• 한국동물생명공학회지
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• 제34권1호
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• pp.10-19
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• 2019

Morphology of cumulus-oocyte-complexes (COCs) at germinal vesicle (GV) stage as one of the evaluation criteria for oocyte maturation quality after in vitro maturation (IVM) plays important roles on the meiotic maturation, fertilization and early embryonic development in pigs. When cumulus cells of COCs are insufficient, which is induced the low oocyte maturation rate by the increasing of reactive oxygen species (ROS) in porcine oocyte during IVM. The ROS are known to generate including superoxide and hydrogen peroxide from electron transport system of mitochondria during oocyte maturation in pigs. To regulate the ROS production, the cumulus cells is secreted the various antioxidant enzymes during IVM of porcine oocyte. Our previous study showed that Mito-TEMPO, superoxide specific scavenger, improves the embryonic developmental competence and blastocyst formation rate by regulating of mitochondria functions in pigs. However, the effects of Mito-TEMPO as a superoxide scavenger to help the anti-oxidant functions from cumulus cells of COCs on meiotic maturation during porcine oocyte IVM has not been reported. Here, we categorized experimental groups into two groups (Grade 1: G1; high cumulus cells and Grade 2: G2; low cumulus cells) by using hemocytometer. The meiotic maturation rate from G2 was significantly (p < 0.05) decreased (G1: $79.9{\pm}3.8%$ vs G2: $57.5{\pm}4.6%$) compared to G1. To investigate the production of mitochondria derived superoxide, we used the mitochondrial superoxide dye, Mito-SOX. Red fluorescence of Mito-SOX detected superoxide was significantly (p < 0.05) increased in COCs of G2 compared with G1. And, we examined expression levels of genes associated with mitochondrial antioxidant such as SOD1, SOD2 and PRDX3 using a RT-PCR in porcine COCs at 44 h of IVM. The mRNA levels of three antioxidant enzymes expression in COCs from G2 were significantly (p < 0.05) lower than COCs of G1. In addition, we investigated the anti-oxidative effects of Mito-TEMPO on meiotic maturation of porcine oocyte from G1 and G2. Meiotic maturation and mRNA levels of antioxidant enzymes were significantly (p < 0.05) recovered in G2 by Mito-TEMPO ($0.1{\mu}M$, MT) treatment (G2: $68.4{\pm}3.2%$ vs G2 + MT: $73.9{\pm}1.4%$). Therefore, our results suggest that reduction of mitochondria derived superoxide by Mito-TEMPO may improves the meiotic maturation in IVM of porcine oocyte.

• International Journal of Industrial Entomology and Biomaterials
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• 제7권1호
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• pp.29-36
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• 2003

Phytoecdysteroids with moulting hormone (MH) activity induce different responses in silkworms when used on different day of final instar, which can be manipulated for maximum benefit like early and uniform spinning behaviour, reducing crop loss and to increase cocoon yield. The results showed that application of this hormone on seed crop viz., CSR2, CSR4 and BL44 and BL67 in early stage of 5$^{th}$ instar i.e., at 72 hrs and 96 hrs though induced early and uniform spinning behaviour, there was an adverse effect by 9-36% on the economic characters like cocoon yield, cocoon weight, cocoon shell weight and also on fecundity etc. Application of this hormone in late stage of $5^th$ instar i.e., at the onset of spinning showed non-significant variations in some of the characters like cocoon weight, cocoon shell weight, cocoon shell ratio and fecundity. The physiological implications of phytoecdysteroid in hastening the maturation events and synchronization of spinning activities in different breeds are discussed.

• Molecules and Cells
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• 제27권2호
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• pp.199-203
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• 2009

The somatic Sertoli cells play an essential role in testis determination and spermatogenesis by providing nutrition and structural support. In the current study, we report on the novel Ankrd7 gene that contains five ankyrin repeat domains. This gene was specifically expressed in Sertoli cells and was regulated in a maturation-dependent manner. Its expression was restricted to testicular tissue, and its mRNA could be detected in testes at as early as 14 dpp (days post partum) using RT-PCR analysis. In both testicular tissue sections and in vitro cultured Sertoli cells, the Ankrd7 protein was localized to the nucleus of the Sertoli cell. Immunohistochemistry and immunocytochemistry investigations showed that the protein was detectable in testicular tissues at 20 dpp, at which time Sertoli cells were gradually differentiating into their mature cellular form. These results suggest that Ankrd7 is probably involved in the process of Sertoli cell maturation and in spermatogenesis.

• 한국수정란이식학회지
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• 제31권1호
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• pp.9-12
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• 2016

Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are oocyte-specific growth factors that regulate many critical processes involved in early folliculogenesis and oocyte maturation. In this study, effects of GDF9 and BMP15 treatment during in vitro maturation of porcine oocytes upon development after parthenogenetic activation were investigated. Neither GDF, BMP15 alone nor in combination affects the number and viability of cumulus cells or the rates of oocyte maturation and blastocyst development. However, the treatment of GDF9 on porcine oocytes increased the number of trophectodermal (TE) cells of blastocysts derived from activated oocytes (P<0.05). The treatment of BMP15 increased the cell numbers of both inner cell mass (ICM) and TE cells (P<0.05). The treatment with the combination of GDF9 and BMP15 further increased the numbers of ICM and TE cells, compared with GDF9 or BMP15 treatment alone (P<0.05). In conclusion, the treatment of GDF9 or BMP15 (or both) enhanced the quality of blastocysts via the increased number of ICM and/or TE cells.

• 한국동물생명공학회지
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• 제35권4호
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• pp.357-365
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• 2020

The aim of present study was to investigate regulatory mechanism of alpha-linolenic acid (ALA) during in vitro maturation (IVM) on nuclear and cytoplasmic maturation of porcine oocytes. Basically, immature cumulus-oocyte complexes (COCs) were incubated for 22 h in IVM-I to which hormone was added, and then further incubated for 22 h in IVM-II without hormone. As a result, relative cumulus expansion was increased at 22 h after IVM and it was enhanced by treatment of ALA compared with control group (p < 0.05). During IVM process within 22 h, cAMP level in oocytes was decreased at 6 h (p < 0.05) and it was recovered at 12 h in ALA-treated group, while oocytes in control group recovered cAMP level at 22 h. In cumulus cells, it was reduced in all time point (p < 0.05) and ALA did not affect. Treatment of ALA enhanced metaphase-I (MI) and MII population of oocytes compared with oocytes in control group at 22 and 44 h, respectively (p < 0.05). Intracellular GSH levels in ALA group was increased at 22 and 44 h after IVM (p < 0.05), whereas it was increased in control group at 44 h after IVM (p < 0.05). In particular, the GSH in ALA-treated oocytes during 22 h of IVM was higher than control group at 22 h (p < 0.05). Lipid amount in oocytes from ALA group was higher than control group (p < 0.05). Treatment of ALA did not influence to absorption of glucose from medium. Cleavage and blastocyst formation of ALA-treated oocytes were enhanced compared with control group (p < 0.05). These findings suggest that supplementation of ALA could improve oocyte maturation and development competence through increasing GSH synthesis, lipid storage, and regulation of cAMP accumulation during early 22 h of IVM, and these might be mediated by cumulus expansion.

• 한국어류학회지
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• 제12권1호
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• pp.46-53
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• 2000

돌돔 Oplegnathus fasciatus의 인위적인 성 성숙 및 산란 유도를 위한 연구에서, 3년생 돌돔 친어를 대상으로 수온 및 광주기 조절에 의한 생식소 발달을 조직학적으로 조사하였다. 수온 및 광주기 조절구 (Exp. I)는 1996년 12월부터 1997년 2월까지 수온은 $14.5^{\circ}C$에서 $21.0^{\circ}C$ 그리고 광주기는 10 : 30 L에서 15 : 30 L로 증가시킨 후 4월까지 계속 유지시켰다. 수온 조절구 (Exp. II)에서 수온은 Exp. I과 동일하게 조절하였고, 광주기는 3월 초순까지는 자연 광주기로 조절하였다가 이후 장일 광주기 (15 : 30L)로 재조절 하였다. 대조구는 1996년 12월부터 1997년 4월까지 해상 가두리의 자연 조건하에서 사육하였다. 생식소 활성은 1 월부터 Exp. I, II 모두에서 시작되었다. 그리고 성숙과 산란은 Exp. I에서는 수온과 광 주기가 각각 $21.0^{\circ}C$와 15 : 30L까지 도달한 2월부터 시작되었으나, Exp. II는 3월 초순까지도 부분적 성숙만 유도되다 장일 광주기 (15 : 30 L)로 조절한 후 3월 하순 부터는 성숙과 산란이 시작되었다.

• Asian-Australasian Journal of Animal Sciences
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• 제31권9호
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• pp.1420-1430
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• 2018

Objective: Epigallocatechin-3-gallate (EGCG) is a major ingredient of catechin polyphenols and is considered one of the most promising bioactive compounds in green tea because of its strong antioxidant properties. However, the protective role of EGCG in bovine oocyte in vitro maturation (IVM) has not been investigated. Therefore, we aimed to study the effects of EGCG on IVM of bovine oocytes. Methods: Bovine oocytes were treated with different concentrations of EGCG (0, 25, 50, 100, and $200{\mu}M$), and the nuclear and cytoplasmic maturation, cumulus cell expansion, intracellular reactive oxygen species (ROS) levels, total antioxidant capacity, the early apoptosis and the developmental competence of in vitro fertilized embryos were measured. The mRNA abundances of antioxidant genes (nuclear factor erythriod-2 related factor 2 [NRF2], superoxide dismutase 1 [SOD1], catalase [CAT], and glutathione peroxidase 4 [GPX4]) in matured bovine oocytes were also quantified. Results: Nuclear maturation which is characterized by first polar body extrusion, and cytoplasmic maturation characterized by peripheral and cortical distribution of cortical granules and homogeneous mitochondrial distribution were significantly improved in the $50{\mu}M$ EGCG-treated group compared with the control group. Adding $50{\mu}M$ EGCG to the maturation medium significantly increased the cumulus cell expansion index and upregulated the mRNA levels of cumulus cell expansion-related genes (hyaluronan synthase 2, tumor necrosis factor alpha induced protein 6, pentraxin 3, and prostaglandin 2). Both the intracellular ROS level and the early apoptotic rate of matured oocytes were significantly decreased in the $50{\mu}M$ EGCG group, and the total antioxidant ability was markedly enhanced. Additionally, both the cleavage and blastocyst rates were significantly higher in the $50{\mu}M$ EGCG-treated oocytes after in vitro fertilization than in the control oocytes. The mRNA abundance of NRF2, SOD1, CAT, and GPX4 were significantly increased in the $50{\mu}M$ EGCG-treated oocytes. Conclusion: In conclusion, $50{\mu}M$ EGCG can improve the bovine oocyte maturation, and the protective role of EGCG may be correlated with its antioxidative property.

• 원예과학기술지
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• 제30권4호
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• pp.345-356
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• 2012

본 실험에서는 국내에서 육성한 조생종 '한아름'과 중생종 '만풍배'를 '신고' 품종과 대조하여 과실생장 기간 중의 과실 품질요인의 변화 및 세포벽 구성물질의 변화를 조사하였다. 세 품종 모두 생육기간 중 과육의 경도는 지속적으로 감소하여 과실성숙기의 경도는 '한아름', '만풍배' 및 '신고'에서 각각 29.4, 33.5 및 27.4N으로 나타났다. 세 품종 모두 성숙기에 에틸렌발생량이 매우 낮았는데 '한아름'에서만 성숙일에 $0.39{\mu}L{\cdot}L^{-1}$를 보였고 '만풍배'와 '신고'는 성숙일에 에틸렌이 측정되지 않았다. 세 품종의 가용성당은 과당이 우점하고 있었고, 과실의 성숙과 더불어 공통적으로 자당의 비율이 증가하였다. 세 품종에서 성숙기에 조사한 알코올불용성 물질(EIS)의 함량은 조생종인 '한아름'의 경우, $10.79mg{\cdot}g^{-1}$ FW로 '만풍배'의 12.72 및 '신고'의 $12.75mg{\cdot}g^{-1}$ FW에 비해 낮았다. 총가용성 펙틴은 '한아름'이 성숙기에 $81.05{\mu}g{\cdot}mg^{-1}$ EIS로 '만풍배'의 57.88 및 '신고'의 $61.81{\mu}g{\cdot}mg^{-1}$ EIS에 비해 높은 수준이었다. 수용성펙틴 분획의 분자량 변화는 과실발육 초기에 공통적으로 유의하였다. 4% KOH 가용성 hemicelluloses 함량은 '신고'에서 $28.05{\mu}g{\cdot}mg^{-1}$ EIS로 세 품종 중 유의하게 그 수준이 낮았고 과실 성숙에 따른 큰 변화를 보이지 않았다. 셀룰로오스 함량은 '한아름'의 경우 성숙기에 $408.00{\mu}g{\cdot}mg^{-1}$ EIS, '만풍배' 및 '신고' 는 각각 538.67 및 $612.33{\mu}g{\cdot}mg^{-1}$ EIS이었다. 세포벽 구성 중성당 함량은 품종에 관계없이 과실이 비대하고 성숙이 진행됨에 따라 그 함량이 감소하는 경향을 보였다. '한아름'의 경우 우점 중성당인 arabinose 및 galactose 함량이 성숙기인 만개 후 127일에 급격히 감소하였다. 조생종인 '한아름'에서 성숙과 더불어 4% KOH 가용성 헤미셀룰로오스 및 xyloglucan의 분해가 유의하였고 '만풍배' 및 '신고'는 과실발육 초기에만 변화가 나타났다. Chelator 및 $Na_2CO_3$ 가용성펙틴과 24% KOH 가용성 헤미셀룰로오스 분획의 분자량 변화는 품종에 관계 없이 매우 제한적이었다.

• 생명과학회지
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• 제25권10호
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• pp.1184-1195
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• 2015

새로운 실험 동물로 대두되고 있는 제브라피쉬는 척추동물 생식생물학 연구에서도 중요한 역할을 한다. 제브라피쉬의 난자 성숙은 maturation inducing hormone (MIH, 17α,20β-Dihydroxy-4-pregnen-3-one)에 의해 촉발된다. 대부분의 동물의 난자성숙에는 cdc2 kinase와 cyclinB 단백질 복합체인 MPF의 활성화가 필요하다. 발톱개구리와 생쥐에서는 MPF 활성이 두 가지 기작에 의해 조절되는데, 하나는 cyclinB 결합이고 또 다른 하나는 Wee1과 Cdc25에 의한 T14/Y15 잔기의 억제성인산화와 탈인산화이다. 발톱개구리나 생쥐와 달리 제브라피쉬를 포함한 대부분의 진골어류(teleost)는 GV 난자에 pre-MPF complex가 존재하지 않으므로 MPF 활성화는 전적으로 cyclinB 단백질의 de novo synthesis에 의존한다. 다른 종과 마찬가지로 제브라피쉬의 모계유래 mRNA도 CPEB, Dazl, Pum1/Pum2, insulin-like growth factor2 mRNA-binding protein 3 등 다양한 RNA binding protein (RBP)의 결합에 의해 번역이 조절된다. 그러나 제브라피쉬 난자에서 단백질 번역 조절에 관여하는 자세한 작용 기작은 확실하게 규명되지 않았다. 그러므로 제브라피쉬 난자의 성숙과정을 연구하는 것은 척추동물 난자 초기 성숙과정에서 단백질 번역 조절의 역할을 규명할 수 있는 새로운 정보를 제공할 것이다.

• Animal cells and systems
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• 제5권1호
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• pp.51-57
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• 2001

We have previously shown that oocyte maturation is induced by an immobilized progesterone, progesterone-3-carboxymethyloxime - bovine serum albumin conjugate (P-BSA) in Rana dybowskii. In this study, we confirmed the maturation inducing activity of P-BSA on Xenopus oocyte and examined the binding character of the immobilized progesterone on the surface of Xenopus oocytes after removal of the vitelline layer. P-BSA induced maturation of Xenopus oocytes but E-BSA failed to do so as observed in Rana. Binding of the immobilized progesterone, fluorescein isothiocyanate-labeled progesterone-3-0-carboxymethyloxime-BSA (P-BSA-FITC) on the devitellined oocytes surface was examined by fluorescence confocal microscopy. The binding affinity of P-BSA-FITC to the devitellined oocyte was higher than that of estrogen-BSA-FITC (E-BSA-FITC) or testosterone-BSA-FITC (T-BSA-FITC). The binding disappeared in the presence of excess free progesterone but not in the presence of free estrogen. Maximum binding occurred after two-hours of incubation with P-BSA-FITC at pH 7.5. Stronger binding occurred in oocytes at stage Vl than stage IV, and in vitro treatment of hCG enhanced the binding. Taken together, these results suggest that a specific receptor for progesterone exists on the plasma membrane of Xenopus oocytes and that progesterone acts initially on this putative receptors and triggers generation of membrane-mediated second messengers during the early stage of oocyte maturation In amphibians.