improve oocyte maturation rates in vitro, especially in more challenging species like the porcine.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.20 no.6
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• pp.509-519
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• 1987

Annual reproductive cycle of the Damselfish, Chromis notatus collected monthly at the four coastal areas around Chejudo, Korea are studied on the bases of histological observations of gonadal tissue and various quantitative variables including gonadosomatic index (GSI), fatness, egg diameter composition and the first maturity. The ovary consisted of a pair of saccular structure with many ovarian sacs. Oogonia proliferated along the germinal epithelium of the ovarian sac. Young oocytes with basophilic cytoplasm showed several nucleoli along the nuclear membrane. When the oocytes reached about $450{\mu}m$ in diameter, nucleus migrate toward the animal pole, nuclear membrane disappeared and most of cytoplasm were filled with yolk materials and oil drops. After ovulation, residual follicle and growing oocytes remaining in the ovarian sacs degenerated. But early young oocytes without follicle layer were not degenerated, and growing continuously till the next year. The testis consisted of a pair of lobular structures in the right and left were united in the posterior seminal vesicle. Cortex of testis was composed of many sperm ducts connected with lobuli. GSI began to increase from March, starting season of longer day length and higher water temperature, and reached the maximum value between June and August. It began to decrease from September with the lowest value appearing between October and February without any evident variation. The annual reproductive cycle could be devided into five successive stage : growing(April to Many), mature(May to August), ripe and spent (June to August) and recovery and resting stage(September to March). The spawning peak occurred from June to August. According to the frequency distribution of egg diameter, Chromis notatus was a polycyclic species to spawn twice or more in a spawning season. Fatness, correlated with gonadal phases, was remarkably decreased by spawning. Percentage of the first maturity . in femate and male fish ranging from 7.0 to 7.9 cm were $50\%$ and from 9.0 to 9.9 cm in total length $100\%$.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.17 no.5
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• pp.423-435
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• 1984

The reproductive cycle of the small filefish, Rudarius ercodes was investigated based on the annual variations of gonadosomatic index(GSI) and hepatosomatic index(HSI) by electronic and photic microscophy. The specimens used were collected at the coastal area of Benden island, Sizuokagen, Japan, from September 1982 to August 1983. GSI began to increase from March, starting season of longer daylength and higher water temperature, and reached the maximum value between June and August. It began to decrease from September with the lowest value appearing between November and February without any evident variation. The annual variations of HSI were not distinct in male filefish and were negatively related to GSI in female : HSI decreased in the summer season when the ovary was getting mature and reached the maximum in the winter season when the ovary was getting retrogressive. The ovary consisted of a pair of saccular structure with numerous ovarian sacs branched toward the median cavity. Oogonia divided and proliferated along the germinal epithelium of the ovarian sac. Young oocytes with basophile cytoplasm showed several scattering nucleoli along the nuclear membrane. when the oocytes growing to about 300 ${\mu}m$, nuclear membrane to disappear with nucleus migrating toward the animal pole. The regions of protoplasm were extremely confined within the animal hemisphere in which most of cytoplasms were filled with yolk materials and oil drops. After ovulation, residual follicles and growing oocytes remaining in the ovarian sacs degenerated. But perinucleatic young oocytes without follicles formed were not degenerated, and growing continuously still in the next year. Mitochondria and endoplasmic reticula in the cytoplasm remarkably increased with oocytes maturing and yolk accumulating. Those were considered to be functionally related to the yolk accumulation. Five or six layers of possible vitellogenin, oval-shaped disc structures with high electron density, appeared in the apex of follicular processes stretching to the microvilli pits of mature oocytes. Testis consisting of a pair of lobular structures in the right and left were united in the posterior seminal vesicle, Cortex of testis was composed of several seminiferous tubules, and medulla consisting of many sperm ducts connected with tubules. Steroid hormone-secreting cells with numerous endoplasmic reticula and large mitochondria of well developed cristae were recognized in the interstitial cells of the growing testis. Axial filament of spermatozoon invaginated deeply in the central cavity of the nucleus and the head formed U-shape with acrosome severely lacking, mitochondria formed large globular paranuclei at the posterior head, and microtubular axoneme of the tail represented 9+9+2 type. The annual reproductive cycles could be divided into five successive stages : growth(March to July), maturation(May to September), Spawning(mid May to early October) and resting stages(October to February). The spawning peak occurred from June to August.