• 농업과학연구
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• 제48권1호
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• pp.163-170
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• 2021

Bacterial infections in the female reproductive tract negatively affect ovarian function, follicular development, and embryo development, leading to the eventual failure of fertilization. Moreover, bacterial lipopolysaccharides (LPS) can interfere with the immune system and reproductive system of the host animal. Therefore, this study examined the effect of LPS on the in vitro maturation (IVM) of pig oocytes. Oocytes were matured in TCM199 medium in the presence of varying concentrations of LPS (0 - 50 ㎍·mL^-1). The maturation rate, cortical granules (CGs) migration, and chromosome alignment were subsequently evaluated during the meiotic development of the oocytes. We observed a dose-dependent and significant decrease in the metaphase II (MII) rate with increasing concentrations of LPS (97.6% control [0 ㎍·mL^-1 LPS] vs. 10.4-74.9% LPS [1 - 50 ㎍·mL^-1], p < 0.05). In addition, compared to the control oocytes without LPS, higher levels of abnormal CGs distribution (18.1 - 50.0% LPS vs. 0% control), chromosome/spindle alignment (20.3 - 56.7% LPS vs. 0% control), and intracellular ROS generation were observed in oocytes matured with LPS (p < 0.05). Nitrite levels were also increased in the maturation medium derived from the oocytes matured with LPS (p < 0.05). These results indicate that LPS induces oxidative stress during IVM and affects oocyte maturation, including CGs migration and chromosome alignment of pig oocytes.

• 한국수정란이식학회지
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• 제21권3호
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• pp.255-262
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• 2006

본 연구는 Open Pulled Straw(OPS)방법에 의해 동결-융해한 돼지 수정란의 체외 생존 능력을 검토하기 위하여 수행되었다. 미성숙 난자의 체외 성숙을 위하여, 돼지 난소는 도축장에서 회수하였으며, 난구세포로 쌓여있는 난자는 직경 $2{\sim}6mm$의 난포로부터 난포액과 함께 흡입에 의하여 회수하였다. 회수된 미성숙 난자의 체외 성숙을 위하여 5 mM hypotaurine, 0.57 mM cysteine, 10% 난포액, 10 IU/ml PMSG 및 10 IU/ml hCG가 함유된 NCSU-23 배양액 내에서 $21{\sim}22$ 시간 배양 후, 호르몬 물질을 제거한 성숙 배양액 내에서 $21{\sim}22$ 시간 동안 추가 배양하였다. 한편 체외 수정을 위하여 동결-융해한 정액은 5.56 mM glucose, 0.33 mM na-pyruvate, 100 IU/ml penicillin, $100 {\mu}g/ml$ streptomycin 및 4 mg/ml BSA가 첨가된 D-PBS 액을 가지고 1,500 rpm에서 10분간 2회 원심분리를 실시하여 세척하였다. 체외 수정을 위한 배양액은 pH $7.2{\sim}7.4$에서 2 mM caffeine과 2 mg/ml BSA가 첨가된 mTBM 배양액을 이용하였다. 체외 수정시 정자의 최종 농도는 $2.5{\times}10^6cells/ml$로 조정하였다. 수정 8시간 후, 체외 발육을 위하여 5.0 mM hypo-taurine, 4 mg/ml BSA 및 10 ng/ml EGF가 첨가된 NCSU- 23액으로 옮겨 7일간 배양을 계속하였다. 그 후 배반포의 여러 단계에서 OPS 법에 의해 동결-보존하였다. 그 결과, 동결-융해 후 형태학적으로 정상적인 수정란의 비율은 초기 배반포(28.3%)보다는 확장 배반포(38.9%)단계에서 동결했을 때 유의적으로 높게 나타났다(p<0.05). 한편, 동결-융해 후 상해를 입은 수정란의 비율은 확장 배반포보다는 초기 배반포 단계에서 동결된 수정란에서 유의적으로 높은 성적을 보였다(p<0.05). 또 다른 실험에서, 수정란의 동결-융해 후 형태학적으로 정상인 수정란을 48시간 추가 배양했을 때, Hatching 된 수정란의 비율은 초기 배반포, 배반포 및 확장배반포기 단계에서 동결-융해한 수정란에서 각각 6.7, 20.0 및 33.3%로 발육 단계가 높을수록 동결-융해 후의 생존율도 높게 나타났다. 본 연구의 결과로부터, 돼지에서 수정란의 동결-융해 후 생존성의 향상을 위해서는 발육 단계가 높은 배반포기 단계에서의 동결이 요구되며 본 연구에서 이용된 OPS 동결 방법이 폭넓게 활용될 것으로 사료된다.

• 한국가축번식학회지
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• 제21권4호
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• pp.335-343
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• 1997

The effect of thiol compounds on development and intracellular glutathione(GSH) concentrations of bovine embryos produced by in vitro maturation and in vitro fertilization(IVM/IVF) was examined in CRlaa medium with or without $\beta$-mercaptoethanol(0, 10, 25 and 50$\mu$MME) and cysteamine(0, 25, 50 and 75 $\mu$M). Numbers of cells comprising blastocysts were also counted using double fluorescence stain and the total glutathione levels(oxidized and reduced form) of morula and blastocyst embryos were than measured by an enzymatic method. Following routine IVM/IVF procedures oocytes and zygotes were cultured for 40 to 44h in CRlaa medium. Then 2 to 8-cell embyos had cumulus cell removed and were allotted randomly to the experimental medium. In Experiment 1, the proportion of embryos developing to and beyond morulae stages in 0, 10, 25 and 50 $\mu$M $\beta$-ME was 42.9%, 50.0%, 53.7% and 65.6%, respectively. Fifty $\mu$M $\beta$-ME group was significantly higher than those of any other groups (P<0.05). In Experiment 2, the percentages of embryos developed beyond morulae stages in 0, 25, 50 and 75 $\mu$M cysteamine was 42.9%, 40.4%, 60.0% and 59.2%, respectively. Fifty and 75$\mu$M cysteamine groups were significantly higher than in 0 and 25 $\mu$M cysteamine groups, but all of culture medium containing cysteamine(52.6%) was not significantly difference in control group(42.9%). In Experiment 3, the intracellular GSH concentrations of morulae and blastocyst embryos in 0 and 50 $\mu$M $\beta$-ME was 42.4 pM and 44.9 pM, 49.5 pM and 67.8 pM, respectively. Morulae embryos were not difference, but blastocyst embryos were significantly difference between treatments(P<0.05). In Experiment 4, the intracellular GSH concentrations of morulae in CRlaa with or without cysteamine were 39.8 pM and 45.6 pM, and blastocysts were 59.3 pM and 66.8 pM, respectively. Cell numbers of blastocysts were similar to in all experimental groups. These experiments indicate that thiol compounds can increase the proportion of embryos that developing to and beyond morulae stage and the intracellular GSH concentrations.

• Asian-Australasian Journal of Animal Sciences
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• 제15권2호
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• pp.172-178
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• 2002

The present study examined whether treatment of in vitro matured pig oocytes with calcium ionophore (A23187) could prevent polyspermic penetration in vitro. When oocytes cultured for maturation for 33, 36 or 44 h were subsequently treated with $50{\mu}M$ A23187 in medium with fetal calf serum (FCS) for 1, 2 and 3 h and then cultured for 12 h without spermatozoa, virtually no activation occurred. In the absence of FCS, however, 31-42, 45-49 and 56-64% of oocytes were activated, respectively. When oocytes treated with $50 {\mu}M$ A23187 in medium with FCS for 3 h were inseminated in vitro, the penetration rates (14-57%) were lower (p<0.01) with a higher (p<0.01) incidence (35-67%) of monospermy compared with untreated oocytes (69-80% penetration and 15-17% monospermy). However, sperm penetration was completely blocked in all oocytes treated with A23187 in the absence of FCS. When oocytes matured for 33 h were treated with different concentrations of A23187 for 3 h and inseminated in vitro, the penetration rate did not change but there was an increased incidence (p<0.05) of monospermy at $10-20{\mu}M$ and $2.5-5{\mu}M$ A23187 in the presence and absence of FCS, respectively, compared with at $0{\mu}M$ A23187. With these lower concentrations of A23187, treatment of oocytes for at least 60 and 30 min in the presence and absence of FCS, respectively, was required to increase the incidence of monospermy without reducing penetration rate. These results indicate that a high concentration ($50{\mu}M$) of A23187 in medium without FCS, but not in medium with FCS, stimulated in vitro matured pig oocytes to induce parthenogenetic activation and a complete block to sperm penetration in vitro. However, treatment of oocytes with lower concentrations of A23187 ( $10-20{\mu}M$ and $2.5-5{\mu}M$) both in the presence and absence of FCS maintained sperm penetration in vitro and increased the incidence of monospermy.

• 한국수정란이식학회지
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• 제21권2호
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• pp.121-128
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• 2006

본 연구는 고품질의 돼지 체외 수정란을 생산하기 위하여 체외 성숙 배지에 첨가하는 polyvinylpyrrolidone(PVP)의 분자량, 첨가 농도 및 시간(실험 1)과 체외 성숙 수정 배양 단계에서 PVP의 첨가(실험 2)가 배 발생과 세포수에 미치는 효과를 검토하였다. 돼지 미성숙 난자의 체외 성숙은 NCSU 23 용액, 체외 수정은 mTBM 용액, 체외 배양은 PZM 3 용액을 이용하였다. 체외 성숙용 배지에서 PVP의 분자량, 농도 및 첨가 시간에 따른 수정율과 배반포 발달율은 차이가 없었다. 그러나 배반포의 부화율은 분자량 40,000(30.1%), 0.5%(28.6%) 및 $0{\sim}44$시간(37.5%) 첨가군이 다른 시험군에 비하여 유의하게 높았다(p<0.05). 분자량 40,000 0.5% PVP를 체외성숙, 수정 및 배양 배지에 각각 첨가(W) 또는 미첨가(W/O)한 결과, 수정율은 체외성숙 수정 배양에서 W-W/O-W 군이 69.3%, 배반포 발달율과 부화율은 W-W/O-W 및 W-W/O-W/O 군이 각각 12.6과 30.0% 및 14.0과 37.5%로서 다른 군에 비하여 유의하게 높았다(p<0.05). 총 세포 수와 Trophoblast 수는 처리군 간에 유사한 경향이었으나, Inner cell mass 수는 W/O-W/O-W 군이 평균 7.4개로서 가장 높았다 (p<0.05).

• 한국수정란이식학회지
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• 제30권4호
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• pp.315-317
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• 2015

In vitro culture of murine embryos is an important step for in vitro production systems including in vitro fertilization and generations of genetically engineered mice. M16 is widely used commercialized culture media for the murine embryos. Compared to other media such as potassium simplex optimization medium, commercial M16 (Sigma) media lacks of amino acid, glutamine and antibiotics. In the present study, we optimized M16 based embryo culture system using commercialized antibiotics-glutamine or amino acids supplements. In vivo derived murine zygote were M16 media were supplemented with commercial Penicillin-Streptomycin-Glutamine solution (PSG; Gibco) or MEM Non-Essential Amino Acids solution (NEAA; Gibco) as experimental design. Addition of PSG did not improved cleavage and blastocyst rates. On the other hand, cleavage rate is not different between control and NEAA treated group, however, blastocyst formation is significantly (P<0.05) improved in NEAA treated group. Developmental competence between PSG and NEAA treated groups were also compared. Between two groups, cleavage rate was similar. However, blastocyst formation rate is significantly improved in NEAA treated group. Taken together, beneficial effect of NEAA on murine embryos development was confirmed. Effect of antibiotics and glutamine addition to M16 media is still not clear in the study.

• 한국수정란이식학회지
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• 제11권3호
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• pp.241-248
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• 1996

This study was conducted to improve the production efficiency of in vitro produced (IVP) embryos in Korean Native cows. The optimal conditions and procedures for in vitro maturation(IVM), in vitro fertilization(IVF) and in vitro culture(IVC) of bovine follicular oocytes and IVP embryos were evaluated. Immature follicular oocytes were collected fiom the follicles of bovine ovaries obtained from abattoirs. The oocytes of Grade I and II for IVM were cocultured with monolayered bovine oviductal epithelial cells(BOEG) or granulosa cells in TCM-199 solution supplemented with follicle stimulating hormone, lutenizing hormone, estradiol-17$\beta$ and heat inactivated fetal calf serum at 39$^{\circ}C$ under 5% $CO_2$ in air for 14 to 24 hours. Most of the oocytes(93%) matured to metaphase II in 24 hours. The cocultured IVM oocytes were fertilized in vitro at significantly(P<0.05) higher rate with BOEC(83.8%) and with granulosa cells(84.6%) than the non-cocultured IVM oocytes(73.6%). The IVM-IVF embryos developed to morula and blastocyst at significantly(P<0.05) higher rate in coculture with BOEC(41.2%) than with granulosa cells(23.1%) or conditioned medium(23.4%).

• Asian-Australasian Journal of Animal Sciences
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• 제11권3호
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• pp.307-310
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• 1998

The present studies were undertaken to evaluate the effects of a low concentration of dimethyl-sulfoxide (DMSO) on in vitro maturation and development of bovine oocytes fertilized in vitro. Significantly more oocytes reached the metaphase stage of the second meiotic division in TCM-199 supplemented with $50{\mu}M$ DMSO than in the control medium (p < 0.05), and the highest rates of development up to the blastocyst stage were obtained when $50{\mu}M$ DMSO was added to the maturation and culture media (p < 0.05). The avarage of cell numbers of the blastocysts, expanded and hatched blastocysts cultured with $50{\mu}M$ DMSO were 81.7, 125.7 and 129.9 cells, respectively. The proportion of blastocysts with normal chromosome numbers was 90.5%. These results suggest that the addition of $50{\mu}M$ DMSO is beneficial for the maturation of bovine oocytes and production of the blastocysts with high quality.

• Clinical and Experimental Reproductive Medicine
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• 제16권1호
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• pp.15-21
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• 1989

An in vitro fertilization assay employing zona-free hamster embryos was used to investigate human spermatozoal fertilitzing ability. Yanaghimarchi et al.(1976) first introduced this cross species fertilzation technique, with its application as a diagnostic tool for male infertility. Human spermatozoa were preincubated for 3 to 4 hrs in B W W medium at concentration of $4{\times}10^6$ sperm/ml prior to the addition to zona-free hamster embryos. After 3 hrs, human sperm was evaluated for fertilizing potential by the presence of swelling or decondencing sperm head in the cytoplasm. The results of penetration rates for sperm were as follow : 1. The average penetration rate of a 7 fertile donor group was $47.8{\pm}27.67%$(Range 14.3-98.0%) 2. The average penetration rate of 12 infertile patients with normal semen analysis was $21.7{\pm}26.9%$(Range 0-38.8%) 3. The average penetration rate of 10 infertile patients with semen abnormalities was $6.1{\pm}8.1%$(Range 0-25%)

• 한국수정란이식학회지
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• 제11권1호
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• pp.71-80
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• 1996

The envelope of the rnannnalian oocyte plays crucial roles in sperm-oocyte interactions by providing sperm receptors, inducing acrosome reaction and preventing polyspermy. Understanding of properties of the zona pellucida (ZP) is essential for the artificial control of fertility in mammals. This study was carried out to produce and characterize monoclonal antibodies(MAbs) to porcine ZP proteins. Approximately 8,000 ZPs were obtained from follicular oocytes and dissolved in 40$\mu$l of double distilled water. Following immunization through foot-pad injections of Balb /c mice with a ZP solution, the popliteal lymph nodes were recovered at 2 weeks after the last injection. Hybridoma cell lines were established by fusing lymph node cells with P3X63 myeloma cells through selection using HAT medium and screening by immunofluorescence(IF) microscopy on the isolated ZP. Secreted MAbs were found to consist k chains and different heavy chains as evidenced by isotyping. Some of the MAbs demonstrated high specificity to the ZP in IF. The Mabs also showed positive cross reactivity with hamster and mouse eggs, while negative with bovine eggs. The results implicate that the MAbs can be used not only for identification of functional regions of the ZP, but also for elucidation of mechanisms involved in fertilization of mammals. The MAbs will provide basic information on biochemical anatomy of the ZP as well as can be candidates for the future contraceptive vaccines.