• Journal of Radiation Protection and Research
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• v.24 no.2
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• pp.87-92
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• 1999

The present study was carried out to evaluate the morphological changes in the degenerating primordial follicles induced by $\gamma$-radiation. The prepubertal female mice of three weeks old ICR strain were whole-body irradiated with a dose of $LD_{80(30)}$ (8.3 Gy). The ovaries were collected at 0 h, 3 h, 6 h, and 12 h post-irradiation. The largest cross sections were prepared with histological semi-thin sections and then observed microscopically. The ratio of normal to atretic follicles was reduced significantly after 6th post-irradiation. At 6 h post-irradiation, the number of degenerated primordial follicles increased. Germinal vesicles disappeared, and lipid droplets increased. No more ooplasmic membranes were seen. Granulosa cells became round in shape, and apoptotic cells started to appear. The ratio of normal to atretic follicles in the control group was 62.50%. The ratio decreased with time after irradiation. The ratio decreased down to 51.61 %, 48.97 %, 11.11 %, and 7.14 % at 0 h, 3 h, 6 h, and 12 h, respectively. Taken together, ionizing radiation acutely induced the degeneration of primordial follicles. The patterns of degeneration are 1) apoptosis of one or more granulosa cells with relatively intact oocyte, 2) apoptosis of oocyte with intact follicle cells, or 3) apoptotic degenerations of both cells. The Present study can provide morphological clues for the identification of degenerating primordial follicles.

• Journal of Embryo Transfer
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• v.29 no.1
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• pp.1-5
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• 2014

Recent 2 decades, including in vitro maturation (IVM), assisted reproductive technologies (ARTs) achieved noteworthy development. However the efficiency of ARTs with in vitro matured oocytes is still lower than that with in vivo oocytes. To overcome those limitations, many researchers attempted to adapt co-culture system during IVM and consequently maturation efficiency has been increased. The beneficial effects of applying co-culture system is contemplated base on communication and interaction between various somatic cells and oocytes, achievement of paracrine factors, and spatial effects of extracellular matrix (ECM) from somatic cell surface. The understanding of co-culture system can provide some information to narrow the gap between in vitro and in vivo. Here we will review current studies about issues for understanding cu-culture system with various somatic cells to improve in vitro maturation microenvironment and provide bird view and strategies for further studies.

• Journal of Embryo Transfer
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• v.19 no.1
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• pp.27-34
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• 2004

This study evaluated the efficiency and compared with different materials of loading vessels for vitrification-plastic/glass, copper grid and nylon. The loading method, vitrification, cryop-reservation and warming method of the oocytes were examined. The loading samples prepared in manual or company-made and sterilized, loaded the COCs selected on each samples and cultured for maturation during 40 hours, and then exposed sequentially to ethylene glycol solution. Thawing method was reversely treated and exposed for warmed oocytes. After oocytes were thawed, fertilized and cultured in vitro for 3-4 hours, rates of development and morphological appearance were examined. The results were as summarized: ㆍOPS from company-made or hand-made of the hematocrit micropipettes, NLS from fishing line and EMG from company-made for EM were used for loading oocytes, respectively. ㆍThe efficiency of freezing method and loading convenience were orderly higher in OPS, NLS and EMG. The optimal capacity per vessel was orderly lowered in NLS, EMG and OPS, respectively. ㆍAfter oocytes were warmed, the recovery rate, morphology and rate of development were orderly higher in OPS, NLS and EMG, respectively. ㆍIn conclusion, OPS has the advantages of achieving a little more survival and preserving results than other two loading methods.

• Korean Journal of Animal Reproduction
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• v.20 no.1
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• pp.63-69
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• 1996

Bovine immature oocytes cultured for various times in TC-199 medium were inseminated with frozne-thawed spermatozoa in TC-199 medium supplemented with caffeine(5mM) and heparin(10$\mu\textrm{g}$/ml). Sperm penetraton was possible in oocytes at any stage of maturation, but penetration rates were lower in oocytes inseminated 0~16h (60~76%) than 20h (98%) after culture. Formation of male and female pronuclei were first observed in oocytes inseminated 8h after cultrue. Formation of male and female pronuclei were first observed in oocytes inseminated 8h after culture. The proportions of polyspermy were high(50~76%) in oocytes inseminated at any stage of maturation. Sperm penetration into oocytes at the GV stage started at 8h after insemination and the penetration rates gradually increased as time after insemination proceeds. The proportion(35%) of oocytes matured beyond metaphase-II 20h after sperm-oocytes incubation was low. When oocytes were incubated without spermatozoa in TC-199 medium, maturation rates were significantly higher (P<0.001) in those without(45 and 84% for 16 and 20 h) than with (0 and 36% for 16 and 20 h) caffeine and heparin. These results indicate that TC-199 medium with caffeine and heparin is not suitable for maturation and fertilization of immature oocytes and may inhibit male pronuclear formation in the cytoplasm.

• Journal of Embryo Transfer
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• v.19 no.2
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• pp.113-119
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• 2004

The purpose of the present study was to examine whether collection time affects results of oocyte recovery from superovulated goats. Fiftyty-one mature Korean native goats, maintained in a pen under natural day length and fed hay ad libitum, were pretreated with progestagen impregnated CIDR for 10 days and then the goats were divided into two groups. One group of the goats received a single intramuscular injection of 1,000 IU PMSG on Day 8 of CIDR insertion. The other group of the goats received twice daily intramuscular injections of a total of 70 mg FSH for 3 days from Day 8 of CIDR. All the gonadotropin treated goats were injected with 10 mg $PGF_{2\alpha}$ on Day 8 and 400 IU hCG in the afternoon on Day 10. For oocyte recovery, donor goats were fasted 24 h before operation. Anesthesia was induced by intravenous injection of 2% xylazine(0.2 mg/kg body weight) and ketamin(11 mg/kg body weight). In vivo oocytes were recovered by follicle aspiration or oviduct flushing at 29 to 34, 35 to 40 and 41 to 50 h after hCG injection through mid-ventral incision. There was no significant difference in the mean number of CL and oocytes recovered. Oocyte collection at 29 to 40 h after hCG increased(P<0.05) the recovery rate of ovulated oocytes in oviducts compared to 41 to 50 h. The same results were also observed in the recovery of follicular oocytes. Oocyte grade was not affected by collection time. When oocytes were collected from follicular oocytes at 41 to 50 h after hCG, the recovery rate of Grade II oocytes was the lowest(P<0.05). From these results, it is suggested that oocyte recovery at 35 to 40 h after hCG will be successful for further use.

• Journal of Embryo Transfer
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• v.21 no.3
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• pp.255-262
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• 2006

The purpose of this study is to investigate the effects of vitrification in open pulled straws (OPS) methods on in vitro survival ability of porcine embryos. For in vitro maturation of immature oocytes, the porcine ovaries were collected from local slaughter-house. The cumulus-oocytes complexes were aspirated from 2 to 6 mm follicles. The collected oocytes were cultured for in vitro maturation in NCSU-23 medium with 5 mM hypotaurine, 0.57 mM cysteine, 10% porcine follicle fluid, 10 IU/ml PMSG and 10 IU/ml hCG for $21{\sim}22$ hrs. Then, the oocytes were more cultured $21{\sim}22$ hrs in vitro maturation in medium removed hormones. The frozen-thawed spermatozoa were washed by centrifugation 2 times for 10 min at 1,500 rpm in D-PBS with 5.56 mM glucose, 0.33 mM Na-pyruvate, 100 IU/ml penicillin, $100 {\mu}g/ml$ streptomycin and 4 mg/ml BSA. The fertilization medium used mTBM with 2 mM caffeine and 2 mg/ml BSA and adjusted to a pH of 7.2 to 7.4. The final concentration of spermatozoa was adjusted to $2.5{\times}10^6$cells/ml motile sperm during fertilization in vitro. At 8 hrs after insemination, the oocytes were transferred into NCSU-23 medium with 5.0 mM hypotaurine, 4 mg/ml BSA and 10 ng/ml EGF and cultured for 7 days. When the blastocysts of different stages were frozen-thawed by OPS methods, the proportions of embryos with normal morphology were significantly (p<0.05) higher in embryos frozen-thawed at expanded blastocyst stage (38.9%) than in early blastocyst stage (28.3%). On the other hand, the proportions of embryos damaged after frozen-thawing were significantly (p<0.05) higher in embryos frozen at early blastocyst stages than in expanded blastocyst stage. In another experiment, the normal embryos morphology after frozen- thawing were further cultured for 48 hrs. After culture, the proportions of embryos hatched were 6.7, 20.0 and 33.3% for embryos frozen-thawed at early blastocyst, mid-blastocyst and expanded blastocyst stages. These finding indicate the possible broader application for OPS methods, as frozen-thawed embryos may be accompanied by developmental stage according to requirements of the survival ability after freezing of blastocyst stage in the pig.

• Clinical and Experimental Reproductive Medicine
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• v.35 no.2
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• pp.111-118
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• 2008

Objective: The purpose of the present study was to examine the hormonal regulation of RGS-2 in the rat ovary. Methods: Immature rats were injected with 10 IU of PMSG to induce multiple growth of preovulatory follicles and 10 IU of hCG to induce ovulation. Northern blot analysis performed for gene expression and in situ hybridization performed for mRNA localization. Results: Northern blot analysis revealed that pregnant mare's serum gonadotropin (PMSG) treatment did not affect RGS-2 mRNA levels. In contrast, human chorionic gonadotropin (hCG) treatment of PMSG-primed rats resulted in an increase in RGS-2 expression within $1{\sim}3\;h$. The major cell-types expressing RGS-2 mRNA were oocytes regardless of follicle size. Interestingly, hCG treatment caused the stimulation of RGS-2 gene expression in granulosa cells of preovulatory and growing follicles. In contrast, cell types expressing RGS-2 protein were theca cells regardless of hCG treatment. Like in vivo, treatment of preovulatory granulosa cells with LH in vitro stimulated RGS-2 levels within 1 h. Interestingly, GnRH antagonist II enhanced the stimulatory action of LH. Conclusion: The present study demonstrates the LH/hCG induction of RGS-2 in preovulatory granulosa cells and suggests a role of RGS-2 in Gq protein signaling pathway during ovulation.

• Journal of Embryo Transfer
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• v.24 no.3
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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.

• Korean Journal of Animal Reproduction
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• v.22 no.4
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• pp.319-329
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• 1998

These experiments were conducted to investigate the optimal maturation stage for cryopreservation of porcine oocyte when the oocytes were frozen-thawed and/or exposed in cryoprotect ant at immature, maturing and mature stages. The results of this research are as follows ; 1. When the oocytes matured for 0, 24 and 44h were exposed in media containing cryoprotectants or without in vitro, the rates of cultured oocytes developed to metaphase II were 44.0, 45.0, 50.3 or 55.0%, respectively. 2. When the oocytes matured for 0, 24 and 44h were exposed in media containing cryoprotectants or without in vitro, the cleavage rates of cultured oocytes were 18.6, 19.7, 47.6 or 50.9%, respectively. 3. When the oocytes matured for 0, 24 and 44h were frozen and thawed using vitrification or not in vitro, the rates of cultured oocytes developed to metaphase II were 4.3, 7.1, 46.7 or 62.4%, respectively. 4. When the oocytes matured for 0, 24 and 44h were frozen and thawed using vitrification or not in vitro, the cleavage rates of cultured oocytes were 2.5, 2.4, 10.2 or 49.6%, respectively.

• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.72-72
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• 2003

대부분의 동물에서 체내 또는 체외수정란의 체외 배양 시 일정한 발육 단계까지 발달한 후 발육지연이나 정지가 되는 체외 발육억제 현상이 나타나게 된다. 특히 돼지에서는 타 가축들과는 달리 4 세포기에서 체외 발육억제 현상이 일어나기 때문에 체외에서의 발육율이 매우 낮아 수정란 생산이 제한되고 있다. 따라서 본 연구는 이러한 돼지 체외발육 억제 현상을 극복하고 돼지 체외 수정란의 체외배양 체계의 기초 자료를 얻고자 돼지 미성숙 난자를 체외에서 성숙, 수정시킨 뒤, 체외 수정란의 배양 시 성장인자와 6탄당의 첨가에 따른 체외 발육율을 검토하였다. 난자의 핵 성숙과 세포질 성숙 및 세포 기능에 영향을 미치는 것으로 알려져 있는 성장인자로는 Insulin-like growth factor-I(IGF-I)과 Epidermal growth factor(EGF)를 사용하였고, 여러 종의 번식기관에 존재하여 배반포 형성을 촉진시키는 것으로 알려진 glucose, mannose, galactose 및 fructose가 6탄당으로 사용되었다. 체외수정란의 발육을 위한 기본 배양액인 NCSU-23에 각각 0, 1, 5, 10 및 20ng/ml의 IGF-I과 EGF를 각각 첨가하여 농도의 차이에 따른 발육율을 검토하였다. 또한 5.56mM의 glucose, mannose, galactose 및 fructose에 5ng/ml의 IGF-I 또는 10ng/ml의 EGF 첨가 유, 무에 따른 초기배 발육율을 검토하였다. 마지막으로, 각각의 6탄당에 위와 같은 농도의 IGF-I와 EGF 공동 첨가 유, 무에 따른 초기배 발육율을 검토하였다. 그 결과 돼지 체외 수정란의 체외 발육 시 배양액 내에 서로 다른 농도의 IGF-I과 EGF를 첨가하였을 때 IGF-I은 5ng/ml(12%)에서, EGF는 10ng/ml(10%)의 실험구에서 가장 높은 배반포기 배의 발육율을 나타냈다. 또한 각각의 6탄당과 IGF-I 또는 EGF 유, 무에 따른 초기배 발육율을 검토한 결과 IGF-I과 EGF 모두 glucose 첨가 시 타 첨가구에 비해 초기 발육 단계의 수정란 발육뿐만 아니라 배반포까지의 배발육(10～11%)이 타 첨가구(3～8%)에 비해 높게 나타났다. 한편, 각각의 6탄당이 첨가된 배양액 내에 IGF-I파 EGF 공동첨가 유, 무에 따른 초기배 발육율을 검토한 결과 모든 실험구에서 EGF와 IGF-I 첨가 시 무첨가보다 높은 초기 배 발육율을 나타냈으며 특히 초기 분열단계 수정란에서는 발육의 차이가 크게 나타났다. 본 연구 결과 성장인자와 6탄당의 첨가는 돼지 수정란의 체외배양 시 초기배 배발육에 효과적인 영향을 미치는 것으로 사료되며, 이는 체외 발육율이 타 가축에 비해 낮은 돼지의 수정란 생산에 있어 체외배양체계의 개선을 위한 기초자료가 될 수 있을 것이라 기대된다.