• Reproductive and Developmental Biology
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• 제34권3호
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• pp.169-173
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• 2010

Correlations between cumulus cells and germinal vesicle (GV) chromatin configuration were examined in porcine oocytes. Cumulus-oocyte complexes (COCs) were collected from 2~6 mm follicles and divided into three categories according to cumulus cell morphology. "A" group was compacted COCs with more than three cumulus cell layers. "B" group was COCs with less cumulus cell layers than "A" group. "C" group was COCs with one or less layer of cumulus cells. Cumulus cells were removed 0.1% hyaluronidase, and denuded oocytes were stained with Hoechst 33342. GV chromatin configuration was classified into GV-Con and GV-Dis. GV-Con meant that a nucleus was surrounded by condensed chromatin in a ring. GV-Dis meant that filamentous chromatin clumps were distributed in nucleus. The proportion (80.2%) of GV-Con in "A" group was significantly higher than "B" (62.0%) or "C" (44.9%). The proportion (55.1%) of GV-Dis in "C" group was significantly higher than "A" (19.8%) or "B" (38.0%). The meiotic competence of COCs was examined after 44 h culture. The proportion (90.0%) of oocytes reaching to metaphase II (M-II) in "A" group was significantly higher than "B" (76.5%) or "C" (45.5%). In conclusion, oocytes with good quality cumulus cell layers are synchronized early GV stage, and early GV stage is important for meiotic competence in pigs.

• 한국수산과학회지
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• 제38권2호
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• pp.94-99
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• 2005

The gametogenesis of the surf clam, Tresus keenae, were investigated by SEM and TEM. Both the testis and the ovary had follicle tubes surrounded by inter-tubal tissue composed of adipogranular cells that provided storage function. In the vitellogenic oocyte, lipid droplets and lipid yolk granules were found in the vacuoles formed by the Golgi apparatus. Proteid yolk granules were formed by the endoplasmic reticulum and cortical granules in the cytoplasm during vitellogenesis. The mature sperm was primitive and resembled a jar with a cover. The sperm heads were approximately $2.00-2.30 {\cal}um$. The acrosomal rod projected in front of the acrosome. In addition, four large mitochondria were in the midpiece.

• 한국가축번식학회지
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• 제20권1호
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• pp.9-17
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• 1996

This study was conducted to investigate freezability of in vitro and in vitro matured rabbit oocytes, possibility of NT using frozen-thawed unfertilized oocytes, and NT efficiency by zona-slit micromanipulation. After freezing of in vitro matured oocytes, 33 to 49% of oocytes appeared normal morphology and 1.0M DMSO and 1.5M glycerol showed slightly high survival rate, but there was no difference in survival between two cryoprotectants. Freezability of in vitro matured oocytes was low in 1.5M glycerol and more sensitive to freezing. Efficiency of enucleation and fusion rate in method B was higher than that in method A and no difference in this efficiency was between 3 groups of oocytes in method B. Cleavage rate and developmental capacity to M＋B stage of fused embryos derived from frozen oocytes was greatly lower than that from fresh oocytes, respectively(39.1% : 79.5% ; 3.1% : 19.3%) and there was no difference in cleavage rate between DC voltages in two group oocytes. Additional incubation in cytochalasin B after electrical stimulation did not affect embryo development. In conclusion, it is suggested that enucleation and nucelar transfer by slitting of zona is more effective method in rabbit and that further study on optimum freezing conditions for in vitro matured oocytes is necessary to use as recipient oocytes.

• 한국수정란이식학회지
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• 제16권1호
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• pp.1-5
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• 2001

A total of 92 unfertilized human oocytes were treated with ethanol (EtOH), calcium ionophore A23187 (CI) or electric pulse (EP) for activating pronuclear formation and subsequent development. In Experiment 1, there was a significant (P=0.0001) treatment effect on the activation of unfertilized oocytes. No spontaneous activation was occurred in the control, but activation treatments induced PN formation with various efficacy. More unfertilized oocytes (UFOs) were activated after EtOH or EP treatment than after CI treatment. EP was as effective (63.6 %) as EtOH, but fragmentation was observed in 43% of UFOs activated by EP. Proportion of UFOs that formed presumptive haploid PN (2 PNs+1 PB or 1 PN +2 PBs) was 33.3, 0 and 28.6% after EtOH, CI and EP treatments, respectively. In Experiment 2, a significant (P=0.0362) effect of immature oocytes (IOs) status on activation was fecund. IOs at the GVBD-MI oocytes had higher potential to form PN than those at the GV stage or with abnormal morphology (25 vs. 77.8%). The results of this study clearly demonstrated that the treatment of 10% ethanol for 5 min effectively induced the activation of UFOs. IOs could form pronucleus with high efficacy by ethanol treatment, as long as they grew beyond the GVBD stage.

• 한국수정란이식학회지
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• 제32권3호
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• pp.81-86
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• 2017

The Somatic cell nuclear transfer (SCNT) method can be applied to various fields such as species conservation, regenerative medicine, farming industries and drug production. However, the efficiency using SCNT is very low for many reasons. One of the troubles of SCNT is that it is highly dependent on the researcher's competence. For that reason, four somatic cell nuclear injection methods were compared to evaluate the effect of hole-sealing process and existence of cytochalasin B (CB) on efficiency of murine SCNT protocol. As a results, the microinjection with the hole-sealing process, the oocyte plasma membrane is inhaled with injection pipette, in HCZB with CB was presented to be the most efficient for the reconstructed in SCNT process. In addition, we demonstrated that the oocytes manipulated in Hepes-CZB medium (HCZB) with CB does not affect the developmental rate and the morphology of the blastocyst during the pre-implantation stage. For this reason, we suggest the microinjection involving hole-sealing in HCZB with CB could improve SCNT process efficiency.

• 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.

• Clinical and Experimental Reproductive Medicine
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• 제29권1호
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• pp.57-66
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• 2002

Objective : This study was designed to investigate the interrelationship and clinical usefulness of sperm morphology by strict criteria (SM), acrosome reaction following ionophore challenge test (ARIC) and sperm penetration assay (SPA) using zona-free hamster ova as prognostic factors in in vitro fertilization. Materials and Methods: Semen samples were provided by 83 patients undergoing IVF. We first evaluated the differences between normal fertilization group and poor fertilization group on three andrologic tests. Secondly, we analyzed the relationship between the three andrologic tests and in vitro fertilization on IVF settings. Finally, we evaluated the effectiveness of the three andrologic tests as the prognostic indicators for fertilizing ability. Results: The fertilization rate of all men in the poor fertilization group was less than 30%; but there was no evidence that this poor fertilization was due to oocyte defects. The results of three andrologic tests were significatly higher in normal fertilization group. Fertilization rate (%) in vitro was highly correlated (p<0.001) with % normal sperm by SM, ARIC value (%), and SPA result. By using Receiver-Operator-Characteristic curve (ROC), we evaluated the effectiveness of these three tests. The sensitivity and specificity of SM, ARIC test and SPA in predicting fertilization potential in IVF setting were 76% and 75%, 84% and 90%, and 76% and 95%, respectively. Conclusion: Our data suggest that the three andrologic tests can be reliable tools as prognostic factors of sperm fertilizing ability. Among these test, ARIC test and SPA gave more accurate information on fertilizing capacity. ARIC test was shown to have a predictive value for fertilizing ability comparable to that of SPA that appears to be a simple and cost-effective addition to current andrology laboratory. Combined application of these three tests may give more information on predicting sperm fertilizing capacity.

• 한국수정란이식학회지
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• 제18권1호
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• pp.27-33
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• 2003

본 연구는 소형 개의 불임 해결과 체외수정란을 생산하기 위한 방안의 하나로써 난자의 형태, 번식주기, 배양시간 및 활성화 처리가 난포란의 체외성숙 및 체외발생에 미치는 영향을 조사하였다. 1. 신선, salt 및 4$^{\circ}C$에 보존한 난소로부터 채취한 난구세포부착 난자와 나화 난자로 각각 체외수정시켰을 때 16세포기로의 발생율은 14.3%, 5.0% 및 7.5%, 2.8%, 5.7% 및 0.0%로써 난구세포 부착난자군의 체외발생율이 나화 난자군에 비해 높게 나타났다. 2. 발정주기를 inactive, follicular, luteal 단계로 구분하여 채취한 난포란을 각각 체외배양시켰을 때 GV 및 MII로의 발생율은 11.3%와 9.4%, 50.7%와 26.7%, 16.9%와 13.8%였고, 16세포기로의 체외발생율은 0.0%, 10.7%, 1.5%였다. 3 신선한 난구세포 부착 난자를 각각 24, 32, 48시간 성숙배양 후 체외수정시켰을 때 분할율은 8.6%, 15.8%, 23.5%였으며, 16세포기로의 체외발생율은 각각 0.0%, 5.3%, 11.8%로써 48시간 배양군이 가장 높은 발생율을 나타냈다. 4. 활성화 처리 및 비활성화 처리 난자를 각각 체외수정시켰을 때 분할율은 각각 42.5%, 22.2%였고 16세포기로의 체외발생율은 각각 15.0%, 6.7%로써 활성화 처리 난자군이 비활성처리 난자군에 비해 높은 발생율을 나타냈다.

• Asian-Australasian Journal of Animal Sciences
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• 제17권5호
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• pp.612-616
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• 2004

This study was carried out to compare the semen characteristics, frozen-thawed sperm viability and testosterone concentration and in vitro fertilization (IVF) and development of in vitro matured pig oocytes between two Yorkshire boars. Semen and blood samples were collected once per week from October to November 2002 from two adult Yorkshire boars at 18 months of age with 170 kg body weight. Sperm were deep frozen in 5 ml maxi-straws with lactose-egg yolk and N-acetyl-D-glucosamine (LEN) diluent and stored in liquid nitrogen. Blood samples were obtained at 10 a.m. by inserting a 21 gauge, hypodermic needle attached to 10 ml syringe into surface veins in the ear. The concentration of testosterone was determined by Competitive Enzyme Immunoassay. Ovaries were collected from prepubertal gilts at a local slaughter house. Cumulus oocyte complexes were aspirated from antral follicles (3 to 6 mm in diameter). The medium used for oocyte maturation was modified TCM 199. After about 22 h of culture, oocytes were cultured without cysteamine and hormones for 22 h at $38.5^{\circ}C$, 5% $CO_2$ in air. For IVF, one frozen 5 ml straw was thawed at $52^{\circ}C$in 40 sec and was diluted with 20 ml Beltsville thawing solution at room temperature. Sperm were washed 2 times in mTLP-PVA and inseminated without preincubation after thawing. Oocytes were inseminated with $2{\times}10^7$/ml sperm concentration. Oocytes were coincubated for 6 h in 500 ${\mu}$l mTBM fertilization medium. At 6 h after IVF, oocytes were transferred into 500 ${\mu}$l NCSU-23 culture medium for further culture of 48 and 144 h. There were no significant differences in the semen volume, motility, normal acrosome morphology and sperm concentration of raw semen between A and B of Yorkshire boar. However, motility and normal acrosome of boar A were higher than those of boar B at 0.5, 2, 3, 4, 5 and 6 h incubations of frozen-thawed sperm. Testosterone concentration (3.75 ng/ml) of boar A was higher than that (2.34 ng/ml) of boar B. The rate of blastocyst formation (15.1%) of boar A was higher than that (10.4%) of boar B. In conclusion, serum testosterone concentration of boar showed very important role for the frozen-thawed sperm viability and the blastocyst formation of pig oocytes matured in vitro.

• 한국가축번식학회지
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• 제25권4호
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• pp.399-407
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• 2001

MII단계 난자의 위란강에 retroviral vector를 주입하여, 형질전환 난자의 생산하려는 연구가 수행되고 있다. 그러나 이러한 난자의 위란강의 크기는 매우 다양하므로, 외래유전자를 위란강에 미세주입을 할 때, 난자의 세포질에 손상을 줄 수가 있다. 이에 본 연구에서는 외래유전자 주입시 발생할 수 있는 난자세포의 손상을 최소화하기 위하여 sucrose처리법을 채택하였다. 즉 난자를 0.5%의 sucrose가 첨가된 배양액으로 처리함으로써 일정한 형태의 세포질을 유지하지 못하는 난자와 일정한 형태의 세포질을 유지하는 난자로 분류할 수 있었으며, 후자의 경우 세포질의 큰 손상 없이 retroviral vector를 난자의 위란강내에 주입할 수 있었다. 그러나 sucrose처리에 의해 선별된 난자의 수정율과 대조군의 그것 사이에는 유의차가 없었다. 또 sucrose처리에 의해 선발된 난자에 있어서 retroviral vector (LN$\beta$-EGFP and LNC-hGH) 주입 후의 분할율과 배반포발달율 같은 양상을 보였다. LN$\beta$-EGFP이 주입된 경우, 분할율과 배반포율이 81 및 25% 보였으며, LNC-hGH이 주입된 경우, 83 및 30%를 보였다. 그 결과 미세주입된 난자는 대조군과 유의적인 차이 없이 발달할 수 있었다. 게다가, hGH-gene의 결합율이 PCR 분석에 의하여 분할된 난자에서 52%를 보였으며, 또한 EGFP-gene의 발현율이 현광현미경을 총해 배반포난자 에서 34%가 관찰되었다. 이상의 결과를 종합할 매, 0.5%의 sucrose처리는 우량난자의 선발을 가능하게 하였으며, 주입유전자의 발현율이 낮아지지 않았을 뿐만 아니라, 외래유전자의 위란강내 주입시 난자에 대한 물리적 손상을 줄일 수 있으므로 발달율을 개선할 수 있는 것으로 사료된다.