• 한국발생생물학회지:발생과생식
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• 제25권2호
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• pp.75-82
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• 2021

We studied steroid metabolites produced from red-spotted grouper ovarian follicles during maturation. Oocytes with 350-500 ㎛ diameter were in vitro incubated in the presence of [³H] 17α-hydroxyprogesterone as a precursor. Steroid metabolites were extracted from incubated media and oocytes. The extracts were separated and identified using thin layer chromatography, high performance liquid chromatography and gas chromatography-mass spectrometry. The identified metabolites were androstenedione (A₄), testosterone (T) and estrone (E₁). The metabolites of A₄ was dominant in all size of oocytes and it was the highest in 480 ㎛ diameter oocytes. The metabolites of two progestins, 17α,20β-dihydroxy-4-pregnen-3-one and 17α,20α-dihydroxy-4-pregnen-3-one were detected in the oocytes less than 480 ㎛ diameter although they were not identified definitely. In the oocytes of 480 ㎛ diameter, metabolite of progestin was the highest, and germinal vesicle (GV) was still in the middle of cytoplasm. In the oocytes of 500 ㎛ diameter, GV was began to migrate and the major metabolites were A₄ and E₁. The metabolite of E₁ was detected in all size of oocytes and it was higher than that of E₂. These results suggest that oocytes of 480 ㎛ diameter are the transitional stage involving steroidogenic shift to final oocyte maturation and potential function of E₁ during maturation process.

• Biomolecules & Therapeutics
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• 제31권3호
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• pp.330-339
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• 2023

Liver kinase B1 (LKB1) is a crucial tumor suppressor involved in various cellular processes, including embryonic development, tumor initiation and progression, cell adhesion, apoptosis, and metabolism. However, the precise mechanisms underlying its functions remain elusive. In this study, we demonstrate that LKB1 interacts directly with malic enzyme 3 (ME3) through the N-terminus of the enzyme and identified the binding regions necessary for this interaction. The binding activity was confirmed to promote the expression of ME3 in an LKB1-dependent manner and was also shown to induce apoptosis activity. Furthermore, LKB1 and ME3 overexpression upregulated the expression of tumour suppressor proteins (p53 and p21) and downregulated the expression of antiapoptotic proteins (nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and B-cell lymphoma 2 (Bcl-2)). Additionally, LKB1 and ME3 enhanced the transcription of p21 and p53 and inhibited the transcription of NF-κB. Moreover, LKB1 and ME3 suppressed the phosphorylation of various components of the phosphatidylinositol-4,5-bisphosphate 3-kinase/protein kinase B signaling pathway. Overall, these results suggest that LKB1 promotes pro-apoptotic activities by inducing ME3 expression.

• International Journal of Industrial Entomology and Biomaterials
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• 제37권1호
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• pp.21-28
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• 2018

CRISPR/Cas9 gene editing system is an efficient method to mutation in a sequence specific manner. Here we report the direct transfection of the Cas9 nuclease and gene specific guide RNA can be used in BM-N cell line derived from Bombyx mori ovarian tissue to enfeeble function of endogenous gene in vitro. We have used gene editing system to negative regulation components of major signaling cascade, the Toll pathway, which controls B. mori resistance to microbe infections, such as fungi and gram positive bacteria. We demonstrate that the $I{\kappa}B-like$ protein Cactus may controls the activation of transcription factors such as Rel A and Rel B. The direct transfection of Cas9 nuclease and Cactus-specific guide-RNA complex may be used in BM-N cells to disrupt the function of endogenous genes in vitro. A mutation frequency of 30-40% was observed in the transfected cells, and various mutations caused the target region. Moreover, RT-PCR analysis revealed that Cactus gene was down regulated after these mutations. More importantly, mutation of BmCactus stimulated expression of lysozyme, moricin, and lebocin genes. These results suggest that the CRISPR/Cas9 systems are expected to efficiently induce site-specific mutations and it was possible to produce antimicrobial peptide through the gene editing.

• BMB Reports
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• 제36권6호
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• pp.603-607
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• 2003

Abstract Plant lipid transfer proteins (LTPs) are a class of proteins whose functions are still unknown. Some are proposed to have antimicrobial activities. To understand whether LTP110, a rice LTP that we previously identified from rice leaves, plays a role in the protection function against some serious rice pathogens, we investigated the antifungal and antibacterial properties of LTP110. A cDNA sequence, encoding the mature peptide of LTP110, was cloned into the Impact-CN prokaryotic expression system. The purified protein was used for an in vitro inhibition test against rice pathogens, Pyricularia oryzae and Xanthomonas oryzae. The results showed that LTP110 inhibited the germination of Pyricularia oryzae spores, and its inhibitory activity decreased in the presence of a divalent cation. This suggests that the antifungal activity is affected by ions in the media; LTP110 only slightly inhibited the growth of Xanthomonas oryzae. However, the addition of LTP110 to cultured Chinese hamster ovarian cells did not retard growth, suggesting that the toxicity of LTP110 is only restricted to some cell types. Its antimicrobial activity is potentially due to interactions between LTP and microbe-specific structures.

• Clinical and Experimental Reproductive Medicine
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• 제11권2호
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• pp.41-49
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• 1984

임신직후 자궁조직은 난소 스테로이드 호르몬에 의하여 분화한다. 착상전 여러 단계에 있는 자궁조직내의 cAMP의 함량을 측정함으로서 cAMP와 호르몬과의 관계를 규명할 목적으로 흰쥐를 재료로 해서 본 실험을 행하였다. 임신제 2일에 난소를 제거하고 즉시, 제3, 또는 제 4일에 estradiol과 progesterone 을 각각 혹은 동시에 주사한 뒤 대조군인 정상임신군, 난소제거군, 난소제거후 호르몬주사군의 자궁조직에서 cAMP농도를 competitive protein-binding assay로 측정하여 이를 각각 비교하였다. 본 연구결과는 다음과 같았다. 정상임신군인 경우 자궁조직이 분화해 갈수록 자궁조직내 cAMP의 농도는 낮아지고 있다. 난소제거군에서는 임신기간이 길수록 자궁조직내의 cAMP농도가 높아진다. 난소제거후 progesterone 혹은 progesterone 과 estradiol을 주사한 실험군에서는 임신 제 6일이 되면 cAMP 농도가 정상임신군과 난소제거군의 중간수치를 보여준다. 한편, estradiol 단독주사인 경우는 임신제3, 제6일의 자궁조직내의 cAMP의 농도는 크게 감소한다. 이처럼 estradiol의 영향아래 세포분열등 분화가 왕성한 자궁조직에서는 cAMP 농도가 크게 낮아지지만 자궁조직의 분화가 억제된 상태에서는 cAMP의 농도가 반대로 높아진다. 난자, 배아 또는 여러 형태의 세포에서처럼 cAMP의 농도는 세포의 대사에 크게 영향을 주고 있다는 것을 알 수 있었다.

• 한국발생생물학회지:발생과생식
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• 제12권2호
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• pp.195-202
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• 2008

1998년 3월부터 1999년 2월까지 전라남도 대흑산도 연안에서 채집한 비단가리비를 대상으로 생식소중량지수, 생식세포 분화 및 난소주기를 조직, 세포학적 관찰에 의해 조사하였다. 초기 난황형성 난모세포에서, 골지체, 미토콘드리아 및 조면소포체들은 지방적 형성에 관여하였다. 후기난황형성난모세포에서 생식상피상에 존재하는 외인성 물질들 즉, 글리코겐 입자들 및 지방 과립상 물질들이 난황막의 미세융모를 통해서 난모세포의 난질로 통과해 들어갔다. 후기난황형성난모세포에서, 난황과립들과 다포체들은 단백질성 난황과립형성에 관여하였다. 난황형성과정은 내인성 자율합성과 외인성 타가합성에 의해서 일어난다. 보조세포들은 초기단계인, 전난황형성 난모세포 및 초기 난황형성 난모세포들의 형성 및 발달에 영양세포로서 기능을 한다. 생식소 중량지수의 월별 변화는 난소 발달단계들과 밀접한 관련을 가지며 변하였다. 본 종의 생식주기는 초기활성기(1~3월), 후기활성기(3~4월), 완숙기(4~8월), 부분산란기(6~8월), 퇴화 및 비활성기(8~12월)의 5단계로 구분되었다. 산란은 6~8월 사이에 일어나며, 주산란기는 수온이 높은 7~8월이었다.

• 한국발생생물학회지:발생과생식
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• 제4권1호
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• pp.45-52
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• 2000

Membrane-type matrix metalloproteinase(MT-MMP)는 세포막에 부착된 채로 작용하는 단백질 가수분해효소로서 최근들어 정상 및 암세포 등 각종 조직세포의 재구성에 중요한 역할을 하는 것으로 알려지고 있다. 본 연구에서는 RT-PCR 방법을 사용하여 생쥐 난자와 초기배아에서의 MT1-MMP와 MT2-MMP 유전자 발현 양상을 조사하였다. 생후 3주 및 8주된 생쥐의 난소로부터 얻은 미성숙난자와 체외 및 체내에서 성숙시킨 난자에서의 MTl-MMP와 MT2-MMP mRMA의 발현을 조사하였다. 그 결과 미성숙난자와 체외 및 체내에서 성숙시킨 난자 모두에서 MT1-MMP 및 MT2-MMP의 mRNA가 발현되었으나 미성숙난자에서는 매우 약하게 발현되는 것이 관찰되었다. 2세포기 배아, 4세포기 배아, 상실배, 포배 및 탈각한 포배를 대상으로 MT2-MMP mRNA의 발현양상을 조사한 결과 2세포기에서는 발현이 일어나지 않았고 4세포기에 이르러서야 뚜렷한 발현이 관찰되었다. 상실배와 포배에서는 현저히 강한 발현이 관찰되었다. 생쥐의 난소조직에서도 MT1-과 MT2-MMP모두 발현되는 것이 관찰되었는데 각각은 배란을 전후로 한 시기에 상관없이 항상 같은 정도의 mRNA발현을 나타내었다. 생쥐의 수란관조직도 난소조직과 유사하게 배란시기에 상관없이 같은 수준의 MT1-과 MT2-MMP mRNA 발현양상을 보여주었다. 이러한 결과들로 미루어 생쥐 배아에서 발현되는 MT2-MMP는 초기배아의 분화과정에서 중요한 역할을 할 것으로 사료된다. 난소와 수란관의 조직재구성과 관련한 MT1-과 MT2-MMP의 역할은 앞으로 더 연구되어야 할 것이다.

• 생명과학회지
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• 제30권5호
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• pp.483-490
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• 2020

유비퀴틴 시스템은 ubiquitin ligases와 deubiquitinases (DUBs)에 의해 타겟 기질의 ubiquitination 유무에 따라 안정화, 활성화, 국소화 및 상호작용을 변화시키고 암 발병의 관여하는 다양한 생물학적 과정을 조절한다. DUBs는 촉매 domain에 따라 6그룹으로 나뉘어지는데, 그 중에서 OTU 그룹 내 대부분의 DUB는 세포의 연속적 신호반응(cell signaling cascade)을 조절할 수 있다. 특이하게도 OTU 그룹에 속하는 otubain 1 (OTUB1)은 정규적(canonical) 활성과 비정규적(non-canonical) 활성을 모두 가지고 있다. 본 보고에서는 OTUB1의 canonical, non-canonical 활성 조절에 있어서 다양한 신호전달경로 및 OTUB1의 역할에 대해 기술하였다. OTUB1은 이 두 종류의 활성 경로를 통하여, OTUB1은 암 관련 신호 전달 체계에 중요한 FOXM1, ERα, KRAS 및 EMT를 조절하고 암세포 증식 및 전이 능력 향상 및 항암제에 대한 내성을 나타낸다. 또한 임상적으로 전이성 및 종양 분화도가 높은 암 조직에서 OTUB1의 발현이 높으며, 이에 따라 환자의 생존율이 감소하는 등 나쁜 예후를 나타낸다. 따라서, 임상적으로 적용할 수 있는 OTUB1 억제제의 개발이 이루어진다면 OTUB1은 종양 치료에 있어 중요한 진단마커이자 치료적인 타겟이 될 수 있다.

• Clinical and Experimental Reproductive Medicine
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• 제21권2호
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• pp.137-147
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• 1994

Controlled ovarian hyperstimulation(COH) for in vitro fertilization and embryo transfer(IVFET) often results in the production of more embryos than can be efficaciously transferred at one time. However, embryo cryopreservation provides a mechanism by which additional embryos can be stored for later thawing and transfer. From November, 1990 to October, 1992, we completed 42 transfer cycles of cryopreserved pronucleus(PN) l-cell embryos using the fixed protocol of hormonal replacement therapy in a physiological manner regardless of individual ovarian function. Artificial endometrial stimulation was performed with only exogenous estradiol and progesterone(E-P) in 36 transfer cycles (Group I) and with gonadotropin-releasing hormone agonist(GnRHa) and exogenous estradiol and progesterone(GEEP) in 6 transfer cycles(Group II ). The results were as follows. 1. The Survival rate of total cryopreserved-thawed embryos was 64.9%(198/305): 64.9% (172/265) in Group I and 65.0% (26/40) in Group II. 2. Total 168 embryos were transferred with an average of 4.7 per ET in Group I and total 26 embryos were transferred with an average of 4.3 per ET in Group II. 3. The pregnancy rate(PR) per cryopreserved-thawed ET and the implantation rate was 33.3 %(14/42) and 6.7%(13/194), respectively. The PRs per cryopreserved-thawed ET were 30.6% (11/36) in Group I and 50.0% (3/6) in Group II without significant difference. 4. The take home baby rate was 11.1%(4/36) in Group I and 33.3% (2/6) in Group II.

• Clinical and Experimental Reproductive Medicine
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• 제31권2호
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• pp.111-117
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• 2004

Objectives: To investigate the effect of LH receptor in folliculogenesis, we confirm the expression level of LH receptor (LH-R) mRNA in human granulosa cells (GCs) and its expression levels were analyzed by comparison to embryo developmental rate and pregnancy rate. Materials and Methods: GCs were obtained at the time of oocyte retrieval from the patients undergoing IVF-ET program. The patients were divided into two groups: Group I (n=20) is poor responder (retrieved oocyte(s)$\leq$3ea), Group II (n=80) is normal responder (retrieved oocytes>3ea). After the extraction of total RNA, semiquantitative RT-PCR was performed and the expression level of LH-R mRNA was normalized by $\beta$-actin. Statistical analysis was performed by using $X^2$ test, Student's t-test and Pearson correlation. Results: In Group II, the relative values of LH-R mRNA (0.680 vs. 0.463, p<0.005) and pregnancy rate (54.7% vs. 23.1%, p<0.05) were significantly higher than in Group I. Number of retrieved oocyte(s) was gradually increased when the expression of LH-R mRNA was increased (p<0.05). But the quality of retrieved oocyte and transferred embryo were not related with the expression of LH-R mRNA. When the pregnancy rate was compared with FSH only group and FSH combined with hMG group in the ovarian stimulation protocol, FSH combined with hMG group was significantly higher than FSH only group in Group I (37.5% vs. 0%), and the expression of LH-R mRNA was significantly higher in hMG combined group than FSH only group (p<0.05). Conclusion: Expression level of LH-R mRNA has important role in ovarian function related with the response to gonadotrophin in human folliculogenesis. Furthermore these data might provide the evidence that additional use of hMG is helpful to poor responders.