• Clinical and Experimental Reproductive Medicine
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• v.25 no.2
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• pp.207-216
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• 1998

Certain types of somatic cells, as well as follicular cumulus cells associating with mammalian oocytes, are known to produce beneficial effects on in vitro fertilization and pre implantation development of mammalian eggs when they are present in oocyte culture medium. To investigate the nature of the effects of somatic cells, the resistance of mouse oocytes against chymotrypsin treatment was examined after culture within various cell conditioned media. When mouse oocytes matured for 17-18 hr in the presence of cumulus cells were treated with 1 % chymotrypsin, half of them remained still alive even after 240 min $(t_{50}>240.0)$. In contrast half of mouse oocytes cultured without cumulus cells underwent degeneration within 65.0 min $(t_{50}=65.0{\pm}13.2min)$ of the same treatment. To see if the effects were duc to the secretory products of cumulus cells, mouse cumulus cells were cultured for 20 hr in medium containing 0.4% BSA and the supernatant of culture medium (conditioned medium) was taken. After maturation in the cumulus cell conditioned medium, mouse oocytes exhibited $t_{50}=190.0{\pm}10.8$ min upon chymotrypsin treatment whereas half of oocytes cultured without conditioned medium degenerated within 25.5 min. Human granulosa cell conditioned medium gave similar effects such that oocytes matured in conditioned medium exhibited $t_{50}=183.3{\pm}19.1$ min while $t_50$ of control group oocytes was $60.0{\pm}6.8$ min, Oocytes matured in vero cell conditioned medium exhibited $t_{50}=196.7{\pm}8.8$ min. On the other hand, amniotic cell conditioned medium resulted in the chymotrypsin resistance of $t_{50}=80.0{\pm}8.4$ min which was not statistically different from the control value of $t_{50}=48.0{\pm}13.2$ min. Based upon these results, it is suggested that certain somatic cell types including cumulus cells might change the biochemical properties of mouse oocyte membrane during meiotic maturation as revealed by the enhanced resistance against chymotrypsin treatment. Such effects of somatic cells appear to be mediated via the secretory products rather than direct communication between somatic cells and oocytes.

• Asian-Australasian Journal of Animal Sciences
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• v.27 no.5
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• pp.635-647
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• 2014

Unfertilized oocytes age inevitably after ovulation, which limits their fertilizable life span and embryonic development. Rapamycin affects mammalian target of rapamycin (mTOR) expression and cytoskeleton reorganization during oocyte meiotic maturation. The goal of this study was to examine the effects of rapamycin treatment on aged porcine oocytes and their in vitro development. Rapamycin treatment of aged oocytes for 24 h (68 h in vitro maturation [IVM]; $44h+10{\mu}M$ rapamycin/24 h, $47.52{\pm}5.68$) or control oocytes (44 h IVM; $42.14{\pm}4.40$) significantly increased the development rate and total cell number compared with untreated aged oocytes (68 h IVM, $22.04{\pm}5.68$) (p<0.05). Rapamycin treatment of aged IVM oocytes for 24 h also rescued aberrant spindle organization and chromosomal misalignment, blocked the decrease in the level of phosphorylated-p44/42 mitogen-activated protein kinase (MAPK), and increased the mRNA expression of cytoplasmic maturation factor genes (MOS, BMP15, GDF9, and CCNB1) compared with untreated, 24 h-aged IVM oocytes (p<0.05). Furthermore, rapamycin treatment of aged oocytes decreased reactive oxygen species (ROS) activity and DNA fragmentation (p<0.05), and downregulated the mRNA expression of mTOR compared with control or untreated aged oocytes. By contrast, rapamycin treatment of aged oocytes increased mitochondrial localization (p<0.05) and upregulated the mRNA expression of autophagy (BECN1, ATG7, MAP1LC3B, ATG12, GABARAP, and GABARAPL1), anti-apoptosis (BCL2L1 and BIRC5; p<0.05), and development (NANOG and SOX2; p<0.05) genes, but it did not affect the mRNA expression of pro-apoptosis genes (FAS and CASP3) compared with the control. This study demonstrates that rapamycin treatment can rescue the poor developmental capacity of aged porcine oocytes.

• Proceedings of the KSAR Conference
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• 2000.10a
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• pp.9-9
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• 2000

The mammalian ovary has a large number of primordial and preantral follicles, which are a potential source of oocytes for the in vitro mass production of embryos. Several in vitro culture systems have been developed to support the growth and development of oocytes from mouse preantral follicles. Under the appropriate condition, meiotically incompetent oocytes from preantral follicles can grow to final size and complete nuclear maturation in vitro. Furthermore, the successful production of live young from in vitro grown and matured oocytes demonstrates that oocytes from preantral follicles are able to acquire full developmental capacity in vitro. However, the efficiency of in vitro production of embryos from mouse preantral follicles is still low. In farm animals as well as human, the growth of oocyte from preantral follicle to the meiotic competence stage has yet to be demonstrate. Therefore, further studies to improve the culture condition or to develope new culture system should be needed in the future. In addition, the visible progress in the establishment of the in vitro culture system for preantral follicles of farm animals and human could help to enlarge the populations of valuable agricultural, phamaceutical product-producing, and endangered animals, and to rescue the oocytes of women about to undergo clinical procedures that jeopardize oocytes.

• Clinical and Experimental Reproductive Medicine
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• v.1 no.1
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• pp.49-54
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• 1974

In the present studies, effect of progesterone on the germinal vesicle break-down of the mouse oocytes cultured in the micro tube was investigated. The results obtained are as follows: As dose of progesterone in the medium rose, accordingly the break-down of the germinal vesicle was suppressed. It was found that $ED_{50}$ was 15.7 ${\mu}g$/ml, and $ED_{90}$ 60.7 ${\mu}g$/ml of progesterone. The dose suppressing the oocyte maturation was apparently higher than that on the rabbit or on the mouse embryonal development. The inhibiting effect of progesterone on the GVBD was reversible. The germinal vesicle of the oocytes were broken down immediately in the medium upon removal of the hormone. Progesterone stops meiosis at any stage upon administration, while dbe AMP or theophylline supresses only the break-down of the nuclear membrane. Recovering of the meiotic division of the oocytes once exposed to progesterone was delayed a little. The inhibiting action of progesterone was not altered by adding more pyruvate or in the presence of higher concentration of the mineral ions in the culture medium.

• Development and Reproduction
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• v.16 no.1
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• pp.59-67
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• 2012

Previously, we found that $Zap70$ (Zeta-chain-associated protein kinase) expressed in the mouse oocytes and played significant role in completion of meiosis specifically at MI-MII (metaphase I-II) transition. Microinjection of $Zap70$ dsRNA into the cytoplasm of germinal vesicle oocyte resulted in MI arrest, and exhibited abnormalities in their spindles and chromosome configurations. The purpose of this study was to determine the mechanisms of action of $Zap70$ in oocyte maturation by evaluating downstream signal networking after $Zap70$ RNAi (RNA interference). The probe hybridization and data analysis were used by Affymetrix Gene Chip Mouse Genome 430 2.0 array and GenPlex 3.0 (ISTECH, Korea) software, respectively. Total 1,152 genes were up (n=366) and down (n=786) regulated after $Zap70$ RNAi. Among those genes changed, we confirmed the expressional changes of the genes involved in the regulation of actin cytoskeleton and MAPK (mitogen-activated protein kinase) signaling pathway, since the phenotypes of $Zap70$ RNAi in oocytes were found in the changes in the chromosome separation and spindle structures. We confirmed the changes in gene expression in the actin skeletal system as well as in the MAPK signaling pathway, and concluded that these changes are main cause of the aberrant chromosome arrangement and abnormal spindles after $Zap70$ RNAi.

• Journal of Animal Reproduction and Biotechnology
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• v.35 no.4
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• pp.347-356
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• 2020

Gangliosides are glycolipids in which oligosaccharide is combined with sialic acids. Our previous studies have suggested an interplay between ganglioside GD1a/GT1b and meiotic maturation capacity in porcine oocyte maturation. Furthermore, ganglioside GD1a and GT1b are known for its antioxidant activity, but it is still unclear whether possible antioxidant role of GD1a and GT1b is involved in porcine embryos development competence during in vitro culture (IVC). Here, the effects of ganglioside GD1a and GT1b on the embryonic developmental competence during in vitro culture of porcine were investigated. The effects of ganglioside GD1a and GT1b on the expression of ST3GAL2 were confirmed during embryos development (2-cell, 4-cell, 8-cell and blastocyst) using immunofluorescent staining (IF). As a result, the fluorescent expression of ST3GAl2 was higher in embryos at 4-8 cells stage than blastocysts. Blastocyst development rate significantly increased in only 0.1 μM GD1a and GT1b treated groups compared with control group. To investigate the cellular apoptosis, we analyzed TUNEL assay. In case of only 0.1 μM GD1a and GT1b treated groups, the total number of cells in blastocyst compared with control group, but there was no significant difference in the rate of apoptotic cells. We identified the intracellular ROS levels using DCF-DA staining. According to the result, ROS production significantly decreased in blastocysts derived from the 0.1 μM GD1a and GT1b treated groups. These results suggest that ganglioside GD1a and GT1b improve the developmental competence of porcine embryos via reduction of intracellular ROS during preimplantation stage.

• Journal of Embryo Transfer
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• v.14 no.1
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• pp.17-22
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• 1999

To investigate the maturational and development competece of porcine oocytes of different diameter groups, oocytes were obtained by aspiration from slaughterdhouse ovaries. After washing three times in NCSU23 medium, each cumulus-oocyte complex was transferred into a $8{mu}ell$ drop of the maturation medium (one oocyte per drop) under paraffin oil. The diameter without zona pellucida of oocytes was measured with micor-calibrator (Mikrometer, E. Leitz) on a screen connected to a VCR on an inverted microscope $(200\times)$. After being measured, the oocytes were divided into 6 groups according to their diameter size : <105, 105 to < 110, 110 to < 115, 115 to < 120, 120 to < 125 and > $125{\mu}{\textrm}{m}$, and in vitro maturation (IVM), fertillzation (IVF) and production (IVP) of oocytes / embryo was performed. The rates of in vitro maturation on oocytes in the greater 105 ${\mu}{\textrm}{m}$ size groups(91.8~100%) were significantly (P<0.05) higher than in the < 105 ${\mu}{\textrm}{m}$ group(66.7%). The rates of sperm penetration were significantly (P<0.05) low in < $105{\mu}{\textrm}{m}$ group (50.0%) than others groups (81.6~85.5%). But the plyspermic fertilization rate was significantly (P<0.05) higher in < $110{\mu}{\textrm}{m}$ oocytes groups than in the $110\leq{\mu}{\textrm}{m}$ size groups. The rates of cleavage and development to blastocysts rose as oocytes diameter increased, however, while oocytes over $120{\mu}{\textrm}{m}$ in diameter failed to develop to blastocysts. There results suggest that porcine oocytes have acquired full meiotic competece at a diameter of $105{\mu}{\textrm}{m}$ but not yet attended full development competence to blastocyst and that oocytes have acquired full development competence at a diameter of $110{\mu}{\textrm}{m}$.

• Korean Journal of Animal Reproduction
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• v.25 no.2
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• pp.101-111
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• 2001

The present study was conducted to develop an in vitro culture system that would support bovine follicle growth from preantral to antral stage, oocyte maturation, fertilization, and embryonic development. Bovine preantral follicles (150$\pm$1.2 ${\mu}{\textrm}{m}$) surrounded by theca cell were isolated ezymatically and mechanically from ovarian cortical slides in Leibovitz L-l5 medium containing 1 mg/$m\ell$ collagenase and 0.2 mg/$m\ell$ DNase I and cultured for 25 days in the presence of different concentrations of bovine FSH and LH in $\alpha$MEM medium. The survival and growth rates of follicles cultured in the presence of FSH (10~150 ng/$m\ell$) were significantly higher than those of control group (P ＜ 0.001), but no significant differences were observed in survival and growth rates of follicles between the LH treatment groups (1~125 ng/$m\ell$) and the control. The survival (40%) and growth (244 $\pm$ 0.5 $\mu\textrm{g}$) of follicles cultured with FSH (90 ng/$m\ell$) and LH (25 ng/$m\ell$) were higher than those of control (25%, 160 $\pm$1.0 $\mu\textrm{g}$). Finally, 50% percent of healthy antral follicles were obtained, and almost 60% of them has complete meiotic division with 1st polar body (18.1%) and 10.0% have developed to the cleaved embryo and blastocyst stage. These results suggest that bovine preantral follicle with intact theca cell can grow to the antral stage using these culture conditions, and that oocytes from in vitro-matured bovine preantral follicle may acquire meiotic competence and can undergo fertilization and development.

• Journal of Embryo Transfer
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• v.19 no.2
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• pp.185-189
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• 2004

The purpose of this study was to determine the possibility of in vitro maturation of tiger oocytes. Immature oocytes were recovered from a pair of ovaries. A total of 78 oocytes was collected, of which forty three were classified as good oocytes with compact cumulus cells and uniform cytoplasm. Forty three COCs were in vitro matured at $39^{\circ}C$, 5% CO2 in air atmosphere for 48 h in a IVM medium (TCM-199 supplement with 10% FBS, 0.6 mM cysteine, 0.2 mM pyruvic acid and 10 IU/mL HMG). Experiment I: the morphologic evaluation was conducted by measuring the diameter of oocytes with or without ZP, the thickness of ZP and the diameter of cytoplasm by microeyepiece at the same magnification (${\times}$100). Experiment II: the evaluation of meiotic development was conducted of the nuclear development stage of tiger oocytes. The results were summarized as follows: 1. The diameter of tiger oocytes $(176.5\pm6.1{\mu}m)$ with ZP was significantly (p<0.05) bigger than that of bovine oocytes $(150.7\pm4.9{\mu}m).$ The ZP thickness of tiger oocytes $(20.4\pm2.9{\mu}m)$ was significantly (p<0.05) bigger than that of bovine oocytes $(12.0\pm2.6{\mu}m;$ p<0.05). However, there was no significant difference in the diameter of cytoplasm (without ZP) between tiger $(122.1\pm9.7{\mu}m)$ and bovine oocytes $(118.7\pm7.5{\mu}m).$ 2. The rates of meiotic development of tiger oocytes were achieved GV (12.5 %) and MII (50.0%), respectively. These results indicated that tiger oocytes could be developed to MII in in vitro culture system.

• Reproductive and Developmental Biology
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• v.35 no.1
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• pp.33-45
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• 2011

Heat shock protein 90 (Hsp90) is ATPase-directed molecular chaperon and affects survival of cancer cell. Inhibitory effect of Hsp90 by inducing cell cycle arrest and apoptosis in the cancer cell was reported. However, its role during oocyte maturation and early embryo development is very insufficient. In this study, we traced the effects of Hsp90 inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), on meiotic maturation and early embryonic development in pigs. We also investigated several indicators of developmental potential, including structural integrity, gene expression (Hsp90-, cell cycle-, and apoptosis-related genes), and apoptosis, which are affected by 17-AAG. Then, we examined the roles of Hsp90 inhibitor on viability of primary cells in pigs. Porcine oocytes were cultured in the NCSU-23 medium with or without 17-AAG for 44 h. The proportion of GV arrested oocytes was significantly different between the 17-AAG treated and untreated group (78.2 vs 34.8%, p<0.05). After completion of meiotic maturation, the proportion of MII oocytes was lower in the 17-AAG treated group than in the control group (27.9 vs 71.0%, p<0.05). After IVF, the percentage of penetrated oocytes was significantly lower in the 17-AAG treated group (25.2%), resulting in lower normal pronucleus formation (2PN of 14.6%). Therefore, the inhibition of meiotic progression by Hsp90 inhibitor played a critical role in fertilization status. Porcine embryo were cultured in the PZM-3 medium with or without 17-AAG for 6 days. In result, significant differences in developmental potential were detected between the embryos that were cultured with or without 17-AAG. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) showed that the number of containing fragmented DNA at the blastocyst stage increased in the 17-AAG treated group compared with control (7.5 vs 4.4, respectively). Blastocysts that developed in the 17-AAG treated group had low structural integrity and high apoptotic nuclei than those of the untreated control, resulting in decrease the embryonic qualities of preimplantation porcine blastocysts. The mRNA expressions of cell cycle-related genes were down-regulated in the 17-AAG treated group compared with control. Also, the expression of the pro-apoptotic gene Bax increased in 17-AAG treated group, whereas expression of the anti-apoptotic gene Bel-XL decreased. However, the expression of ER stress-related genes did not changed by 17-AAG. Cultured pESF cells were treated with or without 17-AAG and used for MIT assay. The results showed that viability of pESF cells were decreased by treatment of 17-AAG ($2{\mu}M$) for 24 hr. These results indicated that 17-AAG decreased cell proliferation and increased cell death. Expression patterns Hsp90 complex genes (Hsp70 and p23), cell cycle-related genes (cdc2 and cdc25c) and apoptosis-related genes (Bax and Bcl-XL) were significantly changed by using RT-PCR analysis. The spliced form of pXbp-1 product (pXbp-1s) was detected in the tunicamycin (TM) treated cells, but it is not detected in 17-AAG treated cells. In conclusion, Hsp90 appears to play a direct role in porcine early embryo developmental competence including structural integrity of blastocysts. Also, these results indicate that Hsp90 is closely associated with cell cycle- and apoptosis-related genes expression in developing porcine embryos.