• 한국동물학회지
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• 제32권3호
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• pp.281-289
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• 1989

포유동물의 생식주기중 대부분의 여포는 폐쇄되고 극히 일부만 배란된다. 돼지의 폐쇄여포액내에는 다량의 androgen이 존재하며 이들이 폐쇄의 한 요인이라는 결과로 보아 폐쇄여포액내에는 다량의 활성적 testosterone이 존재할 것으로 추론하고 이를 정량하고져 하였다. 사람의 여포액내 progesterone, testosterone, estradiol, androstenedion과 dihydrotestosterone의 농도를 본 연구에서 정립된 섬광면역측정법 및 방사면역측정법으로 측정한 결과는 다음과 같았다. 사람의 폐쇄된 여표액내 T,A.DHT의 농도는 소, 중, 대 여포 공히 정상의 여포액내 이들 스테로이드의 농도보다 현저히 증가되어 있었다. 그러나 소여포의 경우 폐쇄여포액내 A의 농도는 정상에 비하여 현저히 낮은 농도를 나타내었다. 반면 P와 E의 폐쇄여포액내 농도는 정상에 비해 현저히 낮은 농도를 나타내었다. 돼지의 소, 중, 대의 폐쇄여포액내 P의 농도는 정상과 통계적으로 유의한 차이가 없었다. T의 경우 역시 각 각의 크기를 가진 폐쇄여포액에서 정상 여포액 농도보다 높게 나타났으나 통계적인 유의성은 없었다. 그러나 E의 경우는 폐쇄여포액내 농도가 정상에 비해 편저히 낮게 나타났다. 한편 여포액내 활성적 T의 점유율은 혈청에 비해 현저히 높았다. 즉 bioavailable T는 정상과 폐쇄여포액내에서 각기 90% 이상을 나타내었다. 위의 결과를 종합해 볼 때 동물중, 크기에 따라 폐쇄의 요인은 다른 것으로 사료되며 폐쇄여포액내 활성적 androgen의 농도는 정상에 비해 높고 또한 E의 농도는 현저히 낮은 것으로 나타났다. 또한 여포액내 대부분의 스테로이드는 활성형의 상태로 존재하며 활성적 T는 폐쇄여포의 판정에 주요기준이 될 수 있을 것으로 판단된다.

• Clinical and Experimental Reproductive Medicine
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• 제24권2호
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• pp.267-271
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• 1997

Apoptosis, programmed cell death, is posulated to occur in granulosa cells in ovarian follicular atresia. bcl-2 gene serves as protector from apoptosis and, thus, is associated with increased cell survival. TRPM-2 gene expression has been implicated as a trigger of apoptosis in rat prostate, uterus and mammary gland. Our objective was to determine if bcl-2 and TRPM-2 are expressed in luteinized human GC and, therefore, have regulatory functions for apoptosis in GC. Human GC were obtained via oocyte retrival from the infertile patients stimulated with exogeneous gonadotropins while undergoing IVF. GC were isolated from follicular fluid using Percoll gradient centrifugation. The GC were further purified with anti-CD45 magnetic beads to remove contaminating WBC's. RT-PCR were performed to analyze the mRNA expression of bcl-2 and TRPM-2 in the GC. The PCR primers were designed to amplify a 195 bp fragment of bcl-2 and a 174 bp fragment of TRPM-2. The PCR products were electrophoresed on 4% agarose gel. Three separate experiments indicated that both bcl-2 and TRPM-2 are concurrently expressed in human GC. We cultured granulosa cells with FSH (1 ng/ml) for 1 day to investigate the relative changes of TRPM-2 mRNA level with RNAse protection assay. When we cultured GC with serum free medium for 1 day TRPM-2 mRNA level increased with 1.3 fold, however it was decreased 0.64 fold with FSH. Therefore we conclude that bcl-2 and TRPM-2 are concurrently expressed and that the interaction of their products may be involved in GC apoptosis. And TRPM-2 may be regulated with FSH.

• Asian-Australasian Journal of Animal Sciences
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• 제21권4호
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• pp.532-537
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• 2008

In order to investigate the factors affecting the culture of mouse preantral follicles in vitro, we examined the effect of culture media, protein supplements, and culture period on their growth. The oocyte diameter (initial size: $55.6{\pm}2.5{\mu}m$) was progressively increased during culture, and the maximum size ($72.0{\pm}2.4{\mu}m$) was reached on day 10 of the in vitro culture. The chromatin configuration in the germinal vesicle (GV) oocyte progressively shifted from a non-surrounded nucleolus (NSN) to a surrounded nucleolus (SN). On day 10 of the culture, most of the oocytes progressed to the SN pattern. The survival and metaphase II rates of the oocytes in alpha-minimal essential medium (alpha-MEM) were significantly higher (p<0.05) than those in Waymouth and tissue culture medium (TCM)-199. As a protein source, fetal bovine serum (FBS) was more suitable for the culture of mouse preantral follicles as compared to human follicular fluid (hFF) and bovine serum albumin (BSA); the optimal concentration of FBS was 5%. These results suggest that in a culture of mouse preantral follicles, alpha-MEM and 5% FBS are an optimal medium and a protein source, respectively; further, the 10 days of culture is required for the complete growth of oocytes in this culture system.

• Clinical and Experimental Reproductive Medicine
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• 제15권1호
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• pp.17-24
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• 1988

Follicular fluid(FF) prostaglandin $E_2$(PG$E_2$) and $PGF_{2{\alpha}}$ levels were compared in 3 groups of spontaneously ovulatory women undergoing ovulation induction with clomiphene citrate (CC) alone or with human menopausal gonadotropin(hMG) (14 patients), hMG(9 patients), or pure FSH/hMG(11 patients) for the purpose of in vitro fertilization. FF volume aspirated did not differ significantly according to the maturity of the oocyte. According to hyperstimulation regimens, the volume of FF from which preovulatory occytes were obtained was significantly less in the hMG-treated group than in the other groups. In follicles of preovulatory oocytes, FF PG$E_2$ values were significantly lower in the FSH treated group than in the Cc.treated or hMG-treated group, and FF $PGF_{2{\alpha}}$ values were significantly higher in the hMG-treated group than in the CC-treated or FSH-treated group. In follicles of immature or atretic oocytes, there was no significant difference in FF PG$E_2$ and PG$F_{2{\alpha}}$ concentrations of the similar morphology of the oocyte according to hyperstimulation regimens. In all cycles, FF PG$E_2$ and PG$F_{2{\alpha}}$ values of preovulatory oocytes were not significantly different from those of immature oocytes, but those of atretic oocytes were relatively lower than those of fertilizable oocytes and it was statistically signifincant in PG$E_2$ values of CC-treated group. In all treatment groups, FF PG$E_2$ and PG$F_{2{\alpha}}$ levels did not show and close relationship with the success of fertilization in vitro and of pregnancy after embryo transfer. Above results suggested that FF PG$E_2$ and PG$F_{2{\alpha}}$ be involved in oocyte maturation and ovulation, but their relationship with the success of in vitro fertilization was not found.

• 한국발생생물학회지:발생과생식
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• 제3권1호
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• pp.39-44
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• 1999

성선자극호르몬으로 자극된 난소로부터 회수된 사람의 성숙난자의 전핵형성과 초기발생에 정자의 운동성과 성숙도가 미치는 영향을 조사하였다. 세포질내정자주입 (ICSI)은 HEPES-buffer mTCM-199 배양액에서 실시하였다. 체내에서 성숙된 난구세포부착난자를 ICS에 의하여 사정된 운동성 정자 또는 비운동성 정자로 수정하였을 때 운동성 정자로 수정된 난자가 비운동성 정자로 수정된 난자보다 전핵형성율이 높았다 (79.8% vs 51.7% ; p<0.002)). 그러나 체내에서 성숙된 난구세포부착난자를 ICS에 의해 정소 내 운동성 정자 또는 비운동성 정자로 수정하였을 때 운동성 정자와 비운동성 정자 사이의 전핵형성율은 차이가 없었다. 10.0 mM lactate, 0.5 mM pyruvate, 0.2 mM taurine, 1.0 mM glutamine, 2.22 mM MEM amino acids, vitamin 그리고 10% 사람 난포액이 포함된 수정 Tyrode 배지에서 전핵이 형성된 수정란의 초기 발생은 정자의 채취원과 운동성에 관계없이 9∼16세포기로 발생하였다.

• Clinical and Experimental Reproductive Medicine
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• 제26권1호
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• pp.75-81
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• 1999

The beneficial effect of glucose and phosphate ions in culture medium on the development of human embryos in vitro has not been fully elucidated. The purpose of this study was to evaluate the influence of fertilization and culture of embryos in glucose/phosphate-free m-TALP medium on pregnancy rates in IVF-ET program. The patients in 244 IVF-ET cycles received GnRH agonist + HMG regimens. A does of 10,000 IU HCG was administered when two or more dominent follicles reached 18mm in diameter. Thirty-six hours after HCG, oocytes were recovered transvaginally using ultrasound guidance. Aspirated oocytes were matured for 4 to 6 h in TCM-199 supplemented with 10% follicular fluid (FF). Insemination was carried out with 50,000 motile spermatozoa in TCM-199 + 10% FF or m-TALP + 5% FF + 5% fetal cord serum (FCS) according to experimental design. After 6 h, oocytes were washed 3 to 4 times and cultured in each fresh medium. After 20 h, oocytes were freed from cumulus/corona cells and examined for the presence of pronuclei. Fertilized oocytes were transferred into each co-culture drops and cultured for further incubation. On day 3, embryo transfer was performed with grade 1 and 2 embryos. Monolayers for co-culture of embryos were prepared by plating $1{\times}10^5$ cumulus cells/ml in 10ul drop of TCM-199 + 10% FF or m-TALP + 5% FF + 5% FCS media 24 h prior to the onset of co-culture. Development to 4 to 16 cell stage was observed at 70x magnification following two days of incubation. Pregnancy was confirmed by detecting increasing serum ${\beta}$-hCG concentrations for 11 days following embryo transfer. Data were analyzed by ${\chi}^2$-test. Oocytes from 244 IVF-ET cycles were randomized. The number of cycles and mean age of patients were 97 and 147, 31.3 yrs and 31.2 yrs for TCM-199 (control) and m-TALP groups, respectively. The mean number of retrieved oocytes/cycle, fertilization rates, number of embryos transferred/ET and pregnancy rates were 11.1 and 10.3, 65.1% and 67.3%, 4.1 and 4.7, 28.9% and 43.8% for TCM-199 and m-TALP groups, respectively. Differences in the pregnancy rates were found between control and m-TALP groups (p<0.05). The pregnancy rate of patients divided according to maternal age groups of ${\leq}30$, 31-35, $36{\leq}$ were 44.4% and 49.0%, 26.1% and 41.3%, 29.2% and 41.2% for control and m-TALP groups, respectively. These data indicate that culture of human embryos in glucose/phosphate-free m-TALP medium improves pregnancy rates.

• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2003년도 제3회 국제심포지움 및 학술대회
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• pp.118-118
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• 2003

The aim of this study was to investigate the effect of glucose on embryonic development of mouse embryos. Two cell embryos were recovered from ICR female mice(3-4weeks) at 46~50 hrs after hCG 5 IU injection (mated just after hCG injection) and cultured in 50 $\mu m$ DMEM droplets supplemented with nothing (control: n=46), glucose 0.5mM (Group A; n=46) or glucose 3.15 mM(Group B; n=46) under mineral oil. All experimental media were supplemented with 20% human follicular fluid. Total blastocyst formation rates was lower (NS) in glucose groups (group A: 52.2% : B. 47.8%) than control group (60.9%). ZiB rates was the highest (P<0.05) in control (47.8%) than those in group A (21.7%) and B (28.3%). ZeB rates were the highest (NS) in group A (30.4%) than those in control (13.0%) and group B (19.6%). Blastocysts, cultured in group B (50.5), had the highest (NS) mean cell number compared with the others (control: 39.2 ; group A: (45.6). The ICM proportion (% ICM of total cells) in blastocysts cultured in group A (20.6%) was the highest (NS) than those of other tested groups (control: 15.2 ; group B: 13.9%). This study shows that a low dose of glucose added to culture medium increases the ICM proportion of blastocysts in mice.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2003년도 학술발표대회 발표논문초록집
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• pp.33-33
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• 2003

This study was conduct to compare the efficacy to produce male and female somatic cloned piglets. Maturation of porcine COCs was accomplished by incubation in NCSU-23 medium supplemented with 0.6 mM cysteine, 10% porcine follicular fluid, 1mM dibutyryl cyclic adenosine monophosphate (dbc-AMP, Sigma, USA), and 0.1 IU/ml human menopausal gonadotrophin (hMG, Teikokuzoki, Japan) for 20h and then cultured without dbcAMP and hMG for another 18 to 24 h. Female and male fetal cells were isolated from each fetus, cultured in ES-DMEM medium containing 10% FCS. Enucleated oocytes were fused with fetal fibroblasts (passage 4 to 15). Reconstructed embryos were cultured in NCSU-23 with 4 mg/ml BSA under mineral oil at 39$^{\circ}C$ in 5% $CO_2$ in air. A total of 12,328 nuclear-transferred embryos (1- to 4-cell stage) were surgically transferred into 69 surrogate gilts. Three recipients aborted during the period of conception. Three gilts delivered eleven female piglets, and five recipients gave rise to birth 22 male piglets. The average birth weigh of the cloned piglets was 1.52 kg (1.38~1.83 kg) in female piglets and 0.84 kg (0.45~1.25 kg) in male piglets. Alive cloned pigs was seven in female piglets (63.6%) and four in male piglets (18.2%). The other two recipients is ongoing. This study suggests that female-derived fetal cell as a nuclear donor has more capability on production of cloned piglets than male.

• 한국가축번식학회지
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• 제22권3호
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• pp.229-235
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• 1998

본 실험은 마우스 초기배의 체외배양에 있어서 배양액 첨가제로서 기초배양액에 RPMI 1640 아미노산, MEM 비타민 및 사람의 성숙 난포액(hFF)을 각각 5가지의 농도로 구분하여 첨가했을 경우, 각 첨가제가 배 발달에 미치는 영향과 각 첨가제의 적정 첨가농도를 검토하고자 수행하였다. 그 결과를 요약하면 다음과 같다. 1. 배양액에 RPMI 1640 아미노산을 무첨가, 0.25%, 0.5%, 1% 및 2%의 농도로 첨가했을 때 배반포 발생율은 각각 35.5%, 54.5%, 65.4%, 48.2% 및 57.4%로 나타났으며, 0.5% 첨가구에서 유의하게 높았다. 2. 배양액에 MEM 비타민을 무첨가, 0.25%, 0.5%, 1% 및 2%의 농도로 첨가했을 때 배반포 발생율은 각각 12.8%, 22.4%, 31.1%, 21.9% 및 19.0%로 나타났으며, 0.5% 첨가구에서 유의하게 높았다. 3. 배양액에 hFF를 무첨가 2.5%, 5%, 10% 및 20%의 농도로 첨가했을 때 배반포 발생율은 각각 20.6%, 20.9%, 21.2%, 18.9% 및 29.4%로 나타났으며, 20% 첨가구에서 가장 높았다.

• Clinical and Experimental Reproductive Medicine
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• 제13권1호
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• pp.11-19
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• 1986

Follicular flxid (FF) and their matched oocytes were obtained from 58 follicles of 27 women who underwent an in vitro fertilization (IVF) procedure with ovarian hyperstimulation by clomiphene citrate(n=8), hMG(n=9),FSH/hMG(n=10). Follicular aspiration was performed 36 hours after human chorionic gonadotropin administration. The concentcation of androstendione (ADD), testosterone (T) was correlated with hyperstimulation regimens, the morphology of the oocyte-corona-cumulus complex (OCCC), oocyte fertilization, and the incidence of pregnancy after embryo transfer. The results were as follows. 1. According to hyperstimulation regimens, there was no significant differance in FF ADD and T concentrations of the similar morphology of OCCC. 2. In clomiphene-treated and FSH/hMG-treated cycles, FF ADD concentrations of preovulatory oocytes were 43.09${\pm}$9.53 ng/ml and 59.46${\pm}$9.09 ng/ml, those of immature occytes were 96.98${\pm}$16.55 ng/ml and 116.13${\pm}$36.81 ng/ml, those of atretic oocytes were 246.5 ${\pm}$9.25 ng/ml and 634.25${\pm}$9.25 ng/ml respectively, reflecting the significant relationship between FF ADD level and morphologic maturity of OCCC (p<0.05). But in hMG-treated cycles, such relationship was not found (p>0.1). In clomiphene-treated and FSH/hMG-treated cycles, FF T concentrations of preovulatory oocytes were 11.37${\pm}$2.38 ng/ml and 11.68${\pm}$1.73 ng/ml respectively which were significantly lower than those of atretic oocytes (25.1${\pm}$7.50 ng/ml and 23.25${\pm}$0.95 ng/ml respectively) (p<0.05). But in all cycles, FF T concentrations of immature oocytes were not significantly different from those of preovulatory oocytes, artetic oocytes (p>0.1). 3. In hMG-treated and FSH/hMG-treated cycles, FF ADD concentrations of fertilized oocytes were 32.43${\pm}$4.09 ng/ml and 42.61${\pm}$4.82 ng/ml respectively which were significantly lower than those of non-fertilized oocytes (72.18${\pm}$17.31 ng/ml and 108.09${\pm}$17.32 ng/ml respectively) (p<0.05), but in clomiphene-treated cycles there was no significant difference (p>0.1). In FSH/hMG-treated cycles, FF T concentration of fertilized oocytes was 7.33${\pm}$1.06 ng/ml which was significantly lower than that of non-fertilized oocytes (20.3${\pm}$6.21 ng/ml) (p>0.02), but in clomiphne-treated and hMG-treated cycles there was no significant difference (p>0.1). 4. In all cycles FF ADD and T concentrations did not correlated with the success of pregnancy after embryo transfer. Above results suggested that FF ADD and T may play an important role in oocyte maturation and fertilization, but their relationship with the success of psegnancy was not found.