• Neonatal Medicine
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• 제18권2호
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• pp.310-319
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• 2011

목적: ELBWI의 출생과 생존율이 증가함에 따라 그들의 삶의 질의 향상을 위해 신경학적 발달을 예측 및 평가하고 재활 치료를 통해 발달 지연을 최소화하기 위한 많은 연구가 이루어지고 있다. 뇌 손상의 평가 및 신경학적 발달을 예측하기 위하여 뇌 MRI의 촬영이 많이 이루어지고 있고, 연구를 통해 다양한 프로토콜이 개발되고 있다. 저자들은 Childs 등이 제안한 뇌 성숙도를 점수화 하는 방법을 통해 ELBWI의 주산기에 발생하는 위험 인자와 뇌성숙의 관계, 뇌 성숙과 신경학적 발달 지연과의 관계에 대해 확인하고자 한다. 방법: 교정 주령 만삭 근처에 뇌 MRI를 시행한 ELBWI 124명을 대상으로 의무 기록을 후항적으로 분석하여 주산기적 위험인자를 확인하였고, 뇌 MRI에서 뇌 성숙도를 점수화(TMS) 하고, 동반 되는 이상 소견을 판독하였으며, BSID III를 시행하여 신경학적 발달에 대한 평가를 하였다. 주산기적 위험인자와 TMS의 관계, 뇌 MRI에서 관찰 되는 이상 소견과 TMS의 관계, TMS와 BSID III 결과의 관계에 대한 분석을 하였다. 결과: 대상 환자의 평균 출생 재태 주령은 27.1${\pm}$2.1주, 평균 출생 체중은 781.5${\pm}$143.9 g 이었다. 뇌 성숙도 점수의 평균은 10.8${\pm}$2.0점이었다. 주산기적 위험 인자와의 관계 분석에서는 MRI 검사 당일의 PMA만이 통계학적으로 유의한 관계를 보였고 (P<0.001), BSID III에서 확인되는 신경학적 발달 지연이나 뇌 MRI에서 이상 소견과 뇌 성숙도 점수 간에는 통계학적으로 유의한 상관관계가 없었다. 결론: Conventional 뇌 MRI에서 뇌 성숙도를 평가하여 신경학적 발달을 예측하고, 뇌 성숙에 영향을 주는 주산기적 위험인자를 찾고자 하였으나 MRI 검사 당일의 PMA 외에는 통계학적으로 의미 있는 결과가 없었다. 하지만 수초화 등의 뇌 성숙의 정도가 신경학적 발달에 영향을 줄 것으로 추측되므로 향후 더 다양한 교정 주수에 촬영된 영상과 여러 임상 환자군의 영상에 대한 분석 및 BSID III 등 신경학적 발달 검사와의 관계에 대한 연구가 필요하다.

• 한국가축번식학회지
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• 제27권1호
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• pp.77-85
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• 2003

본 연구는 xanthine(X)-xanthine oxidase(XO) system하에서 돼지 난자의 체외성숙과 체외수정에 대한 catalase의 영향을 검토하였다. 그 결과 돼지 난포난자가 X 또는 XO하에서 배양되었을 때, 난포난자의 성숙율은 다른 배양시간에도 불구하고 catalase 첨가 유무에 따른 유의적인 차이를 나타내지 않았다. 그렇지만, X-XO-catalase system하에서 배양한 경우 유의적으로 높은 성숙율을 얻었다(P<0.05). 퇴행난자의 비율은 배양기간이 늘어남에 따라 증가되었으며, 배양 120시간에서는 catalase 첨가시보다 무첨가시에 유의적으로 높았다. 다른 한편으로, 단위발생 난자들이 배양 72시간에 높은 비율로 관찰되었지만, 다양한 배양시간에서 catalase 첨가유무에 따른 차이는 발견되지 않았다. 또 다른 실험에서, 동결-응해된 돼지 정자가 체외수정을 위해 X-XO system으로 처리되었다. 난자투명대에 대한 정자침입율은 none (P<0.05), XO, X+XO하에서 체외수정시 catalase 무첨가시보다 첨가시에 높게 나타났다. 다른 한편으로, 돼지정자가 none, X, XO, X+XO로 처리되었을 때, lipid peroxidation은 catalase 첨가시보다 무첨가시에 높은 비율로 나타났으며, 그 결과 정자침입과 lipid peroxidation에서의 변화가 상반되는 양상을 보였다. 그렇지만, 모든 조건하에서 정자의 sulfhydry (-SH) group의 함량은 catalase 첨가시에 높게 측정되었다. 난자의 투명대에 대한 정자의 접착 정도는 salt-stored 돼지 난자에 대한 정자접착을 통해서 평가되었으며, control group의 경우 X, XO, X+XO group에 비해 높은 정자접착율이 관찰되었다. 그렇지만, catalase 첨가유무에 따른 유의적인 차이는 인정되지 않았다. 본 연구의 결과는 X-XO-catalase system에 대한 난포난자와 정자의 노출이 돼지에서의 체외성숙과 체외수정을 촉진시키는 것으로 생각된다.

• Reproductive and Developmental Biology
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• 제33권2호
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• pp.85-91
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• 2009

In this study, in vitro maturation system using fetal bovine serum (FBS) or porcine follicular fluid (pFF) was investigated to produce comparable oocytes to those derived from in vivo. Control group of oocytes was cultured in TCM 199 supplemented with 0.1% polyvinyl alcohol (PVA). Other three groups of oocytes were cultured in TCM 199 supplemented with 10% FBS, 10% pFF or 5% FBS + 5% pFF, respectively. After 44 h maturation, oocytes with the first polar body were activated with two electric pulses (DC) of 1.2 kv/cm for 30 ${\mu}sec$. Also, matured oocytes of four groups were reconstructed and fused. Reconstructed embryos were cultured in PZM-3 under 5% $CO_2$ in air at $38.5^{\circ}C$ for 6 days. The oocytes matured in the medium supplemented with FBS or/and pFF showed significantly higher maturation rates (64.0 vs. 73.9 to 85.2%). In PA embryos, cleavage rates (89.7 vs. 77.1 to 86.6%) and blastocysts rates (30.0 vs. 16.2 to 26.2%) were significantly higher in pFF group (p<0.05). In NT embryos, there was no difference among treatments in cleavage rate, but the blastocyst rates (28.5 vs. 15.5 to 24.6%) were significantly higher in pFF group (p<0.05). The apoptosis rate was significantly higher (p<0.05) in the control than other groups (10.8 vs. 4.9 to 8.2% for PA, 3.1 vs. 0.5 to 1.3% for NT). In order to select the comparable oocyte to in vivo oocytes, each group of oocytes was stained with Brilliant cresyl blue (BCB) after 42h maturation. The matured oocytes were separated according to color of cytoplasm; stained group (BCB+) and unstained group (BCB-). The oocytes matured in the presence of FBS or/and pFF showed significantly higher staining rates (70.3 to 72.7 vs. 35.1%) (p<0.05). To verify the fact that the supplementation of FBS or/and pFF can increase the maturation rates, cdc2 kinase activity, the catalytic subunit of MPF, was determined. The cdc2 kinase activity of the oocytes matured in the medium supplemented with FBS or/and pFF was significantly higher than control group (6.7 to 9.3 vs. 3.8). In conclusion, the supplementation of FBS or/and pFF can support in vitro maturation rate of porcine oocytes through the increment of cdc2 kinase activity level in the cytoplasm.

• Clinical and Experimental Reproductive Medicine
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• 제38권4호
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• pp.193-202
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• 2011

Objective: We found previously that $interferon$ $regulatory$ factor ($Irf$)-1 is a germinal vesicle (GV)-selective gene that highly expressed in GV as compared to metaphase II oocytes. To our knowledge, the function of $Irf-1$ in oocytes has yet to be examined. The present study was conducted to determine the relationship between retinoic acid (RA) and RA-mediated expression of $Irf-1$ and the mouse oocyte maturation. Methods: Immature cumulus-oocyte-complexes (COCs) were collected from 17-day-old female mice and cultured $in$ $vitro$ for 16 hours in the presence of varying concentrations of RA (0-10 ${\mu}M$). Rate of oocyte maturation and activation was measured. Gene expression was measured by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) and cytokine secretion in the medium was measured by Bio-Plex analysis. Apoptosis was analyzed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Results: The rates of oocyte maturation to metaphase II and oocyte activation increased significantly with RA treatment (10 nM-1 ${\mu}M$). With 100 nM RA treatment, lowest level of $Irf-1$ mRNA and cumulus cell's apoptosis was found. Among 23 cytokines measured by Bio-Plex system, the substantial changes in secretion of tumor necrosis factor-${\alpha}$, macrophage inflammatory protein-$1{\beta}$, eotaxin and interleukin-12 (p40) from COCs in response to RA were detected. Conclusion: We concluded that the maturation of oocytes and $Irf-1$ expression are negatively correlated, and RA enhances the developmental competence of mouse immature oocytes $in$ $vitro$ by suppressing apoptosis of cumulus cells. Using a mouse model, results of the present study provide insights into improved culture conditions for $in$ $vitro$ oocyte maturation and relevant cytokine production and secretion in assisted reproductive technology.

• 한국동물학회:학술대회논문집
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• 한국동물학회 1998년도 한국생물과학협회 학술발표대회
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• pp.229.1-229
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• 1998

No Abstract, See Full Text

• 한국가축번식학회지
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• 제17권4호
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• pp.279-285
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• 1994

The objective of the present study was to characterize the cell cycles of granulosa cell populations during follicular maturation in cattle by using flow cytometer. Granulosa cells were isolated from bovine preovulatory antral follicles of F1(>10mm), F2(5~20mm), F3(3~4mm) and F4(1~2mm) diameter and fixed and stained with fluorochromes that selectively bine to DNA. Flow cytometer equipped with a laser excitation system was used to analyze the intensity of fluorescence from stained cells. Forward angle light-scatter(FSC) and 90$^{\circ}$light-scatter(SSC) signals were adopted to measure the size and the granularity of granulosa cells. As a results of FSC/SSC analysis, granulosa cell populations(G1 phase of cell cycle) from each follicle were relatively regular in size and granularity, regardless of follicular size. However, their distribution in granularity was greater than that in size. Most of granulosa cell populations collected from each follicle were distributed in G0/G1, S and G2/M phases. As the follicles approached to ovulation the percentage of cells in the proliferative phases of cell cycle (S and G2/M) decreased significantly, but there was a concomitant increase in the percentage of granulosa cells in G1 phase. Therefore, these data indicate the proportion of main populations to cell cycle of granulosa cells may be changed from proliferative phase to G1 phase during follicular maturation in cattle.

• Reproductive and Developmental Biology
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• 제29권4호
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• pp.241-245
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• 2005

Porcine oviduct epithelial cells (POEC) are widely used in co-culture experiments to improve early embryonic development, in vitro fertilization in embryo transfer programs for domestic animals and in vitro maturation of immature germ cells. POEC were mechanically isolated and cultured in tissue culture medium 199. Cells grew continuously, and confluent monolayers were formed after 7 days. After forming confluent monolayer of epithelial cells, supernatant was collected as the condition medium for maturing round spermatids in vitro. Round spermatids were also separated mechanically and cultured in the POEC condition medium. In this study we observed that $20\%$ of round spermatid cultured were matured into elongating spermatid after 24 h, and about $10\%$ of round spermatid cultured showed complete elongation (elongated spermatid) within $24\~48$ h of in vitro culture. No further development was observed within $50\~72$ h and transformed cells lost their viability after 72 h. These preliminary findings suggest that the condition medium from POEC may be possible to overcome the round spermatid block by improving the milieu of culture system.

• 한국수정란이식학회지
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• 제12권2호
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• pp.181-188
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• 1997

This study was carried out to determine the effect of bovine follicular fluid(bFF), hormones, and fetal bovine serum(FBS) supplemented in the medium on the in vitro fertilization and development of bovine embryos. The ovaries were obtained from a local abattoir and placed in physiological saline kept at 30~32˚C and brought to the laboratory within 3~4 hours. The oocytes and follicular fluid were collected by aspiration from visible follicles, and the oocytes of grades I on the basis of the morphology of cumulus cells attached and the homogeneity of cytoplasmic granules were selected and used for maturation. The basal media used for oocyte maturation, fertilization and embryo development in vitro were Ham' F-10, TALP and TCM-199, respectively. The hormones supplemented in maturation medium were consisted of 35 pg /ml FSH, 10 pg /ml LH and 1 pg/mi estradiol-l7$\beta$. The bFF collected from 5~9 mm follicles was centrifuged, filtered and inactivated by heat-treatment at 56˚C for 30 min. FBS also was inactivated with the same method and kept at -20˚C until use. The embryos were co-cultured with the monolayer of bovine oviductal epithelial cells at 39˚C under 5% $CO_2$ in air for 9 days. The results obtained were summarized as follows: The fertilization rate of oocytes was found 87.4% from 10% FBS and hormones treatment for IVM, and 37.1% of these TVF embryos were developed to blastocyst stage in 10% FBS groups. Compared with this control system, the fertilization rate was decreased significantly(P<0.05) in the maturation without either FBS or hormones. These IVF embryos were developed to morula stage at the similar rate, but to blastocyst at significantly(P<0.05) lower rate in the embryo culture with or without FBS supplementation. The fertilization rate(82.9%) in hormones and 10% inactivated bFF was similar with 10% FBS and hormone groups(87.4%), but decreased significantly(P<0.05) in 20 or 30% bFF (61.0 or 66.0%), respectively. In vitro developmental competence to blastocyst stage in 10% FBS and 20% inactivated bFF(37.1% and 31.4%) was higher than in 10 or 30% inactivated bFF(20.0 or 19.2%) or 10, 20 and 30% fresh bFF(19.1, 21.0 and 17.5%) The results indicated that the in vitro fertillzation and development rate of the embryos should be improved in 10% FBS or 20% inactivated culture system and 20% inactivated bFF might be available economically for bovine oocyte maturation and embryo culture instead of fetal bovine serum.

• 한국가축번식학회지
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• 제19권3호
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• pp.181-189
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• 1995

In this study, methods on fabrication of microtool and setting of micromanipulator were examined and relationship between first polar body extrusion rate and maturation time of follicular oocyte, enulceation rae and repetition of trial, and enucleation rate and maturation period were investigated. The results are as follows: 1. Suitable outside diameter of micropipette tube was 1mm. Holding pipette with less than diameter of oocyte was fitred for manipulation, and zona dissection needle was easily operated when its sharp-point had diameter of about 8 ${\mu}{\textrm}{m}$ and length of 300${\mu}{\textrm}{m}$. The injection pipette with 20~35${\mu}{\textrm}{m}$ outside diameter was adequate for injection of blastomere into perivitelline space. 2. Separation of blastomere was effective when zona pellucida had cut with zonadissection needle and the embryo was pipetted gently with the pipette that had narrower diameter than that of embryo until separation of blastomeres had completed. 3. The extrusion rate of first polar body was 78% during 20~24% hours incubation for maturation. 4. According to repetitions of micromanipulation, the enucleation rate was increased to 85% and the time required for enucleation of a oocyte was shortened to 3 min. 5. The extrusion rate of first polar body and enucleation rate were 82 and 76% respectively, in the group of the oocytes cultured for 22 hours. However in the group cultured for 24 hours, the extrusion rate of first polar body and enucleation rate were 53 and 100% respectively.

• Asian-Australasian Journal of Animal Sciences
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• 제32권12호
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• pp.1844-1853
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• 2019

Objective: We investigated how pituitary adenylate cyclase-activating polypeptide (PACAP) affects embryonic development during pre-in vitro maturation (pre-IVM) using porcine oocytes isolated from small follicles. Methods: We divided the follicles into the experimental groups by size (SF, small follicles; MF, medium follicles) and treated with and without PACAP and cultured for 18 hours (PreSF[-]PACAP; without PACAP, Pre-SF[+]PACAP; with PACAP) before undergoing IVM. The gene expression related to extracellular matrix formation (amphiregulin, epiregulin, and hyaluronan synthase 2 [HAS2]) and apoptosis (Bcl-2-associated X [BAX], B-cell lymphoma 2, and cysteine-aspartic acid protease 3) was investigated after maturation. The impact on developmental competence was assessed by the cleavage and blastocyst rate and total cell number of blastocysts in embryos generated from parthenogenesis (PA) and in vitro fertilization (IVF). Results: Cleavage rates in the Pre-SF(+)PACAP after PA were significantly higher than SF and Pre-SF(-)PACAP (p<0.05). The cleavage rates between MF and Pre- SF(+)PACAP groups yielded no notable differences after IVF. Pre-SF(+)PACAP displayed the higher rate of blastocyst formation and greater total cell number than SF and Pre-SF(-)PACAP (p<0.05). Cumulus cells showed significant upregulation of HAS2 mRNA in the Pre-SF(+)PACAP compared to the SF (p<0.05). In comparison to other groups, the Pre-SF(+)PACAP group displayed a downregulation in mRNA expression of BAX in matured oocytes (p<0.05). Conclusion: The PACAP treatment during pre-IVM improved the developmental potential of porcine oocytes derived from SF by regulating cumulus expansion and apoptosis of oocytes.