• Korean Journal of Animal Reproduction
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• 제26권2호
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• pp.165-171
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• 2002

Follicular fluid (FF) contains an oocyte maturation inhibitor with unknown chemical properties. This study was carried out to chemically define the factor(s) inhibiting cumulus cell denudation (CD) and first polar body formation (PBF). Porcine FF (PFF) was extracted with methanol and the extract was serially separated using gel filtration on Superose 12 and Superdex columns. A Superdex fraction was derived with PITC and analyzed with an amino acid analysis column. The results obtained are as follows; PFF had an activity inhibiting both CD and PBF of porcine primary oocytes. Superdex fractions RV2.11 prepared from PFF exhibited an activity inhibiting CD and PBF. By amino acid analysis, the fraction RV2.11 appeared to be proline having the same activity inhibiting CD and PBF. In conclusion, PFF had oocyte maturation inhibitors, of which proline should inhibit CD and PBF.

• Korean Journal of Agricultural Science
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• 제34권1호
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• pp.1-11
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• 2007

This study is aimed at finding an optimum density for 6-Dimethylaminopurine (6-DMAP) and cycloheximide which have an effect on the revitalization of porcine oocytes. The results were summarized as follows: 1. When 6-DMAP was treated with 2 mM for 2 hours, It showed a significantly (P<0.05) different high result in activation rate, cleavage rate and blastocyst growth rate of 51.2%, 52.7% and 25.2% respectively. 2. When Cycloheximide was treated with 5 ug/ml for 6 hours, It showed a significantly (P<0.05) different high result in activation rate, cleavage rate and blastocyst growth rate of 47.7%, 46.8%, and 27.3% respectively. 3. When it was cultured in the culture medium, NCSU, for 7 days after inducing activation with 6-DMAP and cycloheximide, it showed no differences in the number of inner cell mass (ICM) and total cell of blastocysts. To conclude, it has been examined for porcine oocytes to be suitable when 6-DMAP was treated with 2 mM for 2 hours, Cycloheximide with 5 ug/ml for 6 hours.

• Journal of Embryo Transfer
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• 제11권1호
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• pp.71-80
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• 1996

The envelope of the rnannnalian oocyte plays crucial roles in sperm-oocyte interactions by providing sperm receptors, inducing acrosome reaction and preventing polyspermy. Understanding of properties of the zona pellucida (ZP) is essential for the artificial control of fertility in mammals. This study was carried out to produce and characterize monoclonal antibodies(MAbs) to porcine ZP proteins. Approximately 8,000 ZPs were obtained from follicular oocytes and dissolved in 40$\mu$l of double distilled water. Following immunization through foot-pad injections of Balb /c mice with a ZP solution, the popliteal lymph nodes were recovered at 2 weeks after the last injection. Hybridoma cell lines were established by fusing lymph node cells with P3X63 myeloma cells through selection using HAT medium and screening by immunofluorescence(IF) microscopy on the isolated ZP. Secreted MAbs were found to consist k chains and different heavy chains as evidenced by isotyping. Some of the MAbs demonstrated high specificity to the ZP in IF. The Mabs also showed positive cross reactivity with hamster and mouse eggs, while negative with bovine eggs. The results implicate that the MAbs can be used not only for identification of functional regions of the ZP, but also for elucidation of mechanisms involved in fertilization of mammals. The MAbs will provide basic information on biochemical anatomy of the ZP as well as can be candidates for the future contraceptive vaccines.

• Journal of Embryo Transfer
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• 제24권3호
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• pp.213-220
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• 2009

The objective of this study was to examine the effect of eCG and various concentrations (20, 40, and 80 ${\mu}g/ml$) of porcine FSH on nuclear maturation and intracellular glutathione (GSH) level of oocytes, and embryonic development after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) in pigs. Immature pig oocytes were matured in TCM-199 supplemented with porcine follicular fluid, cysteine, pyruvate, EGF, insulin, and hormones (10 IU/ml hCG and 10 IU/ml eCG or $20{\sim}80{\mu}g/ml$ FSH) for the first 22 h and then further cultured in hormone-tree medium for an additional 22 h. Nuclear maturation of oocytes ($85{\sim}89%$) was not influencem foreCG and various concentrations FSH. Embryonic development to the cleavage stage ($86{\sim}94%$) and mean number of cells in blastocyst ($33{\sim}37$ cells) after PA were not altered but blastocyst formation e-treignificaddlor(p<0.05) improvem forthe supplementation eith 80 ${\mu}g/ml$ FSHr(64%) compared to 47%, io8%, iand 47% in oocytes that were treated with eCG, 20,i and 40 ${\mu}g/ml$ FSH,i numectivelo. In SCNT, fusion ($78{\sim}83%$) of cell-cytoplast couplets and siosequent embryo cleavage ($82{\sim}88%$) were not influencem fordifferent gonadotropins but blastocyst formation tended to increase forthe supplementation eith 80 ${\mu}g/ml$ FSHr(25% vs. $11{\sim}18%$). Our nuults demonstrated that oocyte maturation and embryonic development after PA and SCNT e-frinfluencem fortype of gcem fortype of gits concentration. In this study, supplementation of maturation medium eith 80 ${\mu}g/ml$ FSHrimproved preimplantation development of PA and SCNT pig embryos, probably by increasing intracellular GSH concentration of matured oocytes.

• Reproductive and Developmental Biology
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• 제34권1호
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• pp.55-62
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• 2010

Matrix metalloproteinases (MMP) play important roles in extracellular matrix (ECM) remodeling during ovarian follicular development, oocytes development and ovulation. In an attempt to investigate the effect of MMP activation in development cumulus-oocytes complexes, we examined the localization and expression of MMP, and monitored MMP expression profile. Cumulus-oocytes complexes were collected and matured in vitro for 24 hr, 36 hr and 48 hr. A mRNA expression of MMP-2, MMP-9, TIMP-2 and TIMP-3 was detected in all culture medium regardless of CC, DC and CDCs. Activity of MMP-2 in the DC progressively was increased from 24 hr to 48 hr. But MMP-9 was not detected in all culture medium. The localization of MMP-2 was also measured by immunohistochemistry analysis. The MMP-2 and TIMP-2 was detected in cumulus cell and oocyte zone pellucida. Expression of MMP-2 protein in the COCs was progressively increased from 24 hr to 48 hr. However, MMP-9 protein was progressively decreased from 24 hr to 48 hr. And TIMP-2 protein was most highly expressed in the CDCs 36 hr. Expression of TIMP-3 protein in the CDCs was progressively increased from 24 hr to 48 hr. In conclusion, these results suggest that MMP-2 plays a role in maintaining normal maturation and development by controlling the ECM inhibitor concentration on cumulus cell and oocytes.

• Journal of Embryo Transfer
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• 제19권2호
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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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• 제21권2호
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• pp.129-135
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• 2006

This study was conducted to investigate the effect of in vitro maturation (IVM) duration of porcine follicular oocytes on maturation rate, polyspermic rate, and subsequent embryo development. The nuclear maturation rates of oocytes matured for 36, 38, 40, 42 and 44 hr were similar between 68.0, 78.0, 79.5, 73.8 and 81.8% respectively. There was no significant difference in the rates of polyspermy after in vitro feritilization (IVF). The cleavage rate in the group of 36 hr was significantly higher in than that of 40, 44 hr (p<0.05) but not to 38 and 42 hr. The development rate to blastocyst stage was significantly higher in the group of 38 hr (23.1%) than that in the group of 44 hr (15.6%) (p<0.05) but not to 36, 40 and 42 hr. These results suggest that the aged oocytes for 44 hr is not required for the production of bias to cysts derived from porcine IVF embryos.

• Asian-Australasian Journal of Animal Sciences
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• 제20권3호
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• pp.334-339
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• 2007

Reactive oxygen species (ROS) generate during electrical activation of oocytes which has detrimental effects on embryo survival when overwhelmed. The present study was designed to investigate the ability of L-ascorbic acid, a novel water soluble antioxidant, to reduce the ROS level in developing embryos and their subsequent effects on embryo development in vitro. The compact cumulus oocyte complexes (COCs) were cultured in tissue culture medium (TCM)-199 supplemented with 10 ng/ml epidermal growth factor, 4 IU/ml pregnant mare serum gonadotropin (PMSG), and human chorionic gonadotropin (hCG) and 10% (v/v) porcine follicular fluid (pFF) for 44 h. After maturation culture, the denuded oocytes were activated with a single DC pulse of 2.0 kV/cm in 0.3 M mannitol solution containing 0.5 mM of HEPES, 0.1 mM of $CaCl_2$ and 0.1 mM of $MgCl_2$ for $30{\mu}s$ using a BTX Electro-cell Manipulator. The activated oocytes were cultured in modified North Carolina State University-23 (mNSCU-23) medium for 168 h. The level of $H_2O_2$ in each embryo was measured by the dichlorohydrofluorescein diacetate (DCHFDA) method at 48 h after activation. The blastocyst formation rate was significantly higher when culture medium was supplemented with 50 and $100{\mu}M$ L-ascorbic acid (31.2 and 38.7%, respectively) compared to non-supplemented (16.1%) group. Accordingly, significantly more cells in blastocyst were found for 50 and $100{\mu}M$ L-ascorbic acid (50.0 and 56.4, respectively) compared to 0 and $200{\mu}M$ L-ascorbic acid (36.5 and 39.8, respectively). L-ascorbic acid reduces the $H_2O_2$ level in developing embryos in a dose-dependant manner. The $H_2O_2$ level (pixels/ embryos) was 191.5, 141.0, 124.0 and 163.3 for 0, 50, 100 and $200{\mu}M$ L-ascorbic acid, respectively. So, we recommend to supplement 50 or $100{\mu}M$ L-ascorbic acid in porcine in vitro culture medium.

• Korean Journal of Agricultural Science
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• 제43권1호
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• pp.52-60
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• 2016

In animal reproduction, the quality of oocytes and embryos has been evaluated by the expression of specific molecules. Follistatin (FST), which was isolated from follicular fluid, binds and bio-neutralizes the TGF-${\beta}$ superfamily members. Previous studies using the bovine model showed FST could be an important molecular determinant of embryo developmental competence. However, the effect of FST treatment on porcine embryo developmental competence has not been established. In this study, the effect of exogenous FST on porcine embryo developmental competence was investigated during in vitro culture. FST (10 ng/ml) treatment induced a significant decrease in the rate of cell arrest at the 4-cell stage. The expression levels of DNA-methyltransferase 1 (DNMT1), histone deacetylase 1 (HDAC1), and histone deacetylase 2 (HDAC2) were decreased in 4-cell stage embryos. FST treatment also resulted in significant improvements in developmental competence of embryos in terms of blastocyst formation rate and OCT-4 mRNA levels, the latter being related to pluripotency. In conclusion, during in vitro culture, FST treatment significantly ameliorated 4-cell block during embryonic development and improved embryo developmental competence. Therefore, FST treatment may potentially have a functional role in porcine embryogenesis that is broadly applicable to enhance in vitro embryo development.

• Asian-Australasian Journal of Animal Sciences
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• 제26권4호
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• pp.501-508
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• 2013

The effects of amino acids and dipeptides on in vitro production of porcine embryos and accumulation of ammonia in culture medium during developmental stages were examined in this study. The maturation, fertilization and development of embryonic cultures were performed in modified Tissue culture medium (mTCM)-199 supplemented with 10% (v/v) porcine follicular fluid, modified Tyrode's albumin lactate pyruvate (mTALP) medium, and modified North Carolina State University (mNCSU)-23 medium, respectively. In addition, amino acids and dipeptides of different concentrations and combinations were used to treat the embryos. The addition of L-alanyl-L-glutamine (AlnGln)+L-glycyl-L-glutamine (GlyGln) significantly (p<0.05) improved oocyte maturation, fertilization and the incorporation and oxidation of 14C(U)-glucose when compared to the control group and other treatment groups. Additionally, 2-4 cell, 8-16 cell, morula and blastocyst development increased significantly (p<0.05) following treatment with AlnGln+GlyGln when compared to the control group and other treatment groups, while this treatment reduced the accumulation of ammonia. Taken together, these findings suggest that treatment with AlnGln+GlyGln may play an important role in increasing the rate of porcine oocyte maturation, fertilization and embryonic development by reducing the level of accumulated ammonia measured in the culture media.