• Reproductive and Developmental Biology
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• 제35권1호
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• pp.33-45
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• 2011

Heat shock protein 90 (Hsp90) is ATPase-directed molecular chaperon and affects survival of cancer cell. Inhibitory effect of Hsp90 by inducing cell cycle arrest and apoptosis in the cancer cell was reported. However, its role during oocyte maturation and early embryo development is very insufficient. In this study, we traced the effects of Hsp90 inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), on meiotic maturation and early embryonic development in pigs. We also investigated several indicators of developmental potential, including structural integrity, gene expression (Hsp90-, cell cycle-, and apoptosis-related genes), and apoptosis, which are affected by 17-AAG. Then, we examined the roles of Hsp90 inhibitor on viability of primary cells in pigs. Porcine oocytes were cultured in the NCSU-23 medium with or without 17-AAG for 44 h. The proportion of GV arrested oocytes was significantly different between the 17-AAG treated and untreated group (78.2 vs 34.8%, p<0.05). After completion of meiotic maturation, the proportion of MII oocytes was lower in the 17-AAG treated group than in the control group (27.9 vs 71.0%, p<0.05). After IVF, the percentage of penetrated oocytes was significantly lower in the 17-AAG treated group (25.2%), resulting in lower normal pronucleus formation (2PN of 14.6%). Therefore, the inhibition of meiotic progression by Hsp90 inhibitor played a critical role in fertilization status. Porcine embryo were cultured in the PZM-3 medium with or without 17-AAG for 6 days. In result, significant differences in developmental potential were detected between the embryos that were cultured with or without 17-AAG. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) showed that the number of containing fragmented DNA at the blastocyst stage increased in the 17-AAG treated group compared with control (7.5 vs 4.4, respectively). Blastocysts that developed in the 17-AAG treated group had low structural integrity and high apoptotic nuclei than those of the untreated control, resulting in decrease the embryonic qualities of preimplantation porcine blastocysts. The mRNA expressions of cell cycle-related genes were down-regulated in the 17-AAG treated group compared with control. Also, the expression of the pro-apoptotic gene Bax increased in 17-AAG treated group, whereas expression of the anti-apoptotic gene Bel-XL decreased. However, the expression of ER stress-related genes did not changed by 17-AAG. Cultured pESF cells were treated with or without 17-AAG and used for MIT assay. The results showed that viability of pESF cells were decreased by treatment of 17-AAG ($2{\mu}M$) for 24 hr. These results indicated that 17-AAG decreased cell proliferation and increased cell death. Expression patterns Hsp90 complex genes (Hsp70 and p23), cell cycle-related genes (cdc2 and cdc25c) and apoptosis-related genes (Bax and Bcl-XL) were significantly changed by using RT-PCR analysis. The spliced form of pXbp-1 product (pXbp-1s) was detected in the tunicamycin (TM) treated cells, but it is not detected in 17-AAG treated cells. In conclusion, Hsp90 appears to play a direct role in porcine early embryo developmental competence including structural integrity of blastocysts. Also, these results indicate that Hsp90 is closely associated with cell cycle- and apoptosis-related genes expression in developing porcine embryos.

• 한국발생생물학회지:발생과생식
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• 제9권1호
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• pp.49-52
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• 2005

도축된 돼지의 난소에서 난포란을 채취하여 체외 성숙시킨 후 인위적으로 활성화시켜 이배체 배발달을 유기하기 위해 cytochalasin B를 2.5, 5.0, $7.5\;{\mu}g/mL$ 농도로 3, 5, 7 시간 처리한 후 NCSU23 배양액으로 7일간 배양하여 배발달율에 미치는 영향을 검사하였다. 체외 성숙된 돼지 난모세포를 활성화 처리하여 2일째 분할율을 관찰한 결과 각 처리의 분할율은 $39.0{\sim}48.9%$로 나타났으며, 처리별로 유의차가 없었다. 7일간 배양하여 상실배기 이상으로 발달한 난자들의 처리별 발달율 차이를 관찰한 결과는 cytochalasin B를 $5.0\;{\mu}g/mL$ 농도로 3시간 처리구(19.7%)에서 $2.5\;{\mu}g/mL$ 농도로 3, 5시간 처리한 구(9.4%)의 배발달율에 비해 유의적으로 높은 결과를 얻었다. 시간별과 농도별로 분석한 결과 $2.5\;{\mu}g/mL$ 처리구가 5.0, $7.5\;{\mu}g/mL$ 처리구들보다 유의적으로 낮은 배발달율을 보였다. 배반포기까지 발달한 난자들의 처리별 발달율 차이를 관찰한 결과는 cytochalasin B를 $5.0\;{\mu}g/mL$ 농도로 3시간과 5시간 처리구들의 배반포기 발달율은 9.4%와 9.0%로 $2.5\;{\mu}g/mL$ 농도로 3시간 처리구의 배발달율인 0%보다 유의적으로 높은 배발달율을 보였다. 시간별과 농도별로 분석한 결과 농도에 따라 $5.0\;{\mu}g/mL$ 처리구가 2.5와 $7.5\;{\mu}g/mL$ 처리구들보다 유의적으로 높은 배반포기 배발달율을 보였으며, 3, 5, 7시간 처리에 따른 유의적인 차이는 없었다. 이상의 결과로 돼지 난모세포를 65시간 체외 성숙 후 활성화 처리할 때 cytochalasin B $5.0\;{\mu}g/mL$로 $3{\sim}5$ 시간 처리하는 것이 가장 높은 배반포 발달율을 얻을 수 있었으며, 처리 농도가 배발달에 유의적인 영향을 미치는 것을 확인하였다.

• Reproductive and Developmental Biology
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• 제29권3호
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• pp.169-174
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• 2005

본 연구는 배양액의 종류에 따른 돼지 난자의 성숙 및 단위발생란의 배반포 형성율을 검토하였으며, 배양액의 삼투압과 발달 시기에 따른 배양액의 삼투압 변화가 돼지 단위발생란의 발달에 미치는 영향을 검토하였다. 실험 1에서 난포란을 NCSU-23 mWM 및 mKRB에 각각 성숙배양한 결과 성숙률은 $62.1\~71.3\%$로 배양액에 따른 차이가 없었다. 실험 2에서는 각각의 배양액으로 성숙된 난자를 활성화 처리 후 동일한 배양액으로 6일간 배양하여 발달율을 검토한 결과, 배반포 발육율은 NCSU-23에 배양 시 $22.9\%$로 타 그룹($0\~0.6\%$)보다 유의적으로 높게 나타났다(P<0.05). 실험 3에서는 단위발생란을 NaCl 안에 의해 256, 280 및 300 mOsmol(mOsm)로 조정한 NCSU-23에 6일간 배양한 결과, 배반포 발육율은 $11.0\~14.4\%$로 실험군 간에 유의적인 차이는 없었으나 삼투압이 낮을수록 난자의 fragment 비율이 높게 나타났다(P<0.05). 실험 4에서는 단위발생란을 삼투압이 조정된 세 종류의 NCSU-23에 48시간 배양한 후 삼투압이 높거나 낮은 NCSU-23으로 옮겨 4일간 추가 배양한 결과 배반포 형성율은 배양 48시간 후에 배양액의 삼투압을 낮춰 주었을 때($21.0\%$)가 높여 주었을 때 ($11.8\%$) 보다 유의적으로 높게 나타났다(P<0.05).본 실험의 결과는 돼지 단위발생란의 발육이 배양액의 종류 및 삼투압에 의해 영향을 받으며, 배양액의 삼투압은 돼지 단위발생란의 발육 단계별로 영향을 주어, 초기에는 높은 삼투압의 배양액에서 배양하고 일정 시간 후 낮은 삼투압의 배양액으로 배양함으로써 발육이 증진될 수 있음을 시사한다.

• Journal of Animal Science and Technology
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• 제48권6호
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• pp.797-804
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• 2006

본 연구에서는 동결 보호제 EG l에 sucrose 첨가 농도에 따른 생존성의 실험의 결과를 요약하면 다음과 같다. 1.5 M EG와 1.8 M EG 만을 이용하여 동결융해 후 생존서의 조사한 결과 71.1%와 70.2%로 각각 나타났다. 총세포수에 있어서도 127±1.3개와 124±1.6개로 생존율과 총세포수에 있어서도 두 그룹간에는 유의적인 차이를 보이지 않았다. 1.5 M EG와 1.8 M EG에 0.1 M sucrose를 각각 첨가한 후 동결 보존하여 융해 하였을 때 생존율과 총세포수 조사 결과는 1.5 M EG에 0.1 M sucrose 처리구가 73.6% 그리고 1.8 M EG 에 0.1 M sucrose 첨가군은 76.9%의 결과를 보였으며 총세포수 에 있어서도 118±1.2 와 112±1.2 개의 결과를 보여 생존성과 총세포수에 있어서도 두 처리군 모두 유의적인 차이를 나타내지 않았으나 1.5 M EG 처리구에서 총세포수는 다소 높은 경향을 보였다. 1.5 M EG 와 1.8 M EG에 0.3 M sucrose를 첨가하여 각각 생존성과 총세포수 조사 결과는 70.8%와 88.7%의 생존율을 나타내어 1.8 M EG 에 0.3 M sucrose 처리구가 유의적으로(P<0.05) 높은 결과를 보였다. 따라서 소 체외수정란을 conventional slow-freezing 방법으로 동결 보존할 경우는 1.8 M EG 동결보호제에 0.3 M sucrose를 병행하여 사용하는 방법이 수정란을 최상의 상태로 유지할 수가 있어 수정란이식에 적용할 경우 효과적일 것으로 사료된다.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2001년도 춘계학술발표대회
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• pp.8-8
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• 2001

Mouse follicles require the addition of gonadotropins (Gns) to complete maturation and ovulation of oocyte and antrum formation of follicles in vitro. However, we tried examination of in vitro growth of mouse pre-antral follicles in medium without Gns and physiological factors. And also, pre-antral follicles were isolated from ovaries by mechanical method. Our present studies were conducted to evaluate on the growth of follicles and intra-follicular oocytes and antrum formation in vitro of mouse pre-antral follicles in two different media. Pre-antral follicles (91-120${\mu}{\textrm}{m}$) were isolated mechanically by fine 30G needles not using enzymes from ovary of 3-6 weeks old female ICR mice. Isolated pre-antral follicles were cultured in 20 ${mu}ell$ droplets of TCM (n=17; follicles: 107.8 $\pm$ 1.58 ${\mu}{\textrm}{m}$; oocytes: 59.9$\pm$1.2 ${\mu}{\textrm}{m}$) or MEM (n=12; follicles: 109.3$\pm$2.53 ${\mu}{\textrm}{m}$; oocytes: 55.4 $\pm$1.6${\mu}{\textrm}{m}$) under mineral oil on the 60mm culture dish. All experimental media was supplemented with 10% FBS but without Gns and/or physiological factors. Pre-antral follicles were individually cultured in drops for 8 days. Antrum formation and growth of pre-antral follicles and intra-follicular oocytes were evaluated using a precalibrated ocular micrometer at $\times$200 magnifications during in vitro culture. Results between different groups were analyzed using combination of Student's t-test and Chi-square, and considered statistically significant when P<0.05. Antrum formation of pre-antral follicles had started in two culture media on day-2. On day-8, antrum formation had occurred in 58.3%(7/12) of pre-antral follicles cultured in MEM, but only in 23.5% (4/17) of those cultured in TCM (P=0.0364). Growth of pre-antral follicles and intra-follicular oocytes were observed on day-4 and -8. On day-4, follicular diameters was similar (P=0.1338) in TCM (119.4$\pm$2.58 ${\mu}{\textrm}{m}$) and MEM (125.4$\pm$4.52 ${\mu}{\textrm}{m}$). However, on day-8, diameters of pre-antral follicles cultured in MEM (168.9$\pm$17.29 ${\mu}{\textrm}{m}$) was significantly (P=0.0248) bigger than that in TCM (126.7$\pm$4.28 ${\mu}{\textrm}{m}$). On day-4 and -8, diameters of intra-follicular oocytes were similar TCM (67.1$\pm$1.3 and 72.4$\pm$0.9${\mu}{\textrm}{m}$) and MEM (65.2$\pm$1.7 and 73.3$\pm$1.5 ${\mu}{\textrm}{m}$), respectively. We can conform that medium not supplemented with Gns and/or physiological factors can be used for in vitro antrum formation and growth of mouse pre-antral follicles and intra-follicular oocytes. In conclusion, MEM supplemented with FBS can be used for growth in vitro of mouse pre-antral follicles isolated mechanically.

• Clinical and Experimental Reproductive Medicine
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• 제30권3호
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• pp.207-215
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• 2003

Objective: To compare the pregnancy outcomes after in vitro fertilization with intracytoplasmic sperm injection (IVF-ICSI) between obstrucvtive and non-obstrucvtive azoospermia. Methods: From January 1994 to December 2002, 524 patients with obstructive azoospermia (886 cycles) and 163 patients with non-obstructive azoospermia (277 cycles) were included in this study. Microsurgical epididymal sperm aspiration (MESA) or testicular sperm extraction (TESE) in obstructive azoospermia and TESE in non-obstructive azoospermia were perfomed to retrieve sperm, which was used for ICSI and then fertilized embryos were transferred. The results of ICSI - fertlization rate (FR), clinical pregnancy rate (CPR), clinical abortion rate (CAR) and delivery rate (DR) - were statistically analysed in obstructive versus non-obstructive azoospermia. Results: There were no differences in the number of retrieved oocytes, injected oocytes for ICSI and oocyte maturation rate. FR was significantly higher in obstructive than non-obstructive azoospermia (71.7% vs. 61.1%, p<0.001). There was no difference in CPR per embryo transfer cycle. After pregnancy was established, however, CAR was significantly higher in non-obstructive than obstructive azoospermia (25.6% vs. 12.5%, p=0.004). DR per clinical pregnancy cycle was significantly higher in obstructive than non-obstructive azoospermia (78.0% vs. 64.4%, p=0.012). In the karyotype ananlysis of abortus, abnormal karyotypes were found in 75.0% (6/8) of obstructive and 55.6% (5/9) of non-obstructive azoospermia. Conclusion: Our data show significantly higher FR in obstructive than non-obstructive azoospermia. Though there was no differrence in CPR, CAR was significantly higher in non-obstructive than obstructive azoospermia. The abortion may be related to the abnormal karyotype of embryo, but further investigations are necessary to elucidate the cause of clinical abortion in azoospermia.

• Current Research on Agriculture and Life Sciences
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• 제8권
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• pp.45-50
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• 1990

본 실험은 성선(性腺)자극호르몬과 우태아혈청(牛胎兒血淸) 첨가가 돼지 난포란(卵胞卵)의 체외성숙(體外成熟) 및 체외수정(體外受精)에 미치는 영향을 조사하기 위하여 실시하였다. 미경산돈(未經産豚)(체중 80~90kg)의 난소(卵巢)를 도살된 직후에 절취하여 $37{\sim}39^{\circ}C$의 보온병에 담아 실험실로 운반하여 난포(卵胞)직경이 3~5mm되는 것만을 골라 난포(卵胞)를 찔러서 난포란(卵胞卵)을 채취하였다. 성숙모돈(成熟牡豚)(체중 130~150kg)의 정소상체미부정자(精巢上體尾部精子)를 $4{\times}10^8cells/m{\ell}$ 농도로 희석하여 체외수정(體外受精)에 이용하였다. 본 실험의 경과를 요약하면 다음과 같다. 1. m-KRB와 10% FCS를 m-KRB에 첨가한 경우 성숙율(成熟率)은 82.37%이며, 10% FCS가 첨가된 배양액에 PMSG, hCG 그리고 PMSG와 hCG를 각각 $10IU/m{\ell}$ 첨가한 경우 66, 58, 68%로서 성숙율(成熟率)이 향상되었다. 2. 난구세포(卵丘細胞)의 팽화(膨化)는 m-KRB와 10% FCS가 첨가된 배양액에서 일어나지 않았으나 10% FCS가 첨가된 배양액에 PMSG, hCG 그리고 PMSG와 hCG를 각각 $10IU/m{\ell}$ 첨가시 92, 13, 91%로서 팽화율(膨化率)이 향상되었다. 3. m-KRB에서 성숙된 난포란(卵胞卵)의 체외수정(體外受精)에서 정자침입율과 웅성전핵(雄性前核) 형성율은 각각 93.7%였으나 FCS와 성선(性腺)자극호르몬을 첨가한 경우 각각 100, 80%로서 웅성전핵(雄性前核) 형성율이 향상되었다.

• Clinical and Experimental Reproductive Medicine
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• 제31권2호
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• pp.105-110
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• 2004

Objectives: Despite severe oligospermia, males with Y chromosome microdeletion can achieve conception through ICSI (Intracytoplasmic Sperm Injection). However, ICSI may not only result in the transmission of microdeletions but also the expansion of deletion to the offspring. The purpose of this study was to screen vertical transmission, expansion of microdeletions and de novo deletion in male fetuses conceived by ICSI. Materials and Methods: A total of 32 ICSI treated patients with their 33 (a case of twin) male fetuses conceived by ICSI were used to make this study group. Sequence-tagged sites (STSs)-based PCR analyses were performed on genomic DNA isolated from peripheral blood of fathers and from the amniocytes of male fetuses. Ten primer pairs namely, sY134, sY138, MK5, sY152, sY147, sY254, sY255, SPGY1, sY269 and sY158 were used. The samples with deletions were verified at least three times. Results: We detected a frequency of 12.5% (4 of the 32 patients) of microdeletions in ICSI patients. In 4 patients with detected deletions, two patients have proven deletions on single STS marker and their male fetuses have the identical deletion in this region. Another two patients have two and three deletions, but their male fetuses have more than 3 deletions which include deletions to their father's. Meanwhile, seven male fetuses, whose fathers were analyzed to have all 10 STS markers present, have deletions present in at least one or more of the markers. Conclusions: Although the majority of deletions on the Y chromosome are believed to arise de novo, in some cases a deletion has been transmitted from the fertile father to the infertile patient. In other cases the deletion was transmitted through ICSI treatment, it is likely that one sperm cell is injected through the oocyte's cytoplasm and fertilization can be obtained from spermatozoa. Our tests for deletion were determined by PCR and our results show that the ICSI treatment may lead to vertical transmission, expansion and de novo Y chromosome microdeletions in male fetuses. Because the sample group was relatively small, one should be cautious in analyzing these data. However, it is important to counsel infertile couples contemplating ICSI if the male carries Y chromosomal microdeletions.

• Clinical and Experimental Reproductive Medicine
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• 제26권2호
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• pp.137-148
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• 1999

Purpose of the present study was to find the optimal ovulation induction medicine for the maturation and development of immature oocytes and culture media for 2-cell embryos in the mouse model. ICR female mouse aged 6 to 8 weeks, were stimulated with 5 IU PMSG injection. At 47 to 50 hour post-PMSG injection, ovaries were dissected out and oocytes-cumulus complexes were punctured. The oocyte-cumulus complexes were cultured in media containing various ovulation induction medicine, CC, HMG and Metrodin for 18 hours. Female ICR mice were stimulated with 5 IU PMSG and 48 hours later were injected 5 IU of hCG, then female and male mice were mated. At 48 hour post-hCG injection, oviducts were dissected out and 2-cell embryos were flushed. The 2-cell embryos were cultured in various media, Ham's F-10 media of milli-Q water $(3^{\circ})$, Ham's F-10 media of HPLC (high performance liquid chromatography, Baxter) water, Medicult media, HTF (human tubal fluid) media for 96 hours. The results were as follows. 1. When the oocytes-cumulus complexes were cultured in $10^{-9}{\mu}g/ml{\sim}10^{-8}{\mu}g/ml$ of CC, those were suppressed in meiotic maturation $(28.2{\sim}33.7%)$. Whereas the oocytes-cumulus complexes were cultured in $10^{-7}{\mu}g/ml{\sim}10^{-4}{\mu}g/ml$, these were not effected in meiotic maturation $(54.5{\sim}72.7%)$. 2. When the oocytes-cumulus complexes were cultured in $10^{-4}{\mu}g/ml{\sim}10^{-1}{\mu}g/ml$ of Metrodin, those were suppressed in meiotic maturation $(35.7{\sim}41.5%)$. Meanwhile the oocytes-cumulus complexes were cultured in $10^{-7}{\mu}g/ml{\sim}10^{-5}{\mu}g/ml$, those were not effected in meiotic maturation $(54.2{\sim}70.3%)$. 3. When the oocytes-cumulus complexes were cultured in $10^{-5}{\mu}g/ml{\sim}10^{-4}{\mu}g/ml$ of HMG, those were suppressed in meiotic maturation $(48.2{\sim}50.4%)$. As being cultured in $10^{-7}{\mu}g/ml{\sim}10^{-6}{\mu}g/ml$, increased in meiotic maturation $(75.8{\sim}80.7%)$. 4. When the 2-cell embryos were cultured in Ham's F-10 media of milli-Q water $(3^{\circ})$, Ham's F-10 media of HPLC (high performance liquid chromatograpy, Baxter) water, Medicult media, HTF (human tubal fluid) media, developmental rates to blastocyst and hatching for 96 hour were 50.0%, 45.2%, 71.5% and 95.6%, respectively.

• 한국수정란이식학회지
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• 제22권1호
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• pp.1-7
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• 2007

본 연구는 체외 성숙된 난자와 동결 융해 정자를 이용한 돼지의 체외 수정 과정에서 난구 세포의 존재가 정자 침투율, 웅성전핵 형성률 그리고 후기배로의 체외 발육에 미치는 영향을 알아보기 위하여 수행되었다. 돼지 난소로부터 난자-난구세포 복합체를 채취하여 eCG/hCG, 10% 돼지 난포액, epidermal growth factor 등이 첨가된 TCM 199 배양액에서 44시간 배양하여 체외 성숙을 유도하였다. 성숙 배양 후 난구 세포를 제거한 난자와 난구 세포가 부착되어 있는 난자를 돼지 동결 융해정액을 이용하여 5mM caffeine과 10mM calcium chloride를 함유한 mTBM배양액에서 8시간 체외 수정하였다. 체외 수정 후 난자를 고정, 염색하여 정자 침투율과 웅성전핵 형성률을 조사하였고(실험 $1{\sim}3$) 일부 수정란을 North Carolina State University-23 배양액에서 체외 수정 후 156시간 배양하여 후기배로의 발육능을 검토하였다(실험 3). 실험 1에서는 정자 농도를 $7.5{\times}10^5/ml$로 조정하여 나화 난자와 난구 세포 부착난자에서 정자 침투율 및 웅성전핵 형성률을 조사하였다. 실험 2에서는 난구 세포 부착 난자의 체외 수정에 적합한 정자 농도를 구하기 위해 2, 3, 4, 및 $5{\times}10^6/ml$의 농도로 난자를 수정한 후 정자 침투율 및 웅성전핵 형성률을 조사하였다. 실험 3에서는 나화 난자 및 난구 세포 부착 난자를 각각 $7.5{\times}10^5/ml$의 정자 농도로 체외 수정한 후 후기배로의 발육률을 조사하였다. 실험 1의 결과 정자 침투율은 나화 난자에 비해 난구 세포 부착 난자에서 유의적으로 감소되었다(35.2% vs. 77.4%; p<0.01). 실험 2에서 다양한 정자 농도에 의한 정자 침투율과 정상 수정률을 바탕으로 판단했을 때 $4.6{\times}10^6/ml$의 정자 농도가 다른 정자 농도에 비해 난구 세포부착 난자의 체외 수정에 적합한 것으로 나타났다. 체외 수정과정에서 난구 세포 부착된 상태로 수정된 난자는 나화 난자에 비해 유의적으로(p<0.05) 높은 분할률(48.8% vs. 58.9%), 배반포 형성률(11.0% vs. 22.8%)과 배반포 세포수$(22{\pm}2\;vs.\;29{\pm}2)$를 나타내었다. 본 연구의 결과로부터 돼지의 체외 수정과정에서 난구 세포의 존재는 정자 침투를 저해하지만 분할률, 배반포 형성률 및 배반포의 세포수를 증가시키는 것으로 사료된다.