• Clinical and Experimental Reproductive Medicine
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• 제24권1호
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• pp.1-11
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• 1997

It is well known that the zona pellucidae of mouse oocytes become "hardened" when they are allowed to mature in vitro in the absence of serum components. To see if oocytes already undergone meiotic resumption in vivo exhibit similar zona hardening, hardening of ZP of cumulus-enclosed oocytes(CEOs) was examined after culture in vitro since their release from follicles various hours after hCG injection. When CEOs matured in vivo for 3h or longer were subjected to culture in vitro for 14h with BSA alone, zona hardening was significantly reduced compared to those cultured in vitro from the begining of maturation. However, when CEOs matured in vivo for 5h were freed from cumulus cells and then cultured in vitro with BSA alone, little reduction of zona hardening was observed. Preincubation of CEOs for 5h with fetuin, one of the well known inhibitor of in vitro zone hardening, did not prevent zona hardening during its subsequent culture of CEOs for 14h without fetuin. However, when CEOs precultured with both fetuin and PMSG for 5h and then further cultured with BSA alone for 14h, zona hardening was dramatically reduced. Under these conditions, the expansion of cumulus cell was observed. In addition, CEOs cultured with both BSA and dbcAMP to prevent their meiotic resumption showed a significant increase of zona hardening. Whether the observed zona hardening was correlated with the conversion of ZP2 to $ZP2_{f}$ was examined. Zona pellucida, isolated from CEOs matured for 5h in vivo and then further cultured with BSA alone was subjected to SDS-PAGE. Most of ZP2 molecules from these CEOs did not undergo conversion from ZP2 to $ZP2_{f}$. From these results, it is concluded that CEOs undergone meiotic resumption in vivo do not exhibit zona hardening when they were subsequently cultured in vitro without serum components. It appears that cumulus cells play an important role in this phenomenon.

• Clinical and Experimental Reproductive Medicine
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• 제44권1호
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• pp.33-39
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• 2017

Objective: The aim of this study was to assess the changes of follicular fluid (FF) and serum levels of cerebellin precursor protein 1 (cbln1) and betatrophin in patients with polycystic ovary syndrome (PCOS) undergoing in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) with a gonadotropin-releasing hormone (GnRH) antagonist protocol. Methods: Twenty infertile women with PCOS and 20 control women diagnosed as poor responders undergoing ovarian stimulation with a GnRH antagonist were included. Blood samples were obtained during ovum pick-up. Follicular fluid from a dominant follicle was collected from the subjects. Using enzyme-linked immunosorbent assays, FF and serum levels of cbln1 and betatrophin were measured in both groups of participants. Metabolic and hormonal parameters were also determined and correlated with each other. Results: Both groups of women had similar serum and FF betatrophin levels ($55.0{\pm}8.9ng/mL$ vs. $53.1{\pm}10.3ng/mL$, p=0.11). The serum and FF betatrophin levels of poor responders were found to be similar ($49.9{\pm}5.9ng/mL$ vs. $48.9{\pm}10.7ng/mL$, p=0.22). Conversely, the FF cbln1 levels of PCOS women were found to be significantly higher than the serum cbln1 levels ($589.1{\pm}147.6ng/L$ vs. $531.7{\pm}74.3ng/L$, p<0.02). The FF cbln1 levels of control participants without PCOS were significantly higher than their serum cbln1 levels ($599.3{\pm}211.5ng/L$ vs. $525.3{\pm}87.0ng/L$, p=0.01). Positive correlations were detected among body mass index, insulin resistance, serum insulin, total testosterone, and betatrophin levels in the PCOS group. Conclusion: Follicular fluid betatrophin and cbln1 concentrations may play a pivotal role on follicular growth in PCOS subjects undergoing IVF/ICSI with an antagonist protocol.

• 한국패류학회지
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• 제25권2호
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• pp.145-151
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• 2009

담수 복족류인 다슬기, Semisulcospira libertina libertina의 난자형성과정 동안 생식세포의 미세구조 변화를 광학현미경과 전자현미경으로 관찰하였다. 난소는 나탑 후방부의 간췌장의 표면에 위치하며, 성숙시기에는 녹색을 나타내었다. 난소는 다수의 난자형성소낭들로 이루어져 있었다. 난자형성과정은 5단계로, 난원세포기, 난황형성전기, 난황형성개시기, 난황형성활성기, 성숙기로 구분하였다. 난원세포는 직경 $4-6\;{\mu}m$의 원형으로 커다란 핵을 가진다. 난황형성전기의 난모세포는 직경이 약 $20\;{\mu}m$이며, H-E 염색에서 세포질은 호염기성을 나타냈다. 난황형성개시기의 난모세포는 직경 $60-80\;{\mu}m$ 내외로 세포질에는 전자밀도가 낮은 난황과립들을 가지며, 난병에 의하여 난자형성소낭에 연결되어 있었다. 난황형성 활성기의 난모세포는 직경 $100-120\;{\mu}m$로 난황과립의 전자밀도, 크기, 양이 증가하였으며, 세포질에는 골지체, 미토콘드리아, 조면소포체들이 발달하였다. 성숙기 난모세포의 세포질 대부분은 전자밀도가 높은 단백질성의 난황과립들로 채워져 있었으며, 난막에서는 길이 약 $1.1\;{\mu}m$의 미세융모가 관찰되었다.

• 한국발생생물학회지:발생과생식
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• 제7권2호
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• pp.113-118
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• 2003

포유동물의 성숙한 난포의 난포액 속에는 여러 종류의 단백질 분해효소가 있으며 이들은 난포의 형성과 퇴화 및 난자의 성숙과 배란 등의 다양한 변화에 중요한 역할을 하는 것으로 여겨진다. 난포액 속의 단백질 가수분해효소 중에는 serine proteinase가 비교적 잘 알려져 있으나 다른 효소 특히, caseinolytic enzyme에 대해서는 거의 알려져 있지 않다. 본 연구에서는 사람의 난포액을 재료로 하여 caseinolytic enzyme의 존재 여부 및 동 효소의 특성을 알아보고자 하였다. 사람의 난포액과 혈청 그리고 사람의 제대혈을 $\alpha$-casein을 기질로 하는 zymography 방법으로 분석 한 결과 분자량 80 kDa의 매우 강한 caseinolytic activity를 지니는 효소 단백질과 분자량 78 kDa의 비교적 약한 caseinolytic activity를 갖는 단백질 등 두 개의 caseinolytic enzyme이 관찰되었다. 이 caseinase들의 특성을 알아보기 위해 phenylmethylsulfonyl fluoride(PMSF), soybean trypsin inhibitor(SBf), 1,10-phenanthroline, E-64 그리고 ethylenediamine tetraacetic acid(EDTA)를 zymouaphy의 substrate buffer에 처리한 결과 EDTA와 SBTI에 의해서 80 kDa와 78 kDa caseinase의 활성이 억제되었다 이로 미루어 80 kDa와 78 kDa caseinase는 trypsin-like enzyme인 것으로 추측된다. 한편 사람 난포액의 zymouaphy 수행시에 5 mM의 EDTA가 첨가된 substrate buffer에 CaC $l_2$, MgC $l_2$, MnC $l_2$ ZnC $l_2$를 각각 0에서 10 mM의 농도별로 처리한 결과 모두 5 mM 농도에서 가장 높은 caseinase 활성을 보였다. 금속 이온의 첨가없이 EDTA만 처리한 대조군의 경우 caseinase의 활성은 나타나지 않았다. 이로 미루어 80 kDa 및 78 kDa caseinase는 효소 활성을 위해 이가 금속 양이온을 필요로 하는 것으로 여겨진다.

• 한국수정란이식학회지
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• 제10권2호
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• pp.105-113
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• 1995

This study was conducted to examine the efficiency of enucleation and blastomere isolation from recipient oocytes and donor embryos, respectively and to determine the effect of oocyte age and electric voltage on the fusion rate and in vitro development of the fused oocytes in rabbit nuclear transplantation. Immature oocytes collected from ovarian follicles were matured in vivo for 12 h in TCM-199 containing FCS and hormones and in vivo matured oocytes were collected 17 to 18 h post-HCG. The fresh and frozen donor embryos of 8- to 16-cell stage were collected from the oviduct of superovulated does. The proportion of successfully enucleated oocytes was greatly lower in in vitro matured oocytes (42.3%) than that (62.7%) in in vivo matured oocytes The level of cytochalasin B for in vivo matured oocytes did not affect the efficiency of enuleation, but 7.5 $\mu$g /mL cytochalasin B for in vitro matured oocytes showed a high enucleation rate significantly. The isolation efficiency of a single blastomere nucleus did not differ between 8- and 16-cell stage embryos. The percentage of single blastomeres isolated from 16-cell stage fresh embryos after 0.5% pronase treatment was greatly higher at 16-min treatment (94.4%) than at 8-min(78. 1%) and the blastomeres(61.5%) isolated from frozen-thawed embryos after 16-min pronase were significantly fewer than those of fresh embryos. The age of recipient oocytes affected nuclear fusion rate. The reconstituted oocytes fused at 24-h age showed slightly higher fusion rate (77.8%) than those (65.0%)fused at 18-h age. The fusion rate of in vitro and in vivo matured oocytes inserted with fresh blastomere did not differ among electric voltages, but the cleavage rate and development to morula-blastocysts of in vitro matured oocytes was more higher under 0.6 kV/cm than under 0.8 to 1.2 kV/cm, while the cleavage rate and development of in vivo matured oocytes was higher under 0.8 to 1.0 kV/cm than under 1.2 kV/cm. The fusion and cleavage rate fol1owing insertion with frozen-thawed blastomere was not different between the in vitro and in vivo matured oocytes and was similar to those from fresh blastomere insertion.

• 한국수정란이식학회지
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• 제19권3호
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• pp.257-264
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• 2004

멸종위험이 큰 우리나라 재래돼지를 유전자원으로서 안전하게 보존하고 유전적 다양성을 유지하기 위한 수단으로 수정란을 채취하여 동결보존하기 위해서 미경산 재래돼지에서 과배란 유기를 위한 적정 호르몬의 수준과 수정란의 회수 및 동결보존 방법을 확립하고자 수행한 결과는 다음과 같다. 1. hCG 500IU와 PMSG를 500, 750, 1,000IU 및 hCG 750IU와 PMSG 1,000IU를 각각 투여한 재래돼지의 배란황체와 미배란난포의 수는 각각 12.4, 13.6, 30.0 및 23.3개로 PMSG 1,000IU와 hCG 500IU를 투여한 재래돼지가 다른 용량의 처리돼지보다 난소반응이 양호하였으나 유의적인 차이는 없었으며, 배란황체수에 대한 수정란 회수율은 59.4-79.2％ 수준이었다. 2. 과배란처리된 공란돈에서 수정후 4일에 회수된 수정란의 발육단계는 상실기의 수정란이 수정후 5일보다 유의적으로 많이 회수되었으며(P<0.01), 수정후 5일에 회수된 배 반포기의 수정란을 수정후 4일보다 유의적으로 많이 회수되었다(P<0.05). 3. 확장배반포기 수정란을 1.4M glycerol의 항동 해제를 이용하여 관행의 완만동결법으로 동결한 처리에서 생존율은 25.3％였다.

• 한국수정란이식학회지
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• 제15권2호
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• pp.137-145
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• 2000

To investigate fatty acid constituents and relative compositions in the fluid of the follicles, oviducts, uterine ho군 and uterine body in sows, the fluids of the reproductive tract were analyzed using Gas chromatography. The samples were taken from various reproductive tract of 21 sows slaughtered. 1. Caprylic acid(C8: 0), capric acid(C10:0), lauric acid(C12:0), myristic acid(C14:0), palmitic acid(C16:0), plamitolele acid(C16:1), stearic acid(C18:0), oleic acid(C18:1), linoleic acid(C18:2) and arachidonic acid(C20:4) were found in the reproductive tracts of the sows, which made 10 kinds of fatty acid intotal. 2. Two kinds of polyunsaturated fatty acids, linoleic acid and archidonic acid were found inthe reproductive tracts. 3. Palmitic acid among saturated fatty acids and oleic acid among unsaturated fatty acids were the hihgest level in all of the reproductive tracts. 4. Palmitic acid, oleic acid and stearic acid showed higher rate with 44.89%, 23.69% and 14.36%, respectively, and lauric acid, capric acid, palmitoleic acid, arachidonic acid ad myristic acid showed lower rate with 0.62%, 1.13%, 1.65%, 1.97% and 2.24%, respectively in the reproductive fluid. 5. The highest level of arachidonic acid was found in the uterine horn. 6. The sum of the palmitic acid and oleic acid were 66.91%, 70.41%, 66.14% and 73.36% in the fluid of follicle, oviduct, uterine horn and uterine body, respectively. 7. The relative composition of arachidonic acid was higher during the follicular stage than during the luteal phase in the fluid of oviduct and uterine. 8. The long chain fatty acids such as the palmitic acid, stearic acid, oleic acid and linoleic acid showed higher relative compositions during the follicular phase(93.18%∼96.83%) than during the luteal phase(82.56%∼88.37%). 9. Caprylic acid, luric acid and palmitoleic acid were undetected in the fluid of all of the reproductive tracts during the follicular phase. Low relative compositions of capric acid, myristic acid andarachidonic acid were found during the follicular phase, while the low relative compositions (<5%)of capric acid, lauric acid, myristic acid, plamitoleic acid and arachidonic acid were found during the luteal phase.

• 한국동물위생학회지
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• 제27권1호
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• pp.41-52
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• 2004

To establish the differential diagnosis and functional status in ovarian cystic cows, progesterone(P$_4$) and estrogen(E$_2$) level of cystic follicular fluid, ultrasonography for measuring the cystic diameter and thickness of cystic wall, and histological findings were investigated in cystic ovaries from slaughtered Korean native cows. Ovarian follicles were classified as systic if the diameter was greater than 25 mm by ultrasonography. Ovarian cysts < 3 mm of cystic wall thickness, < 10 ng/ml P$_4$ concentration and >10 ng/ml E$_2$ concentration were classified follicular cyst, ovarian cysts 3 mm of cystic wall thickness, 10 ng/ml P$_4$ concentration and <10 ng/ml E$_2$ concentration were classified luteal cyst, and ovarian cysts 3 mm of cystic wall thickness, < 10 ng/ml P$_4$ concentration and <10 ng/ml E$_2$ concentration were classified non-functional ovarian cyst, respectively. Also ovarian cysts were classified 8 types by anatomical and hisctological findings. Ovarian cysts with corpus luteum were 3 of 73 cows and ovarian cysts without corpus luteum were 70 cows. The incidence rates of 8 various types of ovarian cysts were as follows; 2Aa 56.2%, 2Ba 20.5% and 2Ab 15.1%, respectively. The incidence rates of ovarian cysts without corpus luteum were follicular cyst 76.7% and luteal cyst 19.2%. The thickness of cystic wall were lAb 3.9 mm, 2Ab 3.3 mm and 2Bb 3.2 mm, and the cystic fluid P$_4$ concentrations were above 10.0 ng/ml in lAb, 2Ab and 2Bb, respectively. There was significantly correlations between the thickness of cystic wall and cystic fluid P$_4$ concentration in ovarian cysts(p<0.05). The ovarian cyst was classified follicular cysts, luteal cyst and non-functional ovarian cyst by hormone analysis. The luteal cyst was accuratly dignosed by cystic wall thickness. But follicular cysts was misdiagnosed 13 cows of 56 cystic cows. The 13 cystic cows was determined as had non-fuctional ovarian cysts. The cystic fluid P$_4$ concentration was 3.3 ng/ml in follicular ovarian cysts and 30.1 ng/ml luteinized ovarian cysts. There was significantly positive correlations between thickness of cystic wall and serum P$_4$ concentration in follicular(r$^2$ =0.59, p<0.001) and luteal cysts(r$^2$=0.65, p<0.001). These results indicated that ovarian cysts had various stages of degeneration and luteal cyst was accuratly diagnosed measurement of cystic wall thickness by ultrasonography, but follicular cysts was not diagnosed only cystic diameter and cystic wall thickness. In conclusion, it is suggest that ovarian cysts was diagnosed by combination of clinical sign and anatomical cystic features.

• 한국양식학회지
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• 제9권1호
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• pp.73-81
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• 1996

농어목 어류인 Caesio diagramma의 생식주기를 밝히기 위하여 난모세포의 발달과정을 조직학적 방법으로 관찰하였다. 난모세포의 발달과정은 염색인기, 주변인기, 유구기, 제1차 난황구기, 제2차 난황구기 및 제3차 난황구기의 6단계로 구분되었다. 염색인기와 주변인기의 난모세포는 연중 관찰되었으며, 난모세포 발달과정에서의 특징은 난소 내에서 유구가 초기에 형성되며, PAS 염색 결과 난황포는 타어류에 비하여 크기가 작았고 양도 적었다. 4월에 채집된 어류의 난소에서는 난황 형성기의 난모세포가 출현하기 시작하였다. $5\~6$월의 난소에서는 제3차 난황구기 난모세포가 관찰되었으며, 연중 가장 높은 비율을 나타냈다. 또한, 이 시기부터 난소의 일부에서는 배란을 마친 여포와 퇴화중인 난모세포도 다수 관찰되었으며, 7월까지 이어졌다. 9월 이후의 난소에서는 난황 형성기의 난모세포가 관찰되지 않았으며, 12월까지 염색인기 및 주변인기의 난모세포가 주류를 이루었다. 생식소중량지수(GSI)와 간중량지수(HSI)는 4월부터 증가하기 시작하여, $5\~6$월에 최고치에 도달하였으며 GSI와 HSI의 변화는 난모세포의 발달과정과 일치하였다. 따라서 C. diagramma의 주산란기는 $5\~6$월이며, 난소의 발달과정과 여러 형태의 여포가 존재하는 점으로 미루어, 본 종은 비동기발달형의 난소를 가지며 다회산란 어류에 속한다.

• Asian-Australasian Journal of Animal Sciences
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• 제20권4호
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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.