• Toxicological Research
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• 제21권2호
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• pp.167-173
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• 2005

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 mg/kg 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 5.0mg 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 5.0mg 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 5.0mg 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 0.5mg 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 5.0mg 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.

• 한국가축번식학회지
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• 제11권1호
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• pp.73-84
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• 1987

This experiment was carried out to investigate the effects of hormone addition(FSH, HCG, estrogen and progesterone) and composition (BSA and FCS) of mKRB on the in vitro maturation and fertilizability of follicular oocytes of the Korean native cattle. The ovaries were removed at a slaughterhouse, returned to laboratory in a thermostat (30-35$^{\circ}C$) within 4 hr, and collected by aspirating normal follicles which had diameters of 1 to 6 mm. The oocytes with cumulus cells were cultured for 8, 16, 24 and 30 hr in a modified KRB solution containing BSA or FCS and hormones. The in vitro matured oocytes in mKRB containing FCS, FSH and steroids were transferred in the rabbit uterus for examination of their in vivo fertilizability with bovine sperm preincubated 4 to 6 hr in the rabbit uterus. 1. The mean number of oocytes collected per cattle was 6.5 from 1-3mm follicles, 1.3 from 4-6mm follicles, and total was 7.7. 2. The meiotic division at 16hr-cuture in the oocytes from 1-3mm follicles was slightly stimulated by the addition of FSH in mKRB + BSA solution compared with the control. At 30hr-culture, their maturation rates(%Met II) were also increased by FSH of 1 $\mu\textrm{g}$/ml(38.4%) and 5$\mu\textrm{g}$/ml(35.7%) as compared with the control (21.4%). The maturation rate at 30hr-culture in the oocytes from 4-6mm follicles was 53.8% and 57.1% by the FSH addition of 1$\mu\textrm{g}$/ml and 5$\mu\textrm{g}$/ml, respectively. These rates were similar with the control(57.1%), but higher than those of oocytes from 1-3mm follicles. 3. The meiotic division at 16hr-culture in the oocytes from 1-3mm follicles was stimulated by the HCG addition of 1IU/ml and 5IU/ml. However, the maturation rate at 30hr-culture was greatly decreased by the HCG addtion (26.6% and 13.3%) compared with the control(53.3%) and these rates (30.8%) in the oocytes from 4-6mm follicles were also lower than that fo the control(58.3%). 4. Low maturation rate (37.5%) of the oocytes cultured in mKRB containing BSA and 5IU/ml HCG was increased (55.0%) when 15% FCS with HCG was added to mKRB instead of BSA. 5. When 16hr-cultured oocytes in mKRB containing BSA and gonadotropins (5$\mu\textrm{g}$/ml FSH and 5IU/ml HCG) were transferred in the medium without gonadotropins and recultured for 16hr, the maturation rate of HCG-treated oocytes was greatly improved. 6. The maturation rates of oocytes were greatly affected by steroids. The combined addition of FCS+FSH+estrogen or +progesterone to mKRB increased the maturation rate compared with the combination of BSA+FSH or FCS+FSH in mKRB. 7. The fertilization rate, presence of pronuclei, was increased by the combination of FCS+FSH+p in mKRB as compared with that (5.6%) of BSA+FSH and the rates of FCS+FSH+steroids ranged from 12.5 to 17.6%.

• 생명과학회지
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• 제24권11호
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• pp.1252-1257
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• 2014

DMSO는 배양포유류세포 동결보존의 동결보호제로써 일반적으로 사용 되어져 왔지만, DNA 메틸화 및 히스톤의 수식에 의해 일부 세포에서는 분화를 일으키는 것으로도 알려져 있다. 동결보존시의 배양세포의 안정된 분화형질유지에는 메틸화를 일으키는 DMSO 이외의 동결보호제의 사용이 필요하다. 세포독성이 낮고, 동물정자동결보존에 효과적인 것으로 알려진 아미도 화합물이 동일하게 포유류의 배양세포의 동결보존에서 동결보호작용이 있는지를(8종류의 아미드 화합물) 배양 마우스 혈관내피세포를 이용해 조사했다. 조사한 아미드 화합물 중에 아세트아미드와 락트아미드의 2종류가 배양세포에 대해서 동결보호작용이 있고, 가장 효과적인 것은 농도가 1.5 M의 락트아미드이다. 배양세포의 동결보존에 관해서는 삼투압 스트레스를 받지 않을 필요가 있기 때문에, 1.5 M 락트아미드 용액을 제작 시, 용매를 각 희석율의 PBS로 하고, 삼투압을 바꾼 동결 보존액에 동결세포의 생존율을 조사했다. 그 결과, 0.4배 농도의 PBS가 삼투압 스트레스를 가장 낮고 생존율이 가장 높음을 확인했다. 동결보존배지에 고 분자량재료를 첨가하면 세포생존율이 개선되는 것이 알려져 있기 때문에 BSA, HES, 데키스트란의 효과를 조사했다. 그 결과, 락트아미드를 이용한 동결보존배지는 $0.4{\times}PBS$를 이용한 1.5 M 락트아미드용액에 1%의 BSA를 첨가한 경우, DMSO의 동결보호작용에 필적하는 동결보호작용을 나타내는 것을 확인했다.

• Clinical and Experimental Reproductive Medicine
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• 제38권4호
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• pp.222-227
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• 2011

Objective: To evaluate the ability of serum anti-M$\ddot{u}$llerian hormone (AMH), FSH, and age to clinically predict ovarian response to controlled ovarian hyperstimulation (COH) in IVF patients with endometriosis. Methods: We evaluated 91 COH cycles, including 43 cycles with endometriosis (group I) and 48 cycles with male factor infertility (group II) from January to December, 2010. Patients were classified into study groups based on their surgical history of endometriosis-group Ia (without surgical history, n=16), group Ib (with a surgical history, n=27). Results: The mean age was not significantly different between group I and group II. However, AMH and FSH were significantly different between group I and group II ($1.9{\pm}1.9$ ng/mL vs. $4.1{\pm}2.9$ ng/mL, $p$ <0.01; $13.1{\pm}7.2$ mIU/mL vs. $8.6{\pm}3.3$ mIU/mL, $p$ <0.01). Furthermore, the number of retrieved oocytes and the number of matured oocytes were significantly lower in group I than in group II. In group II, AMH and FSH as well as age were significant predictors of retrieved oocytes on univariate analysis. Only the serum AMH level was a significant predictor of poor ovarian response in women with endometriosis. Conclusion: Serum AMH may be a better predictor of the ovarian response of COH in patients with endometriosis than basal FSH or age. AMH level can be considered a useful clinical predictor of poor ovarian response in endometriosis patients.

• Clinical and Experimental Reproductive Medicine
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• 제27권1호
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• pp.23-29
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• 2000

Objective: Zona pellucida (ZP) has been thought to be the barrier of egg to sperm penetration before and after fertilization. The phenomenon of ZP hardening has been considered as a post-fertilization event until now, and it is generally accepted that it is caused by the secretory products of cortical granules released during the cortical reaction. Hardening of ZP could occur "spontaneously" in mammalian oocytes in standard culture conditions, and that it is probably not a consequence of cortical reaction. The purpose of our study was to investigate the effect of human amniotic fluid (HAF) on nuclear maturation (NM) and fertilization ability of mouse immature oocytes. Methods: HAF was obtained from patients undergoing amniocentesis at $16{\sim}20$ weeks of gestation. HAF from five to ten patients was centrifuged and the supernatants was pooled. Cumulusenclosed mouse immature oocytes were incubated in the medium containing HAF, and examined to confirm NM and fertilization. Female ICR mice (about 3 weeks old) were stimulated with 7.5 IU PMSG. Immature oocytes were isolated at $48{\sim}52$ hrs post PMSG injection and cultured in TCM-199 supplemented with 20% HAF for 18 hrs. FBS was used as a control for the examination. Matured oocytes (MII) were fertilized with sperms collected from the epididymis of male mice (over 10 weeks old). Fertilization was in conducted T6 medium containing 15 mg/ml BSA, and confirmed at 6 hrs post-insemination. Fertilization rate was assessed in zona-intact or zona-free oocytes (denuded by trypsin). Evaluation of NM and fertilization was carried out by rapid staining method. ZP hardening was evaluated by incubating cumulus cell-free mature oocytes in 0.001% chymotrypsin at $37^{\circ}C$ for 10 min. Results: There was no significant difference between the effects of HAF (86.6%) and FBS (87.7%) supplements on NM of immature oocytes. When maturation medium was supplemented with HAF, total fertilization rates (7%) were significantly lower (p<0.01) than that of FBS (85.1%). In HAF group, fertilization rate was increased (p<0.01) in zona-free oocytes (7% versus 100%). The resistance of mouse oocyte ZP to digestion by chymotrypsin after maturation in vitro was significantly higher (p<0.01) in HAF group (86.7%) than in FBS (6.7%). To culture oocytes in FBS were very effective in preventing ZP hardening. However cultured oocytes in HAF showed high rate of ZP hardening (p<0.01). Conclusions: These results suggest that HAF can be used as a supplement for the NM of mouse immature oocytes in vitro. However, HAF induces spontaneous hardening of ZP of mouse immaure oocytes during maturation in vitro.

• Clinical and Experimental Reproductive Medicine
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• 제26권2호
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• pp.193-201
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• 1999

본 연구에서는 소 난자 세포질내 원형정자 및 원형정자 핵을 미세주입 후 수정과정과 체외 배 발달을 조사하였다. 원형정자 주입 3시간 전에 인위적 자극을 주고 원형정자를 주입했을 때 난자들의 수정률이 주입 직후에 자극을 준 것들과 자극을 주지 않은 것들 보다 높았다. 원형정자 주입과 원형 정자핵을 주입한 후 전핵 형성률과 전핵 이동률을 조사하였으나 유의차를 발견할 수 없었다. 전핵의 형성과 이동중에 미세소관의 움직임을 간접형광면역법 및 공촛점 현미경을 사용하여 조사해 본 결과, 난활성 직후 난자의 표층에서 미세소관이 발생해서 이것에 의해 웅성 및 자성전핵이 난자 중심부로 이전됨을 볼 수 있었다. 수정 후 원형정자의 세포질내 Mitochondria의 분포상태를 알아보기 위해 MitoTracker에 노출시킨 원형정자를 주입한 후 형광현미경에서 관찰해 보았다. 그 결과 원형정자의 Mitochondria가 주입 후 4-cell 이 전까지 는 관찰되었으나 그 이 후에는 발견 할 수 없었다. 원형정자를 미세주입한 후 소 난자를 체외에서 배양했을 때 2.5%의 난자가 배반포로 발달하였다. 이상의 결과는 체외 배양한 소 난자 내에 원형정자 혹은 원형정자 핵을 미세 주입하여 정상적인 수정과 배발달이 가능한 것을 보여 주는 것이다.

• 한국수정란이식학회지
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• 제18권3호
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• pp.243-248
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• 2003

1. 생쥐 고환으로부터 얻은 세포를 배양하여 군집을 형성하는 것을 관찰할 수 있었으며, AP, SSEA-1, -3, -4과 Integrin $\alpha$6, $\beta$1 및 Oct4의 발현을 확인하였다. 2. 생쥐 생식줄기세포를 3-5일정도 배양하게 되면, 여러 층으로 이루어진 군집을 이루게 되는데 이는 생쥐 배아줄기세포나 배아생식줄기세포의 형태와 같은 것이었다. 3. 생쥐 생식줄기세포를 체외에서 효과적으로 분리, 배양할 수 있는 조건을 확립하였다.

• Animal Bioscience
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• 제34권10호
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• pp.1579-1589
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• 2021

Objective: This study was conducted to generate single stranded DNA oligonucleotides with selective affinity to bovine spermatozoa, assess its binding potential and explore its potential utility in trapping spermatozoa from suspensions. Methods: A combinatorial library of 94 mer long oligonucleotide was used for systematic evolution of ligands by exponential enrichment (SELEX) with bovine spermatozoa. The amplicons from sixth and seventh rounds of SELEX were sequenced, and the reads were clustered employing cluster database at high identity with tolerance (CD-HIT) and FASTAptamer. The enriched nucleotides were predicted for secondary structures by Mfold, motifs by Multiple Em for Motif Elicitation and 5' labelled with biotin/6-FAM to determine the binding potential and binding pattern. Results: We generated 14.1 and 17.7 million reads from sixth and seventh rounds of SELEX respectively to bovine spermatozoa. The CD-HIT clustered 78,098 and 21,196 reads in the top ten clusters and FASTAptamer identified 2,195 and 4,405 unique sequences in the top three clusters from the sixth and seventh rounds, respectively. The identified oligonucleotides formed secondary structures with delta G values between -1.17 to -26.18 kcal/mol indicating varied stability. Confocal imaging with the oligonucleotides from the seventh round revealed different patterns of binding to bovine spermatozoa (fluorescence of the whole head, spot of fluorescence in head and mid- piece and tail). Use of a 5'-biotin tagged oligonucleotide from the sixth round at 100 pmol with 4×10⁶ spermatozoa could trap almost 80% from the suspension. Conclusion: The binding patterns and ability of the identified oligonucleotides confirms successful optimization of the SELEX process and generation of aptamers to bovine spermatozoa. These oligonucleotides provide a quick approach for selective capture of spermatozoa from complex samples. Future SELEX rounds with X- or Y- enriched sperm suspension will be used to generate oligonucleotides that bind to spermatozoa of a specific sex type.

• 한국가금학회지
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• 제48권4호
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• pp.185-191
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• 2021

닭 정자는 저온 환경에서 생존하는 능력을 보유하고 있으나 정자의 운동성과 첨체 손상에 대한 연구는 상세히 알려져 있지 않다고 판단된다. 본 연구에서는 닭 정자를 BPSE 및 Lake 희석액으로 희석하고 보존 용기를 1.5 mL e-tube와 0.5 mL 스트로에 보존하여 정자 장수성을 조사하였다. 밀봉된 0.5 mL 스트로에 보존된 정자의 운동성이 3일동안 저온보존 시 더 높은 것으로 관찰되었다(68.6±3.1% vs. 22.1±5.7%). BPSE로 희석하여 0.5 mL 스트로에 보존된 닭 정자의 운동성 또한 Lake 희석액보다 더 높게 관찰되었다(57.7±5.6% vs. 37.7±5.4%). 첨체 온전성도 0.5 mL 스트로에 보존하는 것이 우수하였고 냉장보존일이 증가함에 따라 손상된 첨체 비율이 증가하였다. 본 연구에서 0.5 mL에 밀봉된 닭 정자는 저온 보존에 이용될 수 있으며 정자의 운동성을 높여주며 첨체 온전성을 증가시키는 것으로 관찰되었다.

• 한국가축번식학회지
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• 제25권4호
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• pp.305-315
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• 2001

본 연구의 목적은 외래 EGFP 유전자를 분화이전의 웅성생식세포에 도입한 후 이를 난모세포내에 미세주입하여 형질전환동물을 생산하는 기술을 개발하는 데에 있다. 이를 위하여 반수체 정자세포에서 특이적으로 발현하는 생쥐의 mTP1과 햄스터의 hPrm2 유전자 발현 시기를 RT-PCR로 조사한 결과 그시기는 생쥐와 햄스터에서 각각 18일령과 20일령으로 확인되었다. 이에 따라 외래 유전자의 침입이 용이한 감수분열 직전단계인 17일령의 생쥐와 19일령의 햄스터 정자세포를 EGFP 유전자가 포함된 배양액에 부유시킨 다음, 전기자극을 부여한 결과 0.18 ㎸/cm의 전기자극을 가한 후 72시간 배양한 정자세포의 28.5%와 32.1%에서 EGFP 유전자가 발현되는 것으로 확인되었다. EGFP유전자가 도입된 반수체 정자의 수정 및 발달 능력을 검증하기 위하여, 이들 정자세포를 햄스터 난자 내에 미세주입 하였으나, 형광현미경하에서는 EGFP유전자의 발현은 관찰할 수 없었다. 이에 이들 난자를 공시하여 PCR분석을 실시한 결과, 약 44%의 수정란에서 EGFP 유전자의 존재가 확인되었다. 이러한 결과로 보아 반수체 정자세포는 외래 유전자를 난자 내에 도입하기 위한 운반체로 이용될 수 있을 것으로 생각된다.