돼지의 난포액 내 단백질인자의 탐색과 변화에 관한 연구

지미란,정희태,양부근,이채식,박춘근,Ji, Mi-Ran,Cheong, Hee-Tae,Yang, Boo-Keun,Lee, Chae-Sik,Park, Choon-Keun 한국동물번식학회 2010 Reproductive & developmental biology Vol.34 No.3

When fully grown oocytes are removed from their follicles, they can resume meiosis and mature spontaneously under in vitro conditions. However, nuclear maturation under in vitro condition is not accompanied by complete cytoplasmic maturation, which is essential for successful fertilization and the initiation of zygotic development. This study analyzed change of proteins in follicular fluids during the porcine follicular development. Follicular fluids were collected from follicles of diameter 1~2 mm, 2~6 mm and 6~10 mm in ovary of slaughtered pigs. Total proteins were extracted from follicular fluids by M-PER Mammalian Protein Extraction Reagent. We confirmed totally 27 same spots, 1 spot from follicle fluid of 2~6 mm follicle and 5 spots from follicle fluid of 6~10 mm in diameter were analyzed by MALDI mass spectrometry and searched on NCBInr. In results, spot No. 28 from 2~6 mm follicle was Ig lambda chain C region, and spot No.32 and 33 from 6~10 mm was Apolipoprotein A-(APOA4). Spot No.29 and 31 were failed to analyze. These results indicate that the porcine oocyte during in vitro maturation depend on specific different expressed proteins may play an important roles in the sequence of molecular events in porcine oocyte maturation and follicular development.

체외에서 돼지 정자-난자의 상호작용시 투명대내 Lectin 결합

황인선,김정익,정희태,양부근,박춘근,Hwang, In-Sun,Kim, Choung-Ik,Cheong, Hee-Tae,Yang, Boo-Keun,Park, Choon-Keun 대한생식의학회 2002 Clinical and Experimental Reproductive Medicine Vol.29 No.3

Objective: Lectins are cell-agglutinating and sugar specific proteins or glycoproteins of non-immune origin that precipitate glycoconjugates having saccharides of appropriate complementarity. Because of these properties, plant lectins have been used to help characterize the carbohydrate moieties of glycoproteins in the zona pellucida (ZP) of several mammalian species including pigs. Treatment of oocytes with various lectins blocks sperm binding to the ZP in various mammalian species. This study was undertaken to examine the distribution of sugar residues in the ZP of pig oocytes matured in vitro and the ability of spermatozoa to bind to ZP and in vitro penetration in oocytes treated with fluorescein isothiocyanate (FITC)-labelled lectins. Materials and Methods: The lectins of Banderiaea simplicifolia (BS-II, bind to $\beta$-D-N-acetylglucosamine), Canavalin ensiformis (Con A, bind to $\alpha$-D-Mannose), Lens culinaris (LCA, bind to a-D-Mannose), Ricinus communis (RCA-I, bind to $\beta$-D-Galactose) and Ulex europaeus (UEA-I, bind to $\alpha$-L-Fucose) were examined for spermatozoa penetration, binding capacity to ZP and distribution of lectins. Results: The penetration rates were significantry (p<0.05) higher in control oocytes (63%) than those treated with all lectins, but penetration rates ($40{\sim}49%$) were simililar in group treated with lectins. The incidence of monospermy was similar in oocytes untreated and UEA-I, but it was higher in oocytes treated with BS-II, Con A, RCA-I and LCA. The porcine oocytes cultured for 48 h in TC-199 medium were freed from cumulus cells and treated for 30 min with fluorescein isothiocyanate-labelled lectins. When examined under fluorescein illumination, higher (p<0.001) proportions of oocytes showed fluorescein of zona pellucida after treatment with Con A (93%), LCA (93%) and RCA-I (100%) than BS-II (37%) and UEA-I (50%). All of the oocytes treated with RCA-I exhibited strong fluorescein in the outer region of the zona pellucida while those treated with LCA exhibited strong fluorescein throughout the zona pellucida. BS-II bounded mainly to the outer region and UEA-I bounded mainly to the inner region of the zona pellucida, with either strong or weak fluorescein. At 120 min after insemination in vitro, fewer spermatozoa were bound to the zona pellucida of the oocytes treated with BS-II, Con-A and RCA-I. Of the lectins, Con A most inhibited sperm binding. Conclusions: These results suggest that $\beta$-D-Galactose residues in the porcine zona pellucida may act as primary sperm receptors and inducers of the sperm acrosome reaction and these sugar residues may be involved in the block to polyspermy.

임신중인 생쥐에 DEHP 투여가 번식현상에 미치는 영향 Ⅱ. 임신중인 생쥐에 DEHP 투여가 자손의 번식특성과 혈액성분에 미치는 영향

박동헌,장현용,박춘근,정희태,김정익,양부근,Park, Dong-Heon,Jang, Hyun-Yong,Park, Choon-Keun,Cheong, Hee-Tae,Kim, Choung-Ik,Yang, Boo-Keun 한국발생생물학회 2004 발생과 생식 Vol.8 No.2

본 연구는 임신한 생쥐에 DEHP의 투여가 자손의 번식특성과 혈액성분에 미치는 영향을 검토하였으며 그결과는 다음과 같다. 임신기에 DEHP의 투여가 웅성 자손의 체중, 번식기관 무게 및 정액 성상에 미치는 결과는 투여구간에 커다란 차이가 없었다. 임신기에 DEHP의 투여가 웅성 자손의 혈액 성상에 미치는 영향을 조사한 결과, WBC, HB, HT, MCH 및 albumin은 투여구간에 차이가 없었으나, RBC MCV, MCHC, PLT 및 total protein은 각 투여구간에 통계적으로 유의적인 차이가 나타났다. 임신기에 DEHP의 투여가 자성 자손의 체중, 자궁 및 오른쪽 난소의 무게에 미치는 영향을 조사한 결과, 각 투여구간에 커다란 차이가 없었지만, 왼쪽 난소의 무게는 10.0mg 투여구가 대조구와 0.5mg 투여구에 비해 통계적으로 유의하게 높은 무게를 나타냈다. 임신기에 DEHP의 투여가 자성 자손의 혈액성상에 미치는 영향을 조사한 결과, RBC, HB 및 HT는 각 투여구간에 커다란 차이가 없었다. 임신기에 DEHP의 투여가 자손의 정소, 난소 및 자궁의 조직에 미치는 영향을 조사한 결과,정소에서는 투여구간에 차이가 없었으며, 난소에서는 10.0mg 투여구가 여타구보다 많은 황체를 가지고 있었으며, 자궁에서는 대조구에 비해 DEHP의 투여농도가 증가할수록 자궁내막층이 감소했으며, 1.0mg 투여구와 10.0mg 투여구는 대조구에 비해 뚜렷한 자궁내막층의 감소가 있었다. The objective of this study was to assess that the effects of DEHP administration on reproductive characteristics and blood hematological and chemical values in pups born after DEHP administration in pregnant mice. DEHP was administrated to pregnant mice by intraperitoneally injection with 0, 0.5, 1.0 and 10.0mg/kg B.W, 5 times at 3 days interval from Day 1 to Day 16 in the gestation period. The body weight and reproductive organ weight(testis, epididymis and coagulating gland) in male pups on 45 day after birth was not affected in all experimental groups, but vesicular gland in DEHP groups was significantly lower than that of control group(P<0.05). The semen characteristics of male pups were not affected in DEHP treatment groups. The WBC, HB, HT, MCH and albumin values in male pups were not affected in all experimental groups, but RBC MCV, MCHC, PLT and total protein values were significantly different among the experimental groups(P<0.05). In female pups, the effects of DEHP administration were not affected the body and uterus weight, but the left ovary in 10.0mg DEHP group was significantly heavier than in control and 0.5mg DEHP group(P<0.05). The WBC, MCV, MCH, MCHC, PLT, albumin, BUN and total protein values in female were not different in all experimental groups. The RBC, HB and HT values were significantly different among the experimental gruop(P<0.05). The historical evaluation of testis in male pups that were grown to 45 days after birth was not different in all experimental groups. The ovary in female pups had many corpus luteum in 10.0mg DEHP group. The endometriosisi of uterus was significantly decreased in DEHP group. There results suggest that low concentration of DEHP administration in pup born after DEHP administration in pregnant mice was not affered on reproductive characteristic, but was affected on blood hematological and chemical values.

소 체외수정란 및 체세포 복제란의 초자화 동결 후 생존성

권대진,박주희,박춘근,양부근,정희태,Kwon, Dae-Jin,Park, Joo-Hee,Park, Choon-Keun,Yang, Boo-Keun,Cheong, Hee-Tae 한국동물번식학회 2007 Reproductive & developmental biology Vol.31 No.1

본 연구는 소 체외수정란과 체세포 복제란의 초자화 동결 및 응해 후 생존능을 검토하였다. 배반포로 발육된 체외수정란 및 체세포 복제란을 초자화 동결법에 의해 동결하였다가 융해하여 생존율 및 배양 후 부화율을 검사하였다. 체외수정란 배반포를 초자화 동결 응해한 결과, 확장배반포가 배반포기 난자에 비하여 생존율(82.1%, 96/117)과 부화율(64.1%, 75/117)에서 모두 유의적으로 높았다(p<0.05). 핵이식 배반포 복제란을 초자화 동결 응해한 경우도 체외수정란과 비슷한 경향을 보여 확장 및 부화배의 생존율과 부화율이 각각 81.1%(30/37)와 78.3%(29/37)로, 배반포(각각 71.8 및 53.8%)에 비하여 다소 높게 나타났다. 본 연구의 결과는 초자화 동결 방법에 의해서 소 체외수정란과 체세포 복제란을 성공적으로 동결할 수 있으며, 특히 후기 배반포기 단계에서 초자화 동결 시 높은 생존율과 부화배 형성율을 얻을 수 있음을 보여준다. This study was conducted to examine the development of in vitro fertilized (IVF) and nuclear transfer (NT) embryos following vitrification IVF and NT embryos developed to the blastocyst stage were equilibrated by 3 steps, vitrified and thawed, and their survival and hatching rates were examined. In IVF embryos, higher survival (82.1%, 96/117) and hatching rates (64.1%, 75/117) were obtained respectively after thawing and culture in expanded blastocysts compared to blastocysts (p<0.05). High survival and hatching rates were also obtained by vitrification of NT blastocysts, especially in expanded and hatching blastocysts (81.1 and 78.3%, respectively). The result of this study shows that IVF and NT blastocysts, especially late stage blastocysts, are successfully cryopreserved by vitrification.

임신중인 생쥐에 DEHP 투여가 번식현상에 미치는 영향 Ⅰ. 임신중인 생쥐에 DEHP 투여가 분만 후 번식특성과 혈액성분에 미치는 영향

박동헌,장현용,박춘근,정희태,김정익,양부근,Park, Dong-Heon,Jang, Hyun-Yong,Park, Choon-Keun,Cheong, Hee-Tae,Kim, Choung-Ik,Yang, Boo-Keun 한국발생생물학회 2004 발생과 생식 Vol.8 No.2

본 연구는 임신한 생쥐에 DEHP의 투여가 분만 후 생쥐의 번식기능과 태아의 성비에 미치는 영향을 검토하였다. 임신한 자성 생쥐에 DEHP을 투여한 후 임신 19일째에 임신한 자성의 체중, 태아의 체중, 태아수 및 태아성비를 조사한 결과, 실험개시시와 실험종료시 임신한 자성의 체중은 각 투여구간에 차이가 없었으며, 자${\cdot}$웅성태아의 체중, 평균 태아수 및 태아의 자${\cdot}$웅성 성비는 투여구간에 커다란 차이가 없었다. 임신기에 DEHP의 투여가 분만한 후 어미의 번식기관무게와 혈구화학치에 미치는 효과를 검사한 결과, 각 투여구간에 커다란 차이는 없었다. 임신기에 DEHP의 투여가 분만한 후 난소와 자궁의 조직에 미치는 영향을 조사한 결과, 난소에서는 모든 투여구간에 커다란 차이는 없었으나, 자궁에서는 0.5mg 투여구가 대조구에 비해 자궁내막층과 부종이 감소했으며, 1.0mg 투여구와 10.0mg 투여구는 일부에서 자궁내막층이 감소했지만 0.5mg 투여구에서의 감소보다는 적었다. 임신중인 생쥐에 저농도의 DEHP의 투여는 임신중인 모체와 태어난 자손의 번식 특성과 혈구화학치에 영향을 미치지 않았다. This study is illustrated that 1) the effects of DEHP administration on body weight, sex ratio and litter size on 19 days in the prenatal period 2) the effects of reproductive organ weight and blood hematological values on 25 days postpartum in pregnant mice. DEHP was administrated to pregnant mice by intraperitoneal injection at 0, 0.5, 1.0 and 10.0mg/kg B.W from day 1 to day 16 in the gestation period with 5 times at 3 days interval. There were no significantly differences in the material body weight, litter size and sex ratio on 19 days in the prenatal period. The body weight of the fatal male mice was slightly lower in 1.0 and 10.0mg DEHP groups than in any other groups. The reproductive organ weight and hematological values in dam mice on 25 days postpartum were not affected by DEHP administration. The histological evaluation of ovary in dam mice on 25 days after dilivery was not different in all experimental groups, but the endometriosis and edema of uterus in dam mice were significantly decreased in 0.5mg DEHP group than that of control group.These results indicate that the administration of low concentration of DEHP was not affected on reproductive characteristic and blood hematological values in pregnant mice.

임신중인 생쥐에 Bisphenol A 투여가 모체의 생식독성과 태아의 성비에 미치는 영향

박동헌(Dong-Heon Park),장현용(Hyun-Yong Jang),김정익(Choung-Ik Kim),정희태(Hee-Tae Cheong),박춘근(Choon-Keun Park),양부근(Boo-Keun Yang) 한국독성학회 2005 Toxicological Research Vol.21 No.2

Bisphenol A (BPA), a environmental endocrine disruptor, is considered to bind to estrogen receptors and to regulate the expressions of estrogen responsive genes. This study was to evaluate the effect of BPA administration on body weight, sex ratio and litter size on 18 days in prenatal periods, the effect of reproductive organ weight and blood hematological values on 24 days postpartum in pregnant mice. The female mice was administrated to low doses of BPA (0, 0.05, 0.5 and 5.0 ㎎/㎏ B.W.) by intraperitoneal injection in gestation days 0~15 with 5 times at 3 days interval. The maternal body weight, litter size and sex ratios were similar to in all experimental groups, but body weights of male and female offspring was significantly lower in 5.0 ㎎ BPA group when compared to any other groups (P<0.05). No treatment-related effects on body weight, ovary weight and blood hematological values were observed in dams on 24 days after delivery. The uterine weight in 5.0 ㎎ BPA group was slightly higher than those of any other groups, but not significantly difference. The histological evaluation of ovary in dam mice on 24 days after dilivery was not difference in all experimental groups, but the endometriosis of uterus in dam mice were significantly increased in 0.5 ㎎ BPA group when compared to control group. These results indicates that low concentration of BPA should not be considered as a selective reproductive toxicant.

임신중인 생쥐에 Bisphenol A 투여 후 태어난 차산자의 생식독성과 혈액성분에 관한 연구

박동헌(Dong-Heon Park),장현용(Hyun-Yong Jang),김정익(Choung-Ik Kim),정희태(Hee-Tae Cheong),박춘근(Choon-Keun Park),양부근(Boo-Keun Yang) 한국독성학회 2005 Toxicological Research Vol.21 No.2

Bisphenol A (BPA) is a monomer used in the manufacture of a multitude of chemical products, including epoxy resins and polycarbonate. The objective of this study was to evaluate the effects of BPA administration on reproductive characteristics and blood hematological and chemical values in offspring of pregnant dams treated with BPA. BPA was administrated to pregnant mice by intraperitoneally injection with 0, 0.05, 0.5 and 5.0 ㎎/㎏ B.W. for 5 times at 3 days interval on gestation days 1~16. There were no treatment-related effects of BPA on reproductive organ weight in male offsprings at 45 days-of-age, but body weight was the lowest in 5.0 ㎎ BPA group when compared to other groups (P<0.05). No differences in semen characteristics (sperm concentration, viability, motility and abnormality) were observed between the control and BPA treatment groups. The WBC, HB, HT, MCV, MCH, MCHC, albumin, BUN and total protein of blood hematological and chemical values in male offsprings were not difference for any treatment groups, but RBC value in BPA groups was significantly increased comparing to the control group (P<0.05). The PLT value was slightly higher in 5.0 ㎎ BPA groups than in any other group, but not significantly difference among the experimental groups. In female offsprings, the effects of BPA didn't affect to the body and ovary weight, but the uterus weight in 5.0 ㎎ BPA group was slightly heavier than that of control group (P>0.05). No statically significant difference in blood hematological values in female offsprings were observed between the control group and BPA groups, but the concentration of albumin and BUN were significantly higher in 0.5 ㎎ BPA group when compared to control and other BPA treatment groups (P<0.05). The histological evaluation of testis and ovary in growing offspring at 45 days-of-age was not difference between the control group and BPA groups, but endometriosis of the uterus in female offspring was dramatically increased in 0.5 and 5.0 ㎎ BPA groups. These founding suggest that low concentration of BPA might not have a important role on reproductive ability or blood metabolite in offspring of pregnant dams treated with BPA.

돼지정자의 운동성, 원형질막 온전성, 미토콘드리아 기능성 및 원형질막 지질과산화에 미치는 arsenite 및 항산화제의 영향

김한수(Han-Su Kim),이유섭(Yu-Sub Lee),이상희(Sang-Hee Lee),정희태(Hee-Tae Cheong),박춘근(Choon-Keun Park),이승형(Seunghyung Lee),양부근(Boo-Keun Yang) 한국생명과학회 2017 생명과학회지 Vol.27 No.5

본 실험은 항산화제인 melatonin, silymarin, curcumin 및 vitamin E가 aresenite에 의해 손상된 돼지의 정자성상능력에 미치는 영향을 조사한 연구이다. 돼지정자는 채취 후 희석하여 실험에 이용하였으며, 100 μM arsenite는 인위적으로 정자를 손상시키는데 사용하였다. 또한 100 nM melatonin, 2 μM silymarin, 10 μM curcumin 및 500 μM Vitamin E를 희석된 돼지정자에 첨가하여 3, 6 및 9시간 동안 배양 후 정자의 운동성, 원형질막 온전성, 미토콘드리아 기능성 및 원형질막 지질과산화를 검토하였다. 그 결과, 정자의 운동성 및 원형질막 온전성은 100 μM arsenite 처리구에서 유의적으로 감소하였으며, 항산화제 단독처리구는 유의적으로 증가하였다. 또한, arsenite에 의해 손상된 처리구는 항산화제에 의해 유의적으로 증가하였다(p<0.05). 정자의 미토콘드리아 기능성은 100 μM arsenite 처리구에서 유의적으로 감소하였고, 정자의 원형질막 지질과산화는 100 μM arsenite 처리구에서 유의적으로 증가하는 것으로 나타났다(p<0.05). 결론적으로, arsenite의해 감소된 돼지정자의 운동성과 원형질막은 melatonin, silymarin, curcumin 및 vitamin E와 같은 항산화제에 의하여 예방될 수 있다고 판단되며, arsenite에 의해 감소된 정자의 수정능력은 항산화제에 의해 회복될 수 있을 것이라 생각된다. This study was to investigate the role of antioxidants on the characteristics of arsenite-damaged boar semen. Collected sperm was diluted with semen extender, and 100 μM arsenite was used for sperm damage. Then melatonin, silymarin, curcumin, and vitamin E were applied for 3, 6, and 9 hr in arsenite-treated boar sperm. Sperm characteristics were then analyzed for motility, plasma membrane integrity, mitochondrial activity, and lipid peroxidation. In the results, sperm motility (control, 77.3± 1.8%) was decreased by arsenite (33.3±1.5%), while the antioxidant treatment groups (100 nM melatonin, 55.8±3.4%; 2 μM silymarin, 48.8±3.4%; 10 μM curcumin, 53.9±2.8%; and 500 μM vitamin E, 54.5±3.1%) showed increases compared to the arsenite group (p<0.05). 100 μM arsenite decreased the sperm plasma membrane integrity (24.5±1.6%) and mitochondrial activity (58.2±2.6%), and increased lipid peroxidation (5.3±0.2%) at 3 hr (p<0.05). However, arsenite-treated samples with 100 nM melatonin, 2 μM silymarin, 10 μM curcumin, and 500 μM vitamin E increased the plasma membrane integrity and mitochondria activity, and decreased lipid peroxidation compared to the arsenite-treated samples. In summary, arsenite may induce sperm damage and oxidation stress, while antioxidants such as melatonin, silymarin, curcumin, and vitamin E are useful for maintaining sperm characteristics. Therefore, antioxidants can protect sperm against damage by arsenite in fresh boar semen.