This study investigated the use of bovine serum albumin (BSA) as alternatives to fetal bovine serum (FBS) in in vitro maturation medium. The oocyte maturation, cumulus cell-oocyte gap junctional communication, and development of bovine embryos were determined by assessing their cell number, lipid content, mitochondrial activity, gene expression and cryo-tolerance. Oocytes were cultured in TCM-199 supplemented with 1 μg/ml estradiol-17ß, 10 μg/ml FSH, 10 ng/ml EGF, 0.6 mM cysteine, 0.2 mM sodium pyruvate and either 8% BSA (BSA group), 10% FBS (FBS group), or neither BSA nor FBS (TCM group), and followed by in vitro fertilization and the zygotes were cultured in SOF-BE1 medium. The differences in embryo development between experimental groups were analyzed by one-way ANOVA. We have shown that the percentages of embryos that underwent cleavage and formed a blastocyst were non significantly different among all experimental groups (37.4 ± 1.5% for FBS group vs. 31.1 ± 3.9% for BSA group and 34.5 ± 1.6% for TCM group, six replicates were performed). Furthermore, there was no significant difference between the percentage of MII oocyte between FBS (71.8 ± 1.9%) and BSA groups (69.3 ± 2.3%). However, culture of oocytes with FBS increased (P < 0.05) the cumulus cell expansion as well as expression of gape junction proteins, CX37 and CX43, at both transcriptional and translation levels. We also found that FBS significantly increased total cell number and decreased the apoptotic index in day-8 blastocyst comparing to BSA group. The beneficial effects of BSA on embryos were associated with significantly reduced intracellular lipid content and increased mitochondrial activity in both oocytes and blastocyst. Taken together, these data suggest that supplementation of maturation medium with BSA, as alternatives to FBS, can be used as defined medium that support consistently the development of IVP bovine embryos.

This study was conducted to investigate the effects of alpha-lipoic acid (aLA) as an antioxidant that decrease the reactive oxygen species (ROS) in bovine embryonic development. Slaughterhouse derived bovine immature oocytes were collected and 4 different concentrations (0, 5, 10 and 20 mM) of aLA was supplemented in bovine in Vitro maturation (IVM) medium. After 20 hrs of IVM, maturation rates, levels of ROS and glutathione (GSH), and further embryonic development after parthenogenetic activation (PA) and in Vitro fertilization (IVF) was investigated according to aLA concentrations. Maturation rate was significantly higher in 10 mM group than other groups (80.5% vs. 62.9, 73.9, 64.2%; P<0.05). In the levels of ROS and GSH in matured oocytes as an indicator of oocyte quality, significantly better results were shown in 5 and 10 mM groups compared with other 2 groups. After IVM, significantly higher rates of blastocyst formation were shown in 10 mM groups in both of PA (27.9% vs. 18.8, 22.3, 14.2%; P<0.05) and IVF (32.6% vs. 23.9, 27.3, 16.2%; P<0.05) embryos. In addition, significantly more cell total cell number and higher inner cell mass ratio in 10 mM PA and IVP blastocysts showed developmental competence in 10 uM groups. Therefore, based on the entire data from this study, using 10 μM of aLA confirmed to be the optimal concentration for bovine oocyte maturation and embryonic development.

Several species show low sensitivity to IVM, and the development of optimized medium possible oocyte quality and stable growth. Furthermore, adding additive to the medium can effectively reducing development cost and leads to easy handling of oocytes. Isoliquritigenin and formononetin are extracts found in licorice. Previous studies reported that isoliquritigenin and formononetin affected the activity of sperm, but the oocytes are unknown. This study adds isoliquritigenin or formononetin to αMEM to mature oocytes under simple IVC conditions. Recovered oocytes are cultured in αMEM, isoliquritigenincontaining medium and formononetin-containing medium. In study we proved that in addition to the medium, above the quality of oocytes cultured when specific additives were added, more stable growth is possible. collection and IVM of oocyte. SD rats at 6 to 8 wks of age are injected is intraperitoneal with 30 IU/mL of PMSG and 48 hrs later, HCG 50 IU/mL is intraperitoneal injected. Oocytes are collected ovary after 17 hrs. Collected oocytes are cultured for 16 hrs with 200 μL αMEM and 200 μL αMEM containing isoliquritigenin or formononetin at 0, 0.01, 0.02, 0.04, 0.1 mg/mL. Also, isoliquritigenin and formononetin were mixed with 200 μL αMEM at a ratio of 0.25: 0.75, 0.50: 0.50, and 0.75: 0.25 mg/mL respectively. Oocytes supplemented with isoliquritigenin and Formononetin had high quality than oocytes cultured with αMEM and showed an increase in the IVF fertility rate. Our experimental results indicate that using isoliquritigenin, formononetin when cell culture, rather than used only in medium, more effective oocyte quality and stable growth.

In vitro maturation (IVM) systems have become indispensable for the production of large numbers of competent oocytes in domestic species. The quality of in vitro matured oocyte is one of the important factors determining the success of assisted reproductive technologies (ARTs) including intracytoplasmic sperm injection (ICSI), in vitro fertilization (IVF), and somatic cell nuclear transfer (SCNT) in human and livestock. Incomplete cytoplasmic maturation of oocytes can lead not only to a failure of fertilization but also to a developmental arrest after ARTs. Thus, establishment of a stable IVM system to produce a large number of high quality oocytes, especially in domestic animals, is essential for improvement of ARTs efficiency by producing high quality embryos. The morphological characteristics are commonly used to predict the developmental potential of oocytes and embryos. Usually, normal oocytes shrink when exposed to a hypertonic medium, and recover their morphology when returned to an isotonic medium. During this process, oocytes show various morphologic changes, such as shrinkage in spherical (SSP) or irregular shapes (SIR). In the first study, we investigated whether the shrinkage pattern of oocytes that was observed after hyperosmotic treatment could be used as a morphologic characteristic to predict the quality of IVM oocytes in pigs. We found that SSP oocytes showed improved developmental competence after PA and SCNT. This improved embryonic development was most likely because of the more advanced nuclear and cytoplasmic maturation in SSP oocytes compared with SIR oocytes. Pig oocytes shows a wide variation in the size of perivitelline space (PVS) after IVM. Based on this finding, we examined in the second study whether or not there was any correlation between the PVS size of IVM oocytes and their developmental competence after PA and SCNT. Our results demonstrated that in vitro developmental competence to the blastocyst stage positively correlated with the size of the PVS of oocytes after IVM. In addition, we observed that mature oocytes with a larger PVS showed higher levels of intracellular GSH content and transcription factor expression. Furthermore, enlargement of the PVS by culturing in reduced NaCl medium improves the embryonic development after PA and SCNT. In the third study, we investigated the effects of a hypotonic medium with reduced NaCl (61.6 mM) compared with an isotonic medium (108.0 mM NaCl) on oocyte maturation and embryonic development after PA and SCNT. In addition, we attempted to optimize our IVM system using a hypotonic maturation medium by examining the effects of hypotonic medium during various stages of IVM on oocyte maturation and subsequent embryonic development. Our results demonstrated that maturation of pig oocytes in hypotonic medium with reduced NaCl during the last 11 hr of IVM increased the developmental competence of oocytes after PA and SCNT. These beneficial effects was also shown in a commercial medium (a minimum essential medium; aMEM) in which the NaCl concentration was reduced to 61.6 mM. In addition, IVM of oocytes in medium with reduced NaCl increases the proportion of SSP oocytes in pigs. In summary, our results demonstrate that IVM of pig oocytes in a hypotonic medium with low-NaCl is better able to support embryonic development after PA and SCNT, most likely by improving the cytoplasmic maturation via increased intraoocyte GSH content and widened PVS. Based on these results, the newly developed IVM system using a hypotonic medium with reduced NaCl can produce high quality oocytes and be considered a new strategy for improving ARTs efficiency in pigs.

체외 배양액에 성장호르몬 및 사이토카인의 첨가는 초기배 발육 및 생산된 배반포의 질에 영향을 미칠 수 있다. 본 연구는 돼지 유도만능줄기세포(porcine induced pluripotent stem cell, piPSC)의 조정배지(conditioned medium, CM)가 돼지 난자의 체외성숙 및 단위발생 후 초기배 발육에 미치는 영향을 검토하기 위하여 수행하였다. 난자-난구세포 복합체(cumulus-oocyte complex, COC)는 0(control), 25, or 50%의 줄기세포 배양액(stem cell medium, SM) 또는 CM이 첨가된 체외성숙 배양액으로 배양하였으며, 성숙된 난자는 활성화 유도 후 같은 농도의 SM 또는 CM을 첨가한 체외배양액에서 배양하였다. 체외 성숙율은 CM-25% 그룹에서 대조구보다 유의적으로 높았으나(p<0.05), 다른 SM 또는 CM 처리구와는 차이가 없었다. 배반포 형성율은 CM-25% 그룹(29.2%)에서 대조구(20.7%), SM-50%(19.6%) 및 CM-50%(23.66%) 처리구보다 유의적으로 높았다(p<0.05). 배반포에서의 세포수 및 세포사 비율은 SM-25% 그룹이 대조구에 비하여 유의적인 차이가 나타났다(p<0.05). 난자의 질과 연관되어 있는 유전자들(Oct4, Klf4, Tert 및 Zfp42)의 발현은 CM-25% 그룹에서 대조구보다 유의적으로 증가되었다(p<0.05). 따라서 본 실험의 결과 체외성숙(IVM) 및 체외발달(IVC) 배양액에 25% 수준의 CM의 첨가는 돼지 단위발생 난자의 배발달과 난자의 질적 향상에 기여하는 것으로 사료된다.

Antioxidants, as reactive oxygen species scavengers, are one of the beneficial additives in serum-free defined culture medium. In this study, three separate experiments were performed to determine the effects of 3-hyroxyflavone added to the culture medium on the developmental competence of follicular bovine oocytes during in vitro maturation (IVM) and/or in vitro culture (IVC). The rate of blastocyst developed from oocytes cultured in IVM medium with 3 hyroxyflavone was significantly higher than that from control oocytes (39.0% vs. 26.3%, p<0.001), respectively. However, oocytes cultured in the medium with addition of 3-hyroxyflavone only at IVC period did not show significance in the blastocyst development when compared with control. When 3-hyroxyflavone was added to both IVM and IVC media, the rate of blastocyst formation was even significantly lower (21.1%) than control (26.5%; p<0.05). The present findings suggested that antioxidative activity of 3-hydroxyflavone added to only IVM medium beneficially affected the developmental competence of follicular bovine

This study was conducted to examine the effects of human follicular fluid and gonadotropin (FSH+HCG+rhEGF) on in vitro maturation, fertilization and development of human immature oocytes. Cumulus-oocyte complexes (COCs) were collected following for in vitro fertilization and embryo transfer (IVF-ET) cycles of the patients. At the time of oocytes collection, oocytes were classified into MII, MI and GV in accordance with their appearance (MII: Fully mature oocyte at metaphase II of meiosis; MI: Nearly mature oocytes at metaphase I of meiosis; GV: Immature oocytes at prophase I of meiosis). After controlled ovarian stimulation using gonadotropin(FSH) and human chorionic gonadotropin (HCG) in 70 ICSI cycles, 158 MI to MII matured oocytes were intracytoplasmic sperm injection (ICSI) h after in vitro culture and 553 MII oocytes were ICSI after denudation. The aspirated MI and GV oocytes were cultured in culture medium containing 10% (v/v) serum protein substitute (SPS), 10% (v/v) human follicular fluid (hFF) and 10% (v/v) serum protein substitute (SPS)+1 IU/ml FSH+10 IU/ml HCG+10 ng/ml recombinant human epidermal growth factor (rhEGF). The maturation rate of immature oocytes was similar among the three group. When maturation medium was supplemented with 10% SPS, 10% hFF or gonadotropins, the fertilization rate of in vitro matured oocytes was higher in 10% SPS (80.0%), but there was no statistical significance (78.2%; hFF, 76.9%; gonadotropin, p>0.05). The development rate of human embryos developed to cells were not significant difference in the medium containing SPS, hFF and gonadotropins (65.6%, 65.9% and 66.7%). The results of these study suggest that human follicular fluid and gonadotropins supplemented in the culture medium was not effected on the in vitro maturation, fertilization and development of human immature oocytes.

본 연구는 돼지 수정란의 체외 성숙 및 체외 배양액의 retinol 첨가 효과를 규명하기 위하여 체외 성숙 및 체외 배양액에 retinol을 첨가하여 수정란의 체외 발달에 미치는 영향을 구명하고자 수행되었다. 체외 성숙 배양액에 retinol을 첨가한 결과 성숙율은 으로 각 처리구 간의 유의적인 차이가 없었다(p>0.05). 체외 수정 후 배반포로의 발달율은 첨가구에서 의 발달율을 나타내어 타 처리구에 비하여 유의적으로(p<0.05) 높게 나타났으며,

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.

본 연구는 한우 난포란이 체외성숙된 환경의 변화를 단백질 측면으로부터 검토하기 위하여 체외성숙 배지와 세포질내 단백질 변화와 종류를 검토하였다. 그 결과 배지 내의 단백질 발현량은 배양 4.5시간째까지 감소하였고, 배양 13.5시간째까지는 변화가 없었다. 그러나 배양 13.5~18시간 사이에 증가한 후 배양 18시간 이후에 다시 감소하는 경향이었다. 세포질내의 단백질 발현량은 배양 4.5시간째까지 증가하였고 배양 9시간째까지 급격히 감소하였다. 배양 9시간째부터 18시간째 까지는 단백질 발현량이 유사한 경향이었으나 배양 18시간째부터 24시간째까지 다시 증가하였다. 한편 체외성숙한 배지와 세포질을 2차원 전기영동하여 각각 298개 및 35개의 단백질 spot을 확인하였고, 그 중 배지에서는 28개, 세포질에서는 5개의 spot이 유의적인 변화를 확인하였다. 이들 spot 대한MALDI-TOP분석으로 배지와 세포질에서 각각 8개 및 1개의 단백질을 동정하였다. 그 종류는 aldose reductase, alpha enolase, apolipoprotein A-1 precursor, 43M1a collectin precursor, heat shock 27kDa protein, plasminogen activator inhibitor-1 precursor, thrombospondin 1 transitional endoplasmic reticulum ATPase 및 β-tubulin이었다.

본 연구는 배양액의 종류에 따른 돼지 난자의 성숙 및 단위발생란의 배반포 형성율을 검토하였으며, 배양액의 삼투압과 발달 시기에 따른 배양액의 삼투압 변화가 돼지 단위발생란의 발달에 미치는 영향을 검토하였다. 실험 1에서 난포란을 NCSU-23 mWM 및 mKRB에 각각 성숙배양한 결과 성숙률은 62.1~71.3%로 배양액에 따른 차이가 없었다. 실험 2에서는 각각의 배양액으로 성숙된 난자를 활성화 처리 후 동일한 배양액으로 6일간 배양하여 발달율을 검토한 결과, 배반포 발육율은 NCSU-23에 배양 시 22.9%로 타 그룹(0~0.6%)보다 유의적으로 높게 나타났다(P<0.05). 실험 3에서는 단위발생란을 NaCl 안에 의해 256, 280 및 300 mOsmol(mOsm)로 조정한 NCSU-23에 6일간 배양한 결과, 배반포 발육율은 11.0~14.4%로 실험군 간에 유의적인 차이는 없었으나 삼투압이 낮을수록 난자의 fragment 비율이 높게 나타났다(P<0.05). 실험 4에서는 단위발생란을 삼투압이 조정된 세 종류의 NCSU-23에 48시간 배양한 후 삼투압이 높거나 낮은 NCSU-23으로 옮겨 4일간 추가 배양한 결과 배반포 형성율은 배양 48시간 후에 배양액의 삼투압을 낮춰 주었을 때(21.0%)가 높여 주었을 때 (11.8%) 보다 유의적으로 높게 나타났다(P<0.05).본 실험의 결과는 돼지 단위발생란의 발육이 배양액의 종류 및 삼투압에 의해 영향을 받으며, 배양액의 삼투압은 돼지 단위발생란의 발육 단계별로 영향을 주어, 초기에는 높은 삼투압의 배양액에서 배양하고 일정 시간 후 낮은 삼투압의 배양액으로 배양함으로써 발육이 증진될 수 있음을 시사한다.

본 연구에서는 소 체외수정란 생산에 있어서 체외성숙용 배지에 첨가하는 혈청과 호르몬의 효과를 검토하기 위하여 제1극체 출현율과 배발달율을 조사하였고, 생산된 배반포의 품질을 평가하기 위하여 세포수를 검토하였다. 1. 체외성숙용 배지에 혈청 및 성선자극호르몬의 첨가에 따른 한우 난포란의 제1극체 출현율은 비슷한 경향이었다. 배반포까지의 발달율은 혈청 및 성선자극호르몬 공동 첨가군(26.0％)이 대조군과 성선자극호르몬 단독 첨가군보다 유의하게 높았다(P<0

본 연구는 체외에서 한우 난포란의 핵성숙과 그 후의 초기 배발달에 있어서 체외성숙 배지에 아미노산의 첨가가 난포란의 제1극체(PB) 출현율, 배발달율 그리고 배반포의 세포수에 미치는 영향을 검토하였다. 첨가하는 아미노산의 종류와 농도는 각각 MEM 배지의 non-essential amino acids (NEAA)와 BME 배지의 essential amino acids(EAA)는 1, 2, 4배 및 유청중의 lactalbumine hydrolysate(LAH)는 1, 5, 10 ㎎/㎖이었다. 그 결과 NEAA와 EAA의 경우 PB 출현율은 1배 첨가군이 미첨가군보다 유의하게 높았지만(p＜0.05), 첨가량이 증가할수록 오히려 감소하였다. 그러나 배반포로의 배발달율은 모든 군에서 비슷한 경향이었다. 그리고 배반포의 총 세포수와 총 세포수중 TE 세포수는 2배 처리군이 가장 높았으며, ICM 세포수는 아미노산 첨가량이 증가할수록 많아졌다. 한편 LAH의 경우 PB 출현율은 5㎎ 첨가군이 가장높았으며, 배반포로의 발달율은 미첨가군과 1 ㎎ 첨가군이 5 ㎎ 첨가군과 10 ㎎ 첨가군보다 각각 유의하게 높았다(p＜0.05). 그리고 배반포의 세포수는 NEAA와 EAA를 이용하였을 경우와 비슷한 경향이었다. 이상의 결과로부터 체외성숙용 배지에 아미노산의 첨가는 생산된 배반포의 품질을 향상시킬 수 있기 때문에 배반포의 체외생산에 이용할 수 있는 새로운 아미노산의 종류 및 농도를 탐색할 필요가 있다고 사료된다.

성숙배양액과 단백질이 개 미성숙난자의 체외성숙에 미치는 영향