• 한국동물학회지
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• 제36권3호
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• pp.416-424
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• 1993

홍모기 난소에서 일어나는 난황단백질의 합성을 조사하였다. 지방체의 난황단백질합성양과 난소에 축적되는 난황단백질의 양을 rocket immunoelectorphoresis 방법과 in vitro organ culture 방법으로 조사한 결과 지방체에서의 난황단백질 합성을 흡혈 후 6시간째 부터 시작되어 24시간에 최대의 합성양을 나타낸 후 48시간 이내에 완료되었으며, 난소내로의 축적은 6시간부터 시작되어 60시간까지 계속되었다. 흡혈 후 0, 24, 48, 72시간된 난소추출물을 전기영동 및 Western blotting한 결과 24시간된 난소에서는 한종류의 난황단백질이 존재한 반면, 48, 72시간된 난소에서는 두종류의 난황단백질이 나타났다. 흡혈 후 48시간된 난소와 지방체를 $^3$H-leucine이 함유된 배지에서 배양하였을 때 지방체에서는 단백질의 합성이 거의 일어나지 않았으나 난소에서는 난황단백질을 포함한 다량의 단백질합성이 일어났다. Crossed immunoelectrophoreses의 결가 YP1과 YP2는 분자량에서는 차이를 보이나 면역성은 동일한 것으로 나타났다. 이상의 결과 홍모기에서는 난소에서도 난황단백질의 합성이 일어나는 것으로 나타났다.

• Reproductive and Developmental Biology
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• 제34권3호
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• pp.207-211
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• 2010

Current developments in IVF and animal cloning have resulted in increasing demand for large quantities of oocytes and ovarian follicles at specific stages of development. These medical and scientific needs may be met by developing an optimal culture system for preantral follicles. In this study, we investigated the growth of porcine preantral follicle cultures in different media and in the presence and absence of serum. Follicles were manually dissected from ovaries obtained from prepubertal gilts at a local slaughterhouse, and cultured for 3 days in M199 or NCSU23 medium supplemented with porcine FSH, transferrin, L-ascorbic acid and insulin. Follicle diameters were measured on day 1 and 3 of culture. In Experiment 1, the effect of supplementing culture medium with fetal calf serum (FCS) on porcine preantral follicle growth was examined. In the group of cultures supplemented with FCS, follicle diameter after 3 days of culture, survival rate and antrum formation rate in the FCS group were significantly higher than those of the control group. In Experiment 2, the effects of culture medium (M199 and NCSU23) on follicle growth were compared. Follicle diameters were increased in the M199 group, compared with those in NCSU23 (p<0.05), but we observed no significant differences in survival and antrum formation rates between cultures grown in the two media. In conclusion, supplementation of the culture medium with serum enhances preantral follicle growth and antrum formation, and M199 is superior to NUSU23 for porcine preantral follicle culture in vitro.

• Clinical and Experimental Reproductive Medicine
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• 제46권4호
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• pp.152-165
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• 2019

Objective: This study aimed to examine the effect of vitrification on apoptosis and survival in human preantral follicles after thawing. Methods: This experimental study was conducted at an acute tertiary care hospital from March 2012 to April 2013. Ovaries were sliced into 5 × 5 × 1-mm pieces and divided into the following three groups: preantral follicle isolation, ovarian tissue vitrification-warming followed by follicle isolation, and immunohistochemistry of fresh ovarian tissue. For statistical analyses, the Student t-test, chi-square test, Kruskal-Wallis test, and Kaplan-Meier survival analysis were used. Results: A total of 161 preantral follicles (70% secondary) were collected from ovarian cortex tissue of six women between 30 and 37 years of age who underwent oophorectomy due to cervical cancer or breast cancer. There were no significant differences in the follicular morphology of fresh preantral follicles and vitrified follicles after thawing. The mean Fas ligand (FasL) mRNA expression level was 0.43 ± 0.20 (relative to β-actin) in fresh preantral follicles versus 0.51 ± 0.20 in vitrified follicles (p= 0.22). The mean caspase-3 mRNA expression level in fresh preantral follicles was 0.56 ± 0.49 vs. 0.27 ± 0.21 in vitrified follicles (p= 0.233). One vitrified-thawed secondary follicle grew and developed to an antral follicle within 6 days of culture. Conclusion: Vitrification did not affect preantral follicle morphology or mRNA expression of the apoptosis markers FasL and caspase-3. Further studies are required to establish whether vitrification affects the outcomes of in vitro culture and the maturation of preantral follicles.

• Clinical and Experimental Reproductive Medicine
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• 제32권3호
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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.

• 한국수정란이식학회지
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• 제20권1호
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• pp.63-69
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• 2005

본 연구는 개의 미성숙난자의 낮은 성숙율을 개선하기 위하여 자연발정온 개의 생식기관에 미성숙 난자를 이식하여 체내 배양 난자를 회수하여 회수된 난자의 핵 성숙율을 조사한 결과는 다음과 같다. 1. 미성숙난자를 24, 48 및 72시간 동안 체외성숙을 유도하였을 때 성숙시간에 따른 체외성숙율에는 유의적 차이는 인정되지 않았다. 2. 체내 배양 기간에 따른 난자의 회수율은 배양기간이 길어질수록 유의적으로 낮은 회수율을 보였으며, 배양기간이 증가할수록 난자의 생존율은 유의적으로 (P<0.05) 저하되었고 사멸된 난자의 비율도 또한 증가하였다. 3. 회수된 난자는 4, 5, 6일간의 체내 배양기간에 관계없이 대부분 GV상태로 (20/34; 6/15; 4/7)존재하였으며, 성숙된 난자는 체내 배양 4일째 회수된 난자에서 $5.8\%(2/34)$의 난자가 M II까지 발달한 것으로 조사되어 개의 미성숙난자의 체내 배양은 개 난자의 핵성숙을 개선하지 못하였다. 이상의 결과에서와 같이 개의 미성숙난자의 체내 배양은 M II 단계까지의 발달에 기여하지 못한 것으로 판단되며, 이는 개 미성숙난자의 핵발달은 난포에서의 자극과 활성화가 요구되는 것으로 판단된다.

• 한국발생생물학회지:발생과생식
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• 제1권2호
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• pp.157-164
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• 1997

To study the regulation of porcine follicular cell apostosis by gonadotropin, steroid, and nitric oxide, we analyzed DNA fragmentation, the hallmark of apoptosis, and nitrite production of porcine granulosa cells. Dissected indiidual follicles from ovary were separated in size (small, 2-3 mm; medium, 5-6 mm; large, 7-8 mm) and isolated granulosa cells were classified morpholocally as atretic or nonatretic. Nitrite concentration was measured by mixing follicular fluids with an equal volume of Griess reagent. Follicular nitric oxide (NO) concentration of healthy follicles was higher than that of atretic follicles. Apoptotic DNA fragmentation was suppressed in non-apoptotic granulosa cells. Follicular apoptosis was induced by androgen but prevented by gonadotropin in vitro. Apoptosis was confined to the granulosa cells. But it was not clear whether apoptosis of granulosa cells were isolated, incubated with or without gonadotropin, androgen and sodium nitroprusside (SNP), respectively at $37^{\circ}C$ for 24 hrs. Cultured granulosa cells were used to extract genomic DNA and culture media was asssayed for nitrite concentration. Nitrite production of culture media was increased, while apoptotic DNA fragmentation was suppressed in PMSG, hCG, testosterone+SNP and SNP treated groups. Nitrite concentration in culture media was decreased, but apoptotic DNA fragmentation was induced in testosterone treated group. These data suggest that NO production and apoptosis may be involved of granulosa cell apoptosis induced by testosterone.

• Asian-Australasian Journal of Animal Sciences
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• 제15권11호
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• pp.1559-1563
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• 2002

The ovarian follicular fluid was found to contain steroidogenesis stimulatory protein similar to albumin from human and buffalo. Therefore, the albumins from various species, commercial and purified, were studied for their steroidogenic effect on progesterone secretion by granulosa cells from buffalo ovaries, during culture. A dose of $20{\mu}g$ of bovine serum albumin was optimum to exhibit maximum progesterone secretion on day 6 of culture, in medium ($350{\mu}l$) containing $10^5$ cells. Among commercial albumins, chicken albumin showed highest effect on progesterone secretion, which was followed by albumins from goat, bovine, human, sheep and rat, respectively at day 6 of culture. The albumins were also purified from blood serum of buffalo, goat and rat using salt fractionation, ion-exchange chromatography, gel filtration and SDS-PAGE. The highest stimulatory effect on progesterone secretion was shown by albumin purified from buffalo blood serum and lowest by that from rat blood. Comparatively the buffalo and goat albumins were more biologically active than commercial albumins. The presence of some active molecules conjugated with freshly purified albumins may be responsible for better stimulatory effect.

• Clinical and Experimental Reproductive Medicine
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• 제38권3호
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• pp.135-141
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• 2011

Objective: Ovarian angiogenesis plays an important role in folliculogenesis. However, little is known about the expression of angiogenic factors during follicular development according to female age. Stromal cell derived factor-$1{\alpha}$ (SDF-$1{\alpha}$) plays a role in granulosa cell survival and embryo quality as an angiogenic chemokine. Leptin is also involved in folliculogenesis and angiogenesis. This study examined expression of SDF-$1{\alpha}$ and leptin, and their effects on the expression of angiogenic factors in the ovary during follicular development according to female age. Methods: Ovaries were collected from C57BL mice of two age groups (6-9 weeks and 24-26 weeks) at 6, 12, 24, and 48 hours after 5 IU pregnant mare's serum gonadotropin (PMSG) injection. The expression of ovarian SDF-$1{\alpha}$ and leptin mRNA was evaluated by RT-PCR. In the organ culture experiment, the ovaries were cultured in transwell permeable supports with Waymouth's medium treated with various doses of SDF-$1{\alpha}$(50-200 ng/mL) or leptin (0.01-1 ${\mu}g$/mL) for 7 days. Then, mRNA expression of vascular endothelial growth factor (VEGF), endothelial nitric oxide synthase (eNOS), and visfatin were examined in the cultured ovaries. Results: Expression of SDF-$1{\alpha}$ and leptin in the ovary was significantly lower in the aged mouse group compared to the young mouse group ($p$ <0.05). Expression of these two factors increased with follicular development after PMSG administration. SDF-$1{\alpha}$ treatment stimulated visfatin expression in a dose-dependent manner, while leptin treatment significantly increased eNOS expression. Conclusion: These results suggest that decrease of ovarian SDF-$1{\alpha}$ and leptin expression may be associated with aging-related reduction of ovarian function. SDF-$1{\alpha}$ and leptin may play a role in follicular development by regulating the expression of angiogenic factors in mouse ovaries.

• 한국동물학회지
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• 제19권2호
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• pp.85-94
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• 1976

난소 구성성분의 일부인 여포난자와 난구세포가 배양중인 정자의 운동능과 이동능에 미치는 영향을 미세관 배양법을 개량한 배양장치를 고안하여 조사한 결과 다음과 같은 결과를 얻었다. 1. 배양중인 정자의 운동능은 배양시간이 길어짐에 따라 혹은 정자의 농도를 희석시킴에 따라 점차 감소했다. 그러나 온도의 변화 $(22^\\circ C \\sim 36^\\circ C)$에는 거의 영향을 받지 않았다. 2. 난자는 정자의 운동능을 억제하는 경향을 보여주었다. 그러나 이 효과는 8시간 이후에는 나타나지 않았다. 3. 난구세포나 난자-난구 세포의 복합체가 모세관을 통과하는 정자의 이동능을 증진시키는 것으로 보아 난구세포가 어떤 정자유인요소를 분비하는 것으로 추정할 수 있었다.

• Clinical and Experimental Reproductive Medicine
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• 제31권3호
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• pp.155-168
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• 2004

Objective: Melatonin, which is secreted by pineal gland play an important role in the regulation of ovarian function via seasonal rhythm and sleep in most mammals. It also has a role in the protection of cells by removing toxic oxygen free radicals brought about by metabolism. In the present study, effects of melatonin on the mouse oocyte maturation were examined using two different culture conditions provided with 5% or 21% oxygen concentration. Material and Method: Immature mouse oocytes were obtained from the ovarian follicles of $3{\sim}4$ weeks old ICR strain mice intraperitoneally injected with 5 I.U. PMSG 44 hour before. Under stereomicroscope, morphologically healthy oocytes with distinct germinal vesicle (GV) were liberated from the graafian follicles and collected using mouth-controlled micropipette. They were then cultured for 17 hour at $37^{circ}C$, 5% $CO_2$ and 21% $O_2$ (95% air) or 5% $CO_2$, 5% $O_2$ and 90% $N_2$. New modified Hank's balanced salt solution (New MHBS) was used as a culture medium throughout the experiments. Effects of melatonin were examined at a concentration of $0.0001{\mu}M$, $0.01{\mu}M$ or $1.0{\mu}M$. For the prevention of spontaneous maturation of immature oocytes during culture, dibutyryl cyclic AMP (dbcAMP) and/or hypoxanthine were included in the medium. Results: Under 21% oxygen condition, oocytes cultured in the presence of $0.01{\mu}M$ melatonin showed a significantly higher maturation rates, in terms of germinal vesicle breakdown (95.0% vs 89.0%) and polar body formation (88.1% vs 75.4%), compared to those cultured with $0.0001{\mu}M$ or $1.0{\mu}M$ melatonin. However, no difference was observed in oocytes cultured under 5% oxygen whether they were treated with melatonin or not. In the presence of $0.01{\mu}M$ melatonin, oocytes either cultured under 21% or 5% oxygen exhibited no difference in the polar body formation (85.6% vs 86.7%). However, in the absence of melatonin, oocytes cultured under 21% oxygen exhibited lower polar body formation (74.7%). When oocytes were cultured in the presence of dbcAMP alone or with varying concentrations of melatonin, those treated with both compounds always showed better maturation, i.e., germinal vesicle breakdown and polar body formation, compared to those cultured with dbcAMP alone. At the same concentration of melatonin, however, oocytes exposed to 21% oxygen showed poor maturation than those to 5% oxygen. Similar results were obtained from the experiments using hypoxanthine instead of dbcAMP. Conclusion: Based upon these results, it is suggested that melatonin could enhance the meiotic maturation of mouse oocytes under 21% oxygen concentration, and release oocytes from the meiotic arrest by dbcAMP or hypoxanthine regardless of the concentration of oxygen, probably via the removal of oxygen free radicals.