• 한국발생생물학회지:발생과생식
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• 제17권1호
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• pp.63-72
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• 2013

A specific inhibitor of RNA polymerase II, ${\alpha}$-amanitin is broadly used to block transcriptional activities in cells. Previous studies showed that ${\alpha}$-amanitin affects in vitro maturation of cumulus-oocyte-complex (COC). In this study, we evaluated the target of ${\alpha}$-amanitin, and whether it affects oocytes or cumulus cells (CCs), or both. We treated ${\alpha}$-amanitin with different time period during in vitro culture of denuded oocytes (DOs) or COCs in comparison, and observed the changes in morphology and maturation status. Although DOs did not show any change in morphology and maturation rates with ${\alpha}$-amanitin treatment, oocytes from COCs were arrested at metaphase I (MI) stage and CCs were more scattered than control groups. To discover causes of meiotic arrest and scattering of CCs, we focused on changes of cumulus expansion, gap junctions, and cellular metabolism which to be the important factors for the successful in vitro maturation of COCs. Expression of genes for cumulus expansion markers (Ptx3, Has2, and Tnfaip6) and gap junctional proteins (Gja1, Gja4, and Gjc1) decreased in ${\alpha}$-amanitin-treated CCs. However, these changes were not observed in oocytes. In addition, expression of genes related to metabolism (Prps1, Rpe, Rpia, Taldo1, and Tkt) decreased in ${\alpha}$-amanitin-treated CCs but not in oocytes. Therefore, we concluded that the transcriptional activities of CCs for supporting suitable transcripts, especially for its metabolic activities and formation of gap junctions among CCs as well as with oocytes, are important for oocytes maturation in COCs.

• Reproductive and Developmental Biology
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• 제30권1호
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• pp.7-11
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• 2006

본 연구는 호르몬과 Na-pyruvate의 첨가가 개 미성숙난자의 체외성숙에 미치는 영향을 검토하였다. 개의 발정주기와 관계없이 암캐의 난소에서 회수한 난자를 NCSU-37 배양액에서 72시간 체외 배양하였다. 호르몬의 영향을 검토하기 위하여 배양액 중에 다양한 호르몬을 $24{\sim}72$시간 첨가하여 성숙율을 검사하였으며, Na-pyruvate 첨가 농도의 영향을 검토하였다. $E_2$ 단독 첨가구에서는 제2감수분열 중기(MII) 단계까지 성숙이 관찰되었으나(5.7%), 타 처리구에서는 MII기까지의 성숙이 이루어지지 않았다(P<0.05), $E_2$를 $6{\sim}24$시간만 처리하였을 때는 MII기로 성숙되는 난자가 없었으나 72시간 처리하였을 때는 6.0%로 유의적으로 높게 나타났다(P<0.05). Na-pyruvate의 농도를 0.25 mM 첨가한 구보다 2.5 mM 첨가한 구에서 제1감수분열 중기(MI) 단계까지의 성숙율이 증가하는 경향을 보였으나, MII 단계까지의 성숙율은 차이가 없었다. 본 실험의 결과는 개 난자의 체외성숙시 $E_2$가 첨가된 배양액에서 72시간 배양시에 성숙율을 증진시킬 수 있으나, Na-pyruvate의 농도는 개 체외배양에 있어 큰 영향을 미치지 않는 것을 나타낸다.

• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2003년도 제3회 국제심포지움 및 학술대회
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• pp.3-6
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• 2003

The birth of the clone animals is influencing the frontier of research of animal biotechnology. It has effects on research of animal biotechnology itself by necessitating setting of new research subjects, modifications of the strategy of ongoing research projects, and challenges to schemes formerly considered impossible. In my talk, such topics including mass production of fertile ova and oocyte maturation will be discussed. (1) Oocytes are needed for the production of a clone by nuclear transplantation. Mitochondrial DNA inherited via the oocyte are involved also in the morphogenesis. Therefore, oocytes from the same animal must be used as recipients to produce genuine clones by nuclear transplantation. Experimenting on the assumption that selective oogenesis can be avoided, and apoptosis of oocytes can be prevented, by using ovarian angiogenic factos will be introduced. (2) It is important to clarify the factors of oocytes involving in reprogramming of somatic cells. Such factors are thought to be expressed in oocytes during oogenesis and oocyte maturation. Therefore, molecular mechanisms of oogenesis and oocyte maturation must be clarified extensively. Topics in this field including our recent advances will be discussed. (중략)

• 한국가축번식학회지
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• 제25권3호
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• pp.243-250
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• 2001

본 연구는 미성숙 돼지 난포 난자의 체외성숙에 있어서 catalase (0.1 mg/$m\ell$)와 xanthine (5 mM)의 역할에 대하여 검토하였다. 그 결과, 체외에서 성숙배양 48시간 후 metaphase-II 단계로 발육한 난자의 비율은 xanthine (54%) 첨가 보다는 대조구 (72%), catalase (73%) 및 catalase+xanthine (70%) 첨가구에서 유의적으로 높은 성숙율을 나타냈다 (P＜0.05). 한편, 체외에서 30시간 동안 성숙배양한 경우 모든 실험구에서 성숙율의 유의적인 차이는 인정되지 않았으나, 성숙배양 36, 42 및 48시간 후 xanthine의 첨가 여부에 관계없이 catalase 무첨가 (29~50%) 보다는 첨가시 (49~70%)에 유의적으로 높은 성숙율을 나타냈다 (P＜0.05). 체외에서 xanthine의 첨가 또는 무첨가시 배양시간의 연장이 난자의 성숙에 미치는 영향을 검토한 결과 배양 72시간에서 높은 성숙율을 나타냈으며, 퇴행난자의 비율은 배양 120시간에서 catalase무첨가(47%)에 비하여 첨가시 (28%) 유의적으로 낮게 나타났으나 xanthine이 첨가된 배양액내에서 catalase첨가유무에 의한 차이는 인정되지 않았다. 또한 xanthine을 첨가하여 72시간 배양한 경우 단위발생란이 처음으로 관찰되었지만 catalase의 첨가 유무에 의한 차이는 인정되지 않았지만 배양시간이 길어짐에 따라 발생비율이 증가하였다. 이와 같은 결과에서 돼지 난포난자는 catalase와 xanthine을 첨가한 배양액내에서 배양 72시간까지 성숙율이 증가할 수 있으며, 배양기간의 연장시 catalase에 의하여 난자의 퇴행을 억제하며 단위발생란의 증가를 가져오는 것으로 생각된다.

• 한국수정란이식학회지
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• 제30권4호
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• pp.327-333
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• 2015

The objectives of the present study were to select an effective basic medium including its hormone and protein supplementation for IVM of oocytes of indigenous zebu cows. The ovaries of cows were collected from slaughter house and the follicular fluid was aspirated from 2 to 8 mm diameter follicles. The COCs with more than 3 cumulus cell layers and homogenous cytoplasm were selected for maturation. The oocytes were matured in media for 24 hrs at $39^{\circ}C$ with 5% $CO_2$ in humidified air. The maturation of oocytes was evaluated by examining the presence of first polar body under microscope. An efficient basic medium was determined after culturing COCs in either TCM 199 or SOF medium in Experiment 1. An efficient hormone supplementation was determined after culturing COCs in either FSH or gonadotrophin supplemented TCM 199 in Experiment 2. An efficient protein supplementation was determined after culturing COCs in either FBS or Oestrous cow serum (OCS) supplemented TCM 199 in Experiment 3. The oocyte recovery rate per ovary was 3.35. The overall rate of IVM was 74.6%. The maturation rate was $75.5{\pm}3.9$ and $62.2{\pm}20.2%$ in TCM and SOF medium, respectively (P>0.05). The maturation rate of oocytes was significantly higher ($76.6{\pm}13.2%$) in FSH supplemented medium than gonadotrphin supplemented counterpart ($69.7{\pm}10.8%$) (P<0.05). The maturation rates of oocytes were $81.7{\pm}12.9$ and $85.7{\pm}12.7%$ in medium supplemented with FBS and OCS, respectively (P>0.05). In conclusions, both TCM 199 and SOF supplemented with either FBS or OCS, and FSH may be used as medium for IVM of indigenous zebu oocytes in Bangladesh.

• 한국가축번식학회지
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• 제14권2호
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• pp.93-100
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• 1990

These experiments were carried out to investigate the effect of rabbit anti-bovine cumulus cell antibodies on in vitro maturation of bovine follicular oocytes. Antisera to bovine cumulus cell were produced Japanese Ginat rabbit by repeated immunization of intact or solubilized bovine cumulus cell and purified by ammonium sulfate precipitation and Sepharose CL-4B protein-A affinity chromatography. The bovine cumulus cell-specific antibodies were confirmed by indirect ELISA. The results obtained in these experiments were summarized as follows : 1. The titer of the antibodies to cumulus cell determined by indirect ELISA using intact or solubilized bovine cumulus cell coated plates was very high in both intact and solubilized cumulus cells. Namely, the optical density at 1:12,800 dilution of antibodies was still significantly higher than that of non-immunized control serum. 2. When the follicular oocytes were treated with antibody to intact cumulus cells, the maturation rate of cumulus compacted and removed oocytes was ranged 47.6 to 59.1%. These value is significantly lower(p<0.05) than that(78.8%) of follicular oocytes cultured without the antibody. 3. the maturation rate of cumulus compacted and removed oocytes treated with antibody to solubilized cumulus cells was ranged 46.7 to 59.1%, significantly lower(p<0.05) than that(82.1%) of ooyctes cultured in antibody free medium. From above mentioned results, it could be said that cumulus cells promote nuclear maturation of follicular oocytes and that the beneficial effect of cumulus cells to the oocyte maturation is inhibited by the action of antibody to cumulus cells.

• 한국가축번식학회지
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• 제21권1호
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• pp.19-23
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• 1997

This present study was carried out to examine the effect of maturation media and liquid boar semen on in vitro maturation and feritilization of pig oocytes. The results obtained were as follows : When the oocytes were cultured for 36∼42 hours in mTCM-199, Waymouth MB 725/1 and mTLP-PVA medium, the maturation rates were 90%, 92% and 88%, respectively. The sperm penetration rates of pig oocyte matured in vitro were 87%(mTCM-199), 90%(Waymouth MB 725/1) and 86%(mTLP-PVA), respectively. The rates of nuclear maturation and fertilization of pig oocytes among three different media did not differ. However, the rate of male pronucleus formation of pig oocytes was significantly higher in pig oocytes matured in Waymouth MB 725/1(91%) than oocytes matured in mTCM-199(66%) and mTLP-PVA(62%) medium (P<0.05). When the collected sperm-rich fraction without diluent was used fro in vitro fertilization in mTCM-199 fertilization medium, the fertilization rate was 87.9%. However, when the liquid boar semen diluted with B tschwiler diluent was used at day 3 and 5 after dilution, the fertilization rate was 40.8% and 0.0%, respectively.

• 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.

• 한국동물생명공학회지
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• 제37권1호
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• pp.34-41
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• 2022

In vivo oocytes grow and mature in ovarian follicles whereas oocytes are matured in vitro in plastic culture dishes with a hard surface. In vivo oocytes show a superior developmental ability to in vitro counterparts, indicating suboptimal environments of in vitro culture. This study aimed to evaluate the influence of an agarose matrix as a culture substrate during in vitro maturation (IVM) on the development of pig oocytes derived from small antral follicles (SAFs). Cumulus-oocyte complexes (COCs) retrieved from SAFs were grown in a plastic culture dish without an agarose matrix and then cultured for maturation in a plastic dish coated without (control) or with a 1% or 2% (w/v) agarose hydrogel. Then, the effect of the soft agarose matrix on oocyte maturation and embryonic development was assessed by analyzing intra-oocyte contents of glutathione (GSH) and reactive oxygen species (ROS), expression of VEGFA, HIF1A, and PFKP genes, and blastocyst formation after parthenogenesis. IVM of pig COCs on a 1% (w/v) agarose matrix showed a significantly higher blastocyst formation, intra-oocyte GSH contents, and transcript abundance of VEGFA. Moreover, a significantly lower intra-oocyte ROS content was detected in oocytes matured on the 1% and 2% (w/v) agarose matrices than in control. Our results demonstrated that IVM of SAFs-derived pig oocytes on a soft agarose matrix enhanced developmental ability by improving the cytoplasmic maturation of oocytes through redox balancing and regulation of gene expression.

• Animal Bioscience
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• 제37권6호
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• pp.1007-1020
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• 2024

Objective: We increased the nuclear maturation rate of antral follicle derived oocytes by using a pre-in vitro maturation (IVM) culture system and improved the developmental potential of these porcine pathenotes by supplementing with melatonin. Furthermore, we investigated the expression patterns of genes involved in cumulus expansion (HAS2, PTGS2, TNFAIP6, and PTX3) derived from small and medium antral follicles before and after oocyte maturation. Methods: Only the cumulus oocyte-complexes (COCs) derived from small antral follicles were induced with [Pre-SF(+)hCG] or without [Pre-SF(-)hCG] the addition of human chorionic gonadotropin (hCG) during the last 7 h of the pre-IVM period before undergoing the regular culture system. The mature oocytes were investigated on embryonic development after parthenogenetic activation (PA). Melatonin (10^-7 M) was supplemented during in vitro culture (IVC) to improve the developmental potential of these porcine pathenotes. Results: A pre-IVM culture system with hCG added during the last 7 h of the pre-IVM period [Pre-SF(+)hCG] effectively supported small antral follicle-derived oocytes and increased their nuclear maturation rate. The oocytes derived from medium antral follicles exhibited the highest nuclear maturation rate in a regular culture system. Compared with oocytes cultured in a regular culture system, those cultured in the pre-IVM culture system exhibited considerable overexpression of HAS2, PTGS2, and TNFAIP6. Porcine embryos treated with melatonin during IVC exhibited markedly improved quality and developmental competence after PA. Notably, melatonin supplementation during the IVM period can reduce and increase the levels of intracellular reactive oxygen species (ROS) and glutathione (GSH), respectively. Conclusion: Our findings indicate that the Pre-SF(+)hCG culture system increases the nuclear maturation rate of small antral follicle-derived oocytes and the expression of genes involved in cumulus expansion. Melatonin supplementation during IVC may improve the quality and increase the blastocyst formation rate of porcine embryos. In addition, it can reduce and increase the levels of ROS and GSH, respectively, in mature oocytes, thus affecting subsequent embryos.