• Asian-Australasian Journal of Animal Sciences
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• v.17 no.2
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• pp.164-167
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• 2004

This study was carried out to investigate in vitro fertilization and development of in vitro matured pig oocytes inseminated with the Duroc boar sperm by different sperm washing media after thawing of the 5 ml frozen straws. Immature follicular oocytes (30-40) were transferred into each well of a Nunc 4-well multidish containing $500{\mu}l$ mTCM199 maturation medium. The sperm rich portion of ejaculates was collected into a 250 ml insulated vacuum bottle and gradually cooled 22 to $24^{\circ}C$ over a 2 h period. Semen was centrifuged at 800 g for 10 min and the seminal plasma discarded. Sperm were esuspended in a lactose-egg yolk and N-acetyl-Dglucosamine (LEN) diluent to contain $1{\times}10^{9}$ sperm/ml and cooled to $5^{\circ}C$ over a 2 h period. Immediately before freezing, semen was rediluted with an equal volume of LEN+4% glycerol and packed into 5 ml straws. After thawing of the 5 ml straw, the 5 ml semen was diluted with 20 ml Beltsville thawing solution (BTS) at room temperature. Oocytes were inseminated with untreated (unwashed and nonpreincubated) or treated sperm (washed two times in BTS, mTLP-PVA and mTBM media, respectively and nonpreincubated) with $2{\times}10^{7}$ sperm concentration. Oocytes were coincubated for 6 h in $500{\mu}l$ mTBM fertilization. At 6 h after IVF, oocytes were transferred into $500{\mu}l$ NCSU-23 culture medium for further culture of 6 h. Sperm penetration, polyspermy and male pronuclear formation of oocytes at 12 h after IVF and developmental ability of oocytes at 48 h after IVF were evaluated. Sperm penetration rate, male pronuclear formation and rate of cleaved embryos were higher in the BTS, mTLP-PVA and mTBM treatments than the unwashed treatment (p<0.05). The rate of blastocysts from the cleaved oocytes (2-4 cell stage) were higher in the mTLP-PVA treatment than in the unwashed, BTS and mTBM treatments. In conclusion, we recommend the washing of frozen-thawed sperm with mTLP-PVA medium before in vitro fertilization of oocytes in mTBM medium.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.117-117
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• 2002

The oocytes from most of animal species accumulate genetic information and other necessary materials during oogenesis for the later use in the early development. Over the years oocyte maturation has been studied extensively both in vitro and in vivo. Particularly, maturation of follicular oocyte in vitro becomes one of the important tools for the studies of basic cell biology, the in vitro technology of animal production, and in particular, the somatic cell cloning by nuclear transfer. We examined meiotic maturation and cumulus expansion in the presence of translation or transcription inhibitors for varying periods of in viかo maturation (IVM) of pig oocyte. In Experiment 1, the results revealed that translation and transcription inhibitors inhibited cumulus expansion and meiotic maturation during 35h of IVM. However, 50 to 60% of the oocytes underwent nuclear maturation without cumulus expansion during 75h of IVM. The rest of the oocytes were arrested at metaphase I (40-50%) in the presence of the inhibitors. In Experiment II, the OCCs were exposed to the drugs only for 15h to examine translation and transcription inhibitors on cumulus expansion and meiotic maturation. Transcription inhibitors for 15h did not arrest meiotic maturation when the oocytes were cultured for subsequent, necessary period of IVM, whereas cumulus expansion was completely inhibited, suggesting that initial 15h is critical transcription activity far cumulus expansion. Translation inhibitors for 15h exposure did not alter cumulus expansion and meiotic maturation during subsequent culture in the absence of the drugs. In Experiment III, the OCCs were exposed to the drugs only for later 30h to examine the influence of transcription and translation inhibitors on oocyte maturation. Interestingly, all meiotic maturation underwent normally with full expansion of cumulus. Similar results were obtained from Experiment IV where 5h of exposure from 15 to 20h of IVM culture to the drugs was performed and subsequently cultured for same period in fresh medium. Taken there results together, both transcription and translation are necessary for nuclear maturation and cumulus expansion, and first 15h IVM for cumulus expansion is critical. The arrested oocytes by the drugs were still capable of undergoing nuclear maturation, although cumulus expansion was affected.

• Korean Journal of Animal Reproduction
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• v.22 no.1
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• pp.73-80
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• 1998

This study was undertaken to evaluate the interaction between cumulus cells and TGF $\beta$1 on in vitro maturation in porcine oocytes. No differ ences were found in maturation rates when follicular oocytes were cultured in medium with various concentrations of TGF $\beta$. At 24 h after maturation, the oocytes matured to metaphase-II were found in medium with TGF $\beta$ regardless of cumulus cells. On the other hand, the maturation rates were significantly(P < 0.01 higher cumulus-enclosed(70 and 52%) than cumulus-denuded oocytes(35 and 26%) in medium with or without TGF $\beta$ at 48 h after culture. In a another experiment, the same maturation rates (54-71%) were observed when cumulus-enclosed oocytes were cultured with various addition time of TGF $\beta$. However, the maturation rates in cumulus-denuded oocytes were significantly (P < 0.05) higher in medium added at 0~24 h (59%) or 24-48 h(57%) after culture than in medium with(27%) and without(38%) TGF $\beta$ for 48 h. These results indicated that cumulus cells is essential for in vitro maturation in porcine oocytes but TGF $\beta$ can promote oocytes maturation in cumulus-free oocytes.

• The Korean Journal of Zoology
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• v.33 no.2
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• pp.175-182
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• 1990

Progesterone production and oocyte maturation in ovarian follicles of Rana nigromaculata and Rana rugosa were investigated. Addition of frog pituitary homogenate (FPH) to the in utiro cultured follicles of R. nigromaculata stimulated a marked increase in the accumulation and secretion of progesterone (P$_4$) by the follicles and induced their oocyte maturation (germinal vesicle breakdown, GVBD) in a dose dependent manner. The FPH (0.1 pituitary equivalent/2 ml)-inducted P4 peak appeared in 3-6 hours and followed by the oocyte GVBD in 9-12 hours after the hormone stimulation. lncreae of intrafollicular cAMP levels with forskolin (an adenylatecyclase stimulator) and 3-isobutyl-1-methylxanthine (IBMX, a phosphodiesterase inhibitor) mimic the FPH action in the stimulation of P$_4$ production but not in the induction of oocyte maturation. The in uitro cultured follicies of R. rugosa behaved very differently from other amphibian follicles. Addition of FPH-(0. 1 pit. equivl2 ml) to the culture medium neither stimulated P$_4$ production by the follicles nor induced the oocyte GVBD. However, treatment of the follicles with forskolin and IBMX drastically stimulated both the intrafollicular accumulation (800 pg/follicle) and secretion (1700 pg/follicle) of P$_4$ by the follicles during culture period. Thus, the data suggest that the follicles are ready to respond to cAMP increase but not to the FPH stimulation in terms of P$_4$ production.

• Clinical and Experimental Reproductive Medicine
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• v.32 no.3
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• pp.207-216
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• 2005

Objective: To understand the crucial requirement for the normal early folliculogenesis, we evaluated molecular as well as physiological differences during in vitro ovarian culture. Among the important regulators for follicle development, anti-Müllerian hormone (AMH) and FSH Receptor (FSHR) have been known to be expressed in the cuboidal granulosa cells. Meanwhile, it is known that c-kit is germ cell-specific and GDF-9 is also oocyte-specific regulator. To evaluate the functional requirement for the competence of normal follicular development, we investigated the differential mRNA expression of several factors secreted from granulosa cells and oocytes between in vivo and in vitro developed ovaries. Materials and Methods: Ovaries from ICR neonates (the day of birth) were cultured for 4 days (for primordial to primary transition) or 8 days (for secondary follicle formation) in ${\alpha}$-MEM glutamax supplemented with 3 mg/ml BSA without serum or growth factors. The mRNA levels of the several factors were investigated by quantitative real-time PCR analysis. Freshly isolated 0-, 4-, and 8-day-old ovaries were used as control. Results: The mRNA of AMH and FSHR as granulosa cell factors was highly increased according to the ovarian development in both of 4- and 8-day-old control. However, the mRNA expression was not induced in both of 4- and 8-day in vitro cultured ovaries. The mRNA expression of GDF-9 known to regulate follicle growth as an oocyte factor was different between in vivo and in vitro developed ovaries. In addition, the transcript of GDF-9 was expressed in the primordial follicles of mouse ovaries. The mRNA expression of c-kit was not significantly different during the early folliculogenesis in vitro. Conclusion: This is the first report regarding endogenous AMH and FSHR expression during the early folliculogenesis in vitro. In conclusion, it will be very valuable to evaluate cuboidal granulosa cell factors as functional marker(s) for normal early folliculogenesis in vitro.

• Reproductive and Developmental Biology
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• v.29 no.3
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• pp.201-205
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• 2005

The objective of this study was to investigative the effects of amino acids supplementation on maturation, fertilization and embryo development of pig oocytes. Essential amino acids (EA), non-essential amino acids (NA) or both amino acids (EA + NA) were supple-mented to North Carolina State University (NCSU) 23 medium containing porcine follicular fluid (pFF). When the amino acids were supplemented to the maturation medium, the maturation rates were higher (p<0.05) in the NA group than control ($83.3{\pm}0.04\%\;versus\;70.0{\pm}0.05\%$, but the subsequent cleavage rates and development to morula and blstocyst stage between aminoacid supplement groups and control were not different. The developmental rates to morula and blastocysts stage were not significantly different regardless of amino acid supplementation to culture medium. In addition, supplementation of amino acids did not significantly affect the rate of fertilization and polyspermy. When the amino acids were supplement to culture medium, the number of trophectodermal (TE) cells was significantly (p<0.05) higher in amino acid supplement group than that of control ($18.6{\pm}0.5\;versus\;16.1{\pm}0.6$), whereas the numbers of inner cell mass (ICM) cells were not different among the treaonent groups and control ($29.0{\pm}0.9\~31.5{\pm}1.2$). Total cell number was also significantly (p<0.05) higher in EANA group ($50.0{\pm}1.0$) than that of control group ($44.2{\pm}1.1$). These results indicate that the amino acid supplementation to maturation and culture medium may not significantly stimulate early embryo development, but may improve the TE cell number of blastocyst stage in the pig.

• Journal of Aquaculture
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• v.8 no.3
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• pp.171-181
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• 1995

Gonadosomatic index (GSI) of the crucian carp (Carassius auratus) was investigated to clarify annual reproductive cycle from February in 1992 to October in 1994. The values of GSI were high with individual variation from April to July which period was coincided with the breeding season of fish. The GSI was very low in August and September, when follicular atresia developed in the ovaries. GSI value began to increase in October and reached a peak around the following March, which indicated that ovarian follicles may grow during this period. Human chorionic gonadotropin (HCG 10 IU), $17\alpha$, 20\beta-dihydroxyprogesterone\;(1-100{\mu}g/ml)$ and phorbol 12-myristate-13-acetate (TPA, protein kinase C activator, 0.1-10${\mu}M$) induced germinal vesicle breakdown (GVBD), but $4\alpha-phorbol$ 12, 13- didicanoate ($4\alpha-PDD,\;phorbol\; ester\;analogue,\;25{\mu}M$) did not induce germinal vesicle breakdown in the follicular oocytes. Prostaglandin $F_{2\alpha}$ $(0.1-10 {\mu}g/ml)$ and TPA $(0.1-10 {\mu}M$ induced ovulation of the oocytes, but $4\alpha-PDD$ $(25{\mu}M)$ did not induce ovulation of the follicles. $17\alpha-hydroxyprogesterone$ production was examined from the isolated follicles to investigate the steroid production ability in the crucian carp ovaries. HCG (1 lU, 10 lU) and forskolin (adenylate cyclase activator, 0.1-10 ${\mu}M$) stimulated $17\alpha-hydroxyprogesterone$ production. The time course of HCG (10 lU) and forskolin $(10\;{\mu}M)$ stimulated $17\alpha-hydroxyprogesterone$ production within 3 hours, the elevated levels were maintained during the rest of the culture period. The data indicates that cyclic AMP and protein kinase C may play important roles in the oocyte maturation and ovulation in crucian carp.

• Korean Journal of Animal Reproduction
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• v.21 no.3
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• pp.293-302
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• 1997

Follicular oocytes of Grade I and II were collected from 2~6 mm ovarian follicles and matured in vitro (IVM) for 24 hrs in TCM-199 su, pp.emented with 35$\mu\textrm{g}$/ml FSH, 10$\mu\textrm{g}$/ml LH, and 1$\mu\textrm{g}$/ml estradiol-17$\beta$ at 39$^{\circ}C$ under 5% CO2 in air. They were fretilized in vitro (IVF) by epididymal spermatozoa capacitated with heparin for 12 hrs. The zygotes were then co-cultured in vitro with bovine oviducted epithelial cells (BOEC) for 7 to 9 days. The optimal time for IVM, the successful enucleation of IVM oocytes by micromanipulation at different oocyte ages after IVM, and the ideal culture system for IVM for effective IVF and in vitro development of IVM-IVF embryos was examined for in vitro production of nuclear recipient oocytes and nuclear donor embryos. To improve the efficiency of nuclear transplantation (NT) of IVF embryo into IVM follicular oocytes, this study evaluated the optimal electric condition and oocytes age for activation of IVM oocytes and in vitro development of NT embryos. In vitro development of NT embryos with preactivation or non-preactivation in enucleation oocytes, cell number of IVN-IVF embryos, and NT embryos wre also examined. The results obtained were as follows; 1. The most suitable enucleation time was at 24 hpm (83.3%) rather than that of 28 hpm(69.6%) and 32 hpm(50.0%). 2. There was no difference among the fusion rates of NT embryos at the voltages of 0.75, 1.0 and 1.5 kV/cm, but the in vitro development rates to morule and blastocyst were significantly (P<0.05) higher at the voltage of 0.75(12.5%) and 1.0kV/cm (12.6%) compared to 1.5kV/cm(0%). 3. No significant difference in activation rates were seen in NT embryos stimulated for 30, 60 and 120 $\mu$sec (71.7, 85.2 and 71.9%, respectively), but the in vitro development rates to morulae and blastocyst were significantly (P<0.05) higher in the oocytes stimulated for 30 $\mu$sec (11.6%) and 60 $\mu$sec(10.7%) than 120 $\mu$sec(0.0%). 4. The fusion rates (71.0 and 87.3%) and the in vitro development rates (9.1 and 12.7%) to morula and blastocyst were seen in the NT embryos stimulated at 28 and 32 hpm under the condition of 1.0 kV/ml, 60 $\mu$sec. However, at 24 hpm the fusion rates were 64.8% and the in vitro development to morula and blastocyst were not seen. 5. The fusion rates between the 8~12, 13~17 and 18~22-cell stage of IVM-IVF embryos were not significantly different. The in vitro development rates of the fused embryos to morula and blastocyst which were received from a blastomere of 8~12, 13~17 and 18~22-cell stages of IVM-IVF embryos were 14.9, 8.3 and 6.5%, respectively. 6. The in vitro development rate of the enucleated recipient oocytes with preactivation (24.2%) to morula and blastocyst was significantly (P<0.05) higher than that of non-preactivation (12.8%). 7. The cell numbers of NT blastocyst and IVM-IVF blastocyst cultured during 7~9 days were 63$\pm$11 and 119$\pm$23, and then their the mean cell cycle number were 5.98 and 6.89, respectively.

• Journal of Embryo Transfer
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• v.20 no.2
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• pp.105-112
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• 2005

The present study was conducted to examine some factors affecting in vitro development of oocytes from somatic cell nuclear transfer (SCNT) in Korean native goats. Recipient oocytes were surgically collected after superovulation by using CIDR and FSH, PMSG, hCG and estrous synchronization in Korean Native goats. For nuclear transfer, the fibroblasts from caprine ear cells and fetal fibroblasts were surgically harvested and were cultured in vitro until cell confluency in serum-starvation condition (TCM-199 + $0.5\%$ FBS) for 3 to 5 days. The zona pellucidae of matured oocytes were partially drilled by laser irradiation. A single somatic cell was individually transferred into each enucleated oocyte. The reconstructed oocytes were then electrically fused and activated. Activated NT embryos were cultured in mSOF medium supplemented with $0.8\%\;BSA\;6\~7\;day\;at\;39^{\circ}C,\;5\%\;CO_2,\;5\%\;O_2,\;90\%\;N_2$ in air. There were no significant difference in the number of embryos cleaved and 4-cell development between the fibroblast nuclei from mature ear cells and fetal cells, but the rate of 8-cell development was higher (P<0.05) in ear cells $(40.5\%)$ than in fetal cells $(55.5\%)$. However, the embryo development to morula or blastocyst was not significantly different between both the groups$(6.7\%\;vs\;16.0\%)$, respectively. The number of embryo cleaved $(79.0\%)$ were higher (P<0.05) in the oocytes activated with ionomycin+6-DMAP than in the oocytes activated electrically $(9.5\%)$. The development of fused embryos to morula or blastocyst was found $15.6\%$ in ionomycin+6-DMAP, but no morula or blastocysts were developed in electrical stimulation. The development rate of SCNT embryos to morula or blastocyst was love. (P<0.05) in SCNT embryos $(19.0\%\;vs\;0.0\%)$ than that in parthenotes $(66.1\%\;vs\;59.1\%)$. In the parthenotes, the cleavage rate and development to morula or blastocyst were significantly higher (P<0.05) as $86.8\%\;and\;50.0\%$ in ovulated oocytes than in follicular oocytes $(69.0\%\;vs\;23.6\%)$, respectively. These results suggest that some factors Including superovulation treatment, oocyte source, maturation of follicular oocytes, activation method and culture condition may affect in vitro developmental capability of embryos produced by somatic cell nuclear transfer in Korean Native goats, and the fusion rate be greatly low compared with other species.

• Korean Journal of Agricultural Science
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• v.26 no.2
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• pp.19-24
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• 1999

This study was conducted to investigate the influence of media and hormones on in vitro maturation and development of porcine follicular oocytes. Basic media were used to TCM-199, Waymouth MB751/1 and BMOC-II, and hormones were used to hCG and FSH in each medium. The results obtained were summarized as follows : 1. The maturation rates of oocytes cultured in TCM-199 medium containing hCG, FSH and hCG+FSH were 78.05, 72.50 and 67.50%, respectively. The maturation rates of oocytes with hormones were significantly (P<0.05) higher than those of oocytes cultured without hormone. However, the cleavage rate(hCG 46.88%, FSH 31.04%. hCG+FSH 37.04%) of embryo cultured in TCM-199 containing hormone was significantly(P<0.05) lower than that(89.47%) of oocytes cultured without hormone. 2. The maturation rates of oocytes cultured in Waymouth MB751/1 medium containing hCG. FSH and hCG+FSH were 69.77, 71.43 and 80.00%, respectively. The maturation rates of oocytes with hormones were significantly(P<0.05) higher than those of oocytes cultured without hormone. However. the cleavage rate(hCG 46.67%. FSH 36.00%, hCG+FSH 35.71%) of embryo cultured in Waymouth MB751/1 containing hormone was significantly(P<0.05) lower than that(60.00%) of oocytes cultured without hormone. 3. The maturation rates of oocytes cultured in BMOC-II medium containing hormone were 66.67(control). 66.67(hCG). 91.89(FSH) and 81.82(hCG+FSH)%. respectively. showing the highest rate in FSH treatment. And, the cleavage rates of oocytes cultured in BMOC-II medium containing hormone were 81.82 (control, 79.17(hCG), 50.00(FSH) and 66.67(hCG+FSH)%, respectively.