• 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.

• Asian-Australasian Journal of Animal Sciences
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• 제20권5호
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• pp.693-700
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• 2007

The present study was conducted to examine the effect of different levels of essential and nonessential amino acid in NCSU-23 medium on the in vitro-produced porcine embryo as it develops from the zygote to the blastocyst stage. Four experiments were performed, each with a completely randomized design involving 5 to 8 replications of treatments. In order to know the effect of nonessential amino acids in NCSU-23 medium, 0, 5, 10 and $20{\mu}/ml$ MEM were supplemented there to, (Exp. 1) and the medium was supplemented with same level of essential amino acids (Exp. 2). The combined effect of nonessential (0, 5, 10 and $20{\mu}/ml$ MEM) and essential amino acids (0, 5, 10 and $10{\mu}/ml$ MEM) in NCSU-23 medium (Exp. 3), first 72 h with non-essential amino acids (at 0, 5, 10 and $20{\mu}/ml$ MEM), and last 4 d with essential amino acids with the same level as NEAA (Exp. 4) were examined. The embryo development was monitored and the quality of blastocysts was evaluated by counting the number of total cells and determining the ratio of inner cell mass (ICM) to trophoectoderm (TE) cells. When Eagle's nonessential amino acids (MEM) added to NCSU-23 medium, it significantly increased the likelihood of development to the 2- to 4-cell stage and subsequent blastocyst development. Supplementation of different levels of essential amino acids in the NCSU-23 medium decreased cleavage rate, rate of morula and blastocyst development and the number of ICMs. In the case of the combined effect of essential and nonessential amino acids, better and significant results were found for blastocysts, hatching blastocysts and for ICM numbers which were also dose dependent. With respect to the biphasic effect of nonessential and essential amino acids, nonessential amino acids increased cleavage whereas essential amino acids increased the total cell number. Neither the nonessential nor the essential group of amino acids, on their own, affected blastocyst cell number or the differentiation of cells in the blastocyst. In conclusion, this study determined the role of nonessential and essential amino acids in the culture of the porcine embryo and showed that the embryo requires different levels of amino acids as it develops from the zygote to the blastocyst stage.

• 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.

• Journal of Animal Science and Technology
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• 제55권4호
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• pp.237-247
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• 2013

This study was to examine single or combined in vitro effects of environmental endocrine disruptors on boar sperm characteristics, oxidative stress damage in sperm and development of porcine IVF embryos. Addition of various concentration of NP (10, 20, $30{\mu}M$), DBP (10, 50, $100{\mu}M$) and BPA (1, 5 or $10{\mu}g/ml$) on boar sperm characteristics such as percentages of sperm motility, viability, membrane integrity and mitochondrial activity were dose-dependently decreased within 3, 6 or 9 hr incubation period (p<0.05). The overall detrimental effects increased with incubation time increasement. NP, DBP and BPA showed the detrimental effects on sperm membrane and mitochondria of energy production organelles affecting cell viability with the dependancy of dose and incubation time. In combination effects, NP ($10{\mu}M$) + DBP ($10{\mu}M$) significantly decreased boar general sperm characteristics for 3 or 6 hr incubation period compared with control (p<0.05). When both of NP and DBP concentrations (NP; $30{\mu}M$, DBP; $100{\mu}M$) increase, the detrimental effects on sperm characteristics were larger than those of low concentration combination (p<0.05). The inhibitory effects of NP ($30{\mu}M$) + BPA ($10{\mu}g/ml$) on sperm characteristics were larger than those of NP ($10{\mu}M$) + BPA ($1{\mu}g/ml$) (p<0.05). DBP ($100{\mu}M$) + BPA ($10{\mu}g/ml$) decreased sperm characteristics compared with the low concentration combination (DBP $10{\mu}M$ + BPA $1{\mu}g/ml$, p<0.05). This result indicates the detrimental effects of both chemicals on sperm characteristics were dose dependent. Addition of NP ($30{\mu}M$) + DBP ($100{\mu}M$), NP ($30{\mu}M$) + BPA ($10{\mu}g/ml$), DBP ($10{\mu}M$) + BPA ($1{\mu}g/ml$) or DBP ($100{\mu}M$) + BPA ($10{\mu}g/ml$) significantly increased lipid peroxidation for 3 or 6 hr incubation period (p<0.05) compared with no addition control. NP (${\geq}20{\mu}M$) decreased the percentages of IVF embryo development from morulae and blastocyst stages (p<0.05) and its detrimental effects were dose-dependant. BPA 0, 1, 5 or $10{\mu}g/ml$ decreased significantly and dose-dependently the percentage of morulae plus and blastocysts (p<0.05). Combinations of DBP ($100{\mu}M$) plus NP ($30{\mu}M$) and DBP ($100{\mu}M$) plus BPA ($10{\mu}g/ml$) did not affect on morulae and blastocyst development, but NP ($30{\mu}M$) plus BPA ($10{\mu}g/ml$) has significant detrimental effect on embryo development at these stages (p<0.05). These overall results indicate that the partial detrimental effects on boar sperm characteristics and embryo development by NP, DBP, BPA or the combination of these chemicals might be due to the increasement of lipid peroxidation and free radical formation in the cell and there were no specific interaction effects on boar sperm and embryo degeneration among the combined treatments.

• 한국수정란이식학회지
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• 제20권2호
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• pp.123-128
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• 2005

본 연구는 돼지 난포란의 vitrification 동결 시 내동제의 종류 및 농도가 생존율에 미치는 영향과 수정 후 체외발생율을 조사하고자 수행하였다. 1.0, 15, 30 및 40시간 성숙 배양시킨 난포란을 vitirfication 동결보존 후의 MII로의 발생율은 각각 $56.7\%,\;53.3\%,\; 63.3\%,\;65.0\%$였으며, diploid로의 발생율은 $23.3\%,\;18.3\%,\;10.0\%,\;3.3\%$로서 대조군의 ME 단계의 $78.2\%$에 비해 낮게 나타났으며 diploid 단계의 $5.5\%$에 비해서는 높은 체외성숙율을 나타냈다. 체외발생율은 초기의 미숙 난포란일수록 높은 체외성숙율을 나타냈다. 2. 미성숙 난포란을 회수 후 0, 15, 30 및 40시간 성숙 배양시킨 난포란을 vitirfication 동결 후 융해하였을 때 생존율은 각각 $34.0\%,\;26.0\%,\;18.0\%,\; 12.0\%,\;10.0\%$로서 비동결 난포란의 $60.0\%$에 비해 낮게 나타났지만 비교적 양호한 생존율을 나타냈다. 3. 0, 15, 30 및 40시간 성숙 배양시킨 미성숙 난포란을 vitrification 동결 융해 후 수정시켰을 때 체외수정율은 $60.0\%,\;54.0\%,\; 48.0\%,\;38.0\%$였으며, 배반포로의 체외발생율은 각각 $26.0\%,\;18.0\%,\; 8.0\%,\;4.0\%$로서 비동결 대조군의 $78.0\%$와 $38.0\%$에 비해 낮은 체외수정율과 체외발생율을 나타냈다. 4. 돼지 난포란을 EDS와 ETS 액으로 vitrifiestion 동결운해 후 $0\~15$ 시간 배양한 다음 체외수정시켰을 때 체외 수정율과 발생율은 각각 $50.0\%,\;22.0\%$ 및 $46.0\%,\;18.0\%$로서 대조군의 $74.0\%$와 $38.0\%$에 비해 낮게 나타났다.

• Clinical and Experimental Reproductive Medicine
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• 제23권3호
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• pp.303-310
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• 1996

본 연구는 epidermal growth factor (EGF)가 처리된 돼지 체외성숙란의 채외수정 후 배발달을 조사하기 위하여 실시하였다. 난구세포가 치밀한 미성숙란은 TCM 199 배양액에 (1) 무처리군 (2) 10 ng/ml EGF 처리군 (3) 10 ${\mu}g/ml$ FSH와 10% FBS 처리군 (4) 10 ng/ml EGF와 10 ${\mu}g/ml$ FSH 그리고 10% FBS 처리군으로 나누어 42시간 동안 배양하였다. 실험 1은 이들 처리군을 수정 후 NCSU (0.4% BSA) 배양액에서 배양하여 후기 배발달을 조사하였다. 그 결과 (3)과 (4)처리군 (13.4, 18.3%)의 배반포로의 발달율이 (2) 처리군(5.2%, p < 0.005)보다 현저하게 높았지만, (2) 처리군의 경우 (1) 처리군(1.2%, p < 0.005)의 배반포로의 발달보다는 현저하게 높았다. 실험 2는 수정 이후 6일째 이들 처리군에서 생산된 배반포기배를 double staining (propidium iodide and bisbenzimide) 방법을 이용하여 세포수를 조사하였다. 그 결과 (4) 처리군 (58.80 ${\pm}$ 11.90)의 배반포의 total 세포수가 (2)와 (3) 처리군 (42.17 ${\pm}$ 9.97, 49.07 ${\pm}$ 9.77, p < 0.05)보다 현저하게 많았으며, 배반포의 ICM 수에 있어서도 (4) 처리군 (11.69 ${\pm}$ 5.56)이 (2)와 (3) 처리군 (5.00 ${\pm}$ 4.24, 6.77 ${\pm}$ 4.92, p < 0.05)보다 현저하게 많았다. 더 우기 (4) 처리군 (19.0 ${\pm}$ 1.6)의 total 세포수에 대한 ICM의 비율이 (2)와 (3) 처리군 (11.1 ${\pm}$ 3.0, 12.7 ${\pm}$ 2.1) 보다 높았다. 이들의 결과로서, EGF가 단독 처리된 돼지 체외성숙란은 비록 낮았지만 배반포로 발달할 수는 있으나, 체외성숙시 FSH와 FBS의 추가 첨가는 높은 배반포로의 발달과 total 세포수와 ICM의 증가를 가져올 수 있음을 알수 있었다.

• 한국수정란이식학회지
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• 제19권3호
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• pp.219-227
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• 2004

본 연구는 체외 성숙액인 TCM-199 배지를 기초로 무혈청첨가물인 PVA, PVP 및 pFF의 한정배지를 이용하여 돼지 미성숙난자의 체외성숙, 수정 및 배양 후 난자의 수정율과 배발달에 미치는 영향과 나아가 BSA 대체물로서의 이용 가능성을 알아보았다. 1. 무혈청 첨가물을 이용하여 난포란의 체외 성숙을 유기한 결과 PVA, PVP, pFF, BSA의 전체 분할율은 각각 82.4%, 78.6%, 89.4%, 90.0%로 나타났으며, GV, MI-MII율은 PVA 첨가시 각각 15.1%, 84.9%, PVP는 각각 26.5%, 73.5%, pFF는 각각 11.8%, 88.2%, BSA는 각각 11.1%, 88.9%로 PVA 혹은 pFF 첨가시에는 모두 BSA와 유사한 결과를 보였으나 PVP와는 유의적인 차이를 나타내었다 (P<0.05). 2. 체외 성숙된 난자를 수정시킨 후의 배발달율을 확인한 결과 전체 난할율은 PVA가 73%, PVP는 64.1% , pFF가 77.2%, BSA가 73%로 PVA와 pFF는 BSA와 유사하게 나타났으나, PVP는 그 발달율이 다른 처리군 들과 비교하여 유의적(P<0.05)으로 낮은 결과를 보였다. 3. 각 처리구간의 morulae와 blastocyst의 합을 비교한 결과 체외성숙 첨가물 중 PVA와 pFF는 각각 63%, 69%로 BSA(65%)와 유사한 결과를 나타내었으나 PVP는 54%로 유의적으로 낮은 결과를 나타내었다(P<0.05). 4. 전체 성숙율과 수정율을 비교해 본 결과 성숙 율은 PVA, pFF, BSA가 82.4%, 89.4%, 90.0%인 반면 PVP는 72.4%로 유의적으로(P<0.05) 낮은 결과를 보였고, 수정율은 pFF, BSA가 각각 87.1%, 89.1%로 PVA, PVP의 78.0%, 70.6%에 비해 각각 유의적(P<0.05)으로 높았다. 이상의 연구결과 돼지 난자의 체외성숙 및 배양 배지에 있어 PVA, pFF 첨가는 BSA 대체물로서 이용이 가능하나 PVP는 그 이용에 제한이 따를 것으로 판단된다.