• Clinical and Experimental Reproductive Medicine
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• v.32 no.4
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• pp.331-336
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• 2005

Objective: To investigate the effects of female age on in vitro maturation and fertilization of immature oocytes from controlled ovarian hyperstimulation (COH) in human IVF-ET program. Method: A total of 96 immature oocytes (GV & metaphase I) obtained from 40 cycles of IVF-ET (29 patients). The mean age of female patients was $31.8{\pm}3.1years$. Ovulation was triggered by urinary or recombinant hCG. Immature oocytes were cultured with YS medium containing 30% of patients' human follicular fluids, LH (1 IU/mL), FSH (1 IU/mL) and EGF (10 ng/mL), and then matured oocytes were fertilized by ICSI. In vitro maturation and fertilization of immature oocytes were analyzed according to age of female (< 34 or ${\geq}34years$). Results: The maturation rate was similar between two groups (68% vs 64%). The fertilization rate of in?vitro-matured oocytes was higher in patients < 34 years old, but there was no statistical significance (64% vs 50%, p=0.347). The fertilization rate of in-vitro-matured oocytes was significantly lower compared with those of in-vivo-matured oocytes in both age groups (64% vs 79%, p=0.035, 50% vs 86%, p=0.007). Conclusion: In older female group, fertilization rate of in-vitro-matured oocytes seems to be decreased. Further investigations should be warranted to increase fertilization potential of in-vitro-matured oocytes.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.98-98
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• 2002

Recruitment of primordial follicles(PMF) is crucial for female fertility. however, factors and mechanisms that regulate this process is poorly understood. The present study was conducted to obtain an inclusive view of the gene expression and to identify novel factors and their pathways of regulating PMF arrest and/or growth initiation. Ovaries from one-day neonatal(consists of oocyte and PMF) and five-day old(consists of PMF and primary follicles, PRIF) mice were collected, either total RNA or mRNA was isolated, and suppression subtractive hybridization(SSH) was used to isolate and clone genes that differentially expressed in day 1 and day 5 ovaries. Confirmation that some of these genes are differentially expressed in PMF and/or in PRIF was accomplished by using laser captured microdissection(LCM), RT-PCR. in situ hybridization(ISH) and/or immunohistochemistry(IHC). In toto, 357 clones were sequenced and analyzed by BLAST and RIKEN program. Sequences of 330 clones significantly matched database entries while 27 clones were novel. Forty-two and 47 different genes were identified as differentially expressed in day 1 and day 5 ovaries, respectively, while 7 genes were expressed in both stages of ovaries. Day 5-subtracted library included several genes known as markers far growing follicles, such as ZP2, MATER, and fetuin. Among the genes with assigned functions, 23.8% was associated with cell cycle/apoptosis regulation, 7.1% with cellular structure, 11.9% with metabolism, 26.2% with signal transduction, and 31.0% with gene/protein expression in day 1; while 10.6%, 17.0%, 23.5%, 25.5%, and 23.4% in day 5, respectively. Genes such as GDF-8, Lats2, Septin2, and Weel were the highly expressed genes in PMF, while HSP84, Laminin2, MATER, MTi7, PTP, and Wrn were highly expressed genes in PRIF. We have successfully discovered list of genes expressed in day 1 and day 5 ovaries and confirmed that some of them are differentially expressed in PMF and/or PRIF. Gene expression profile from the present study would provide insight for the future study on the mechanism(s) involved in primordial-primary follicular transition. This work was Supported by Korean Health 21 RND Project, Ministry of Health and Welfare, Korea (01-PJ10-PG6-01GN13-0002).

• Journal of Embryo Transfer
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• v.19 no.3
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• pp.275-282
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• 2004

This study was carried out to examine the effects of development media and those change on in vitro development (IVD) of porcine oocytes matured and fertilized in vitro. Putative embryos, which were matured in vitro in modified NCSU-23 (mNCSU-23) supplemented with 10％ porcine follicular fluid (pFF) and were fertilized in mTBM, were developed in vitro as the experimental scheme. The results are as follows. When porcine putative embryos were cultured in vitro in NCSU-23 or CZB/Pig-MEM, the percentage of oocytes cleaved was not different between two systems, but the percentage of blastocysts in NCSU-23 was significantly higher than in CZB/Pig-MEM (P<0.05). And when porcine putative embryos were cultured in vitro in NCSU-23 during 7 days with or without changing media at day 5, which was supplement with or without 10％ fetal bovine serum(FBS), the percentage of oocytes cleaved, blastocysts at day 6, and the cell number of ICM, TE and total of blastocysts at day 7 were not different among three treatments. As a result of this study, it is supposed that NCSU-23 be more favorable, to develop porcine embryos derived from IVM/IVF, than CZB/Pig-MEM, but that demonstration on the effects of changing medium with fresh one stand in need of the more experiments.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.4
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• pp.487-495
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• 2007

The objectives of the present study were to initiate cloning of Korean native goat by somatic cell nuclear transfer (NT) and to examine whether unovulated (follicular) oocytes can support the same developmental ability of NT embryos as ovulated (oviductal) oocytes after hCG injection in stimulated cycles of the goat. The in vivo-matured and immature oocytes were collected from the oviducts and follicles of superovulated does, respectively, and the immature oocytes were maturated in vitro. Ear skin fibroblasts derived from a 3-yr-old female Korean native goat were used as the donors of nuclei or karyoplasts. Following fusion, activation and in vitro culture to a 2- to 4-cell stage, 49 in vitro-derived and 105 in vivo-derived embryos were transferred to 6 and 17 recipient does, respectively. One doe and three does of the respective groups were identified as pregnant by ultrasonography on day 30 after embryo transfer. However, only one doe, which had received in vivo-derived embryos, delivered a normal female kid of 1.9 kg on d 149. The cloned kid gained more weight than her age-matched females as much as 87% during the first 4 mo after birth (17.7 vs. $9.4{\pm}0.8$ kg) and reached puberty at 6-mo age a few months earlier than normal female does. The telomere length of the kid, which was similar to that of the donor fibroblast at 2-mo age, decreased 8% between 2- and 7-mo ages. Moreover, at 7-mo age, she had 21% shorter telomere than her age-matched goats. To our knowledge, this is the first case in which a cloned animal born with a normal weight exhibited accelerated growth and development. The unusually rapid growth and development of the cloned goat may have resulted from SCNT-associated epigenetic reprogramming involving telomere shortening.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.3
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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 Animal Reproduction
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• v.22 no.2
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• pp.187-194
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• 1998

In order to determine suitable conditions for rapid freezing of porcine embryos, the kind and concentration of cryoprotectants, sucrose concentrations, equilibration time and thawing temperature in freezing medium were examined in relation to the survival of frozen-thawed oocyte and embryos. The results obtained are as follows : 1. The suitable concentrations of cryoprotoctant in the freezing medium which consisted of TCM-199+20% FCS were 1.5M for glycerol, 2.0M for DMSO, 2.5M for ethylene glycol, and 2.0M for propanediol. The sucrose concentration of 0.25M in the medium was found to optimal because the survival rate was markedly higher at this concentration when compared to the others. The survival rate was relatively high when the frozen embryos were thawed at 30$^{\circ}C$ in the freezing medium containing 2.5M cryoprotectants. The equilibration periods of 2.0 and 5.0 minutes revealed the higher survival in the media containing 1.5 or 2.1M glycerol when compared to 10 and 15 minutes. 2. The fertilization rates of frozen-thawed follicular oocytes which matured in vitro for 1, 12, 24 and 48 hours were 6.7~26.7% depending on the maturation time, and the rates were relatively high for those matured for a short period of time. The survival rates of frozen-thawed oocytes which matured in vitro for certain periods and fertilized were 10.0~30.0% depending on the maturation time.

• Korean Journal of Animal Reproduction
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• v.24 no.1
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• pp.83-88
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• 2000

This study was conducted to determine the effect of repeated ultrasound-guided transvaginal retrieval of oocytes recovery, estrous cycle and ovarian adhesion in Korean native, Hanwoo heifers. Heifers were at unknown stages of the estrous cycle at the start of experiments in which all follicles $\geq$6mm in diameter were ablated. The results obtained in this study were as follows; Follicle developing number and oocytes collected number were no effected to repeated OPU to nine session, 4 e.a range oocytes collected to repeated OPU session. Oocytes were observated follicles were 8.7$\pm$4.2 e.a, collected oocytes were 4.1$\pm$3.4 e.a to two times collected per week and observated follicles was 10.2$\pm$6.1 e.a, collected oocytes were 4.3$\pm$2.9 e.a to one times collected per week, but no difference significantly(P＜0.05). Ovaries adhesive percentage to repeated OPU was eight ovaries adhisived(20%) of forty ovaries, three ovaries(7.5%) to 1~3 times oocytes collected, four ovaries(l0%) to 4~6 times, one ovaries(2.5%) to 7~9 times oocytes collected session. To repeated OPU effection, ovaries adhisive heifers were long estrous cycle(＞25 day) to 7 heads(87.5%) of 8 head, non-adhesive ovaries heifers were 5 heads(41.7%) were long estrous cycle to repeated OPU 12 heads. Although, now unknown about the dynamics of follicles wave and about functional changes to repeated OPU ovaries, more question about ovaries adhesive cause remain.

• Reproductive and Developmental Biology
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• v.31 no.3
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• pp.181-186
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• 2007

Inhibitor of DNA binding protein or inhibitor of differentiation(Id) is largely considered as positive and/or negative regulators of proliferation, differentiation, angiogeneisis, and apoptosis. The four Id genes(Id1, Id2, Id3, and Id4) were known in mammals. However, little is known about the expression and function of these genes in reproductive physiology. Among them, this study was conducted to analyze the expression pattern of Id3 mRNA on folliculogenesis in rat ovary. After PMSG administration, the ovaries were obtained at 3, 6, 12, 24, 36, and 48hrs, fixed, dehydrated, and paraffin embedded. For in situ hybridization, anti-sense and sense Id3 cRNA probes were prepared and applied to the ovarian section. The ovarian sections were coated with NTB-2 emulsion. After that, the slides were developed and counterstained with hematoxylin and eosin staining. The hybridization signal was estimated on a scale of 1+ to 4+. In oocyte, the intensity of Id3 mRNA in primordial and primary follicles was scored at ${\geq}2+$, but the intensity was less than 1+ in secondary, dominant, and preovulatory follicles. In granulosa cells, the Id3 mRNA was strongly expressed(3+ or 4+) in dominant and preovulatory follicles. Taken together, Id3 mRNA was expressed specifically at follicle stages and follicular tissue and might be closely related with follicle development.

• Clinical and Experimental Reproductive Medicine
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• v.25 no.1
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• pp.71-76
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• 1998

This study was carried out to examine whether the developmental capacity of bovine immature oocytes frozen ultra-rapidly using electron microscope (EM) grids and EFS30 can be obtained. As freezing solution, we used EFS30 which consisted of 30% ethylene glycol, 0.5 M sucrose, 18% ficoll and 10% FBS added in D-PBS. As criterior of oocyte viability, the rates of maturation, fertilization and embryonic development were determined. The results obtained in this experiment were summarized as follows: When ultra-rapidly frozen immature oocytes were thawed, 43.2% of them were survived. The rates of maturation (84.1%) and normal 2 pronuclei formation (57.5%) of frozen immature oocytes were not significantly different when compared to those of control (92.5, 65.0%). In addition, the rates of $\geq2$-cell (65.0%) and blastocyst formation (30.8%) of freezing group were not significantly different when compared to those of control (73.7, 35.7%). These results demonstrate that developmental capacity of frozen-thawed bovine immature oocytes can be successfully obtained when survived from the ultra-rapid freezing method using EM grid and EFS30.

• The Korean Journal of Zoology
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• v.38 no.4
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• pp.523-530
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• 1995

Specific affinity binding sites for atrial natriuretic peptide (ANP) were Investigated in the pig ovarian tissues by in vitro autoradiographic techniques. In the pig ovary, the highest binding sites for 12514abeiled rANP(l~28) were localized in the granulosa cell layer of the forncles. The binding sies on theca layer of the ovarian follicles were mainly localized in the external layer, but none was observed In the Internal layer. In the corpus luteum, the binding site was not observed. The specific bindings of 200 pM of l2Sl4abelled rANP(l~28) to granulosa and theca externa layers were reversed completely by excess concentration (1 ~4) of unlabelled rANP(l~28) but not by 10 ~ of unrelated peptides, human angiotensin II and arginine vasopressin. The binding was also displaced by 1 ~ of desiGIn18, Ser19, Gly2O, leu21, Gly22I ANP(4~2g) (C- ANF) as a spedfic ligand of the ANP clearance receptor. Therefore these results indicate ~hat the biological and the clearance ANP receptors exist in the theca externa and granulosa layer of the pig ovary, and suggest that the ANP receptors may be related with the regulatory lundion of the ovarian follicular development including oocyte maturation.