• Clinical and Experimental Reproductive Medicine
• /
• 제25권3호
• /
• pp.331-340
• /
• 1998

본 연구에서는 돼지 난자내에 돼지정자, 여러 가지 처리된 정자두부 (1% Triton, 0.1% Trypsin, 100mM NaOH)및 이종정자 (소, 쥐, 사람)를 미세 주입한 후 난 활성과 웅성 전핵형성, 전핵의 이동 및 배발달을 관찰하였다. 전자현미경으로 관찰한 결과 Triton X-100을 처리하였을 때 첨체가 제거되었으나 핵 주변 물질은 제거되지 않았고 Trypsin 또는 NaOH를 처리 할 경우 핵주변 물질 (perinuclear material)이 제거됨을 볼 수 있었다. 돼지 난자는 정자, 정자두부 및 Triton X-100을 처리한 정자두부의 주입을 통해 난 활성이 유도되었으며 쥐, 소, 사람의 정자를 주입하였을 때 난 활성이 유도되고 정상적인 전핵형성이 이루어졌다. 그러나 정자꼬리나 Trypsin 또는 NaOH에 의해 정자 핵주변 물질(perinuclear material)이 제거된 정자두부를 주입하였을때는 난 활성은 야기되지 않았다. 유사분열 및 2-세포기까지 정상적인 수정은 동종의 정자 및 정자두부를 주입한 난자에서 관찰할 수 있었으나 이 종정자를 주입한 난자에서는 관찰되지 않았다. 또한 상실배 및 배 반포까지 정상적인 수정은 동종의 정자 및 정자두부를 주입한 난자에서 관찰할 수 있었다. 이러한 결과는 돼지에서 정자 및 정자두부의 미세주입에 의해 수정이 이루어지는 것을 시사하며 수정시 정자유래의 난할성인자는 정자 핵주변 물질(porinuclear material)에 존재하며 종특이적이지 않다는 것을 보여주는 것이다.

• Clinical and Experimental Reproductive Medicine
• /
• 제32권3호
• /
• pp.207-216
• /
• 2005

Objective: To understand the crucial requirement for the normal early folliculogenesis, we evaluated molecular as well as physiological differences during in vitro ovarian culture. Among the important regulators for follicle development, anti-Müllerian hormone (AMH) and FSH Receptor (FSHR) have been known to be expressed in the cuboidal granulosa cells. Meanwhile, it is known that c-kit is germ cell-specific and GDF-9 is also oocyte-specific regulator. To evaluate the functional requirement for the competence of normal follicular development, we investigated the differential mRNA expression of several factors secreted from granulosa cells and oocytes between in vivo and in vitro developed ovaries. Materials and Methods: Ovaries from ICR neonates (the day of birth) were cultured for 4 days (for primordial to primary transition) or 8 days (for secondary follicle formation) in ${\alpha}$-MEM glutamax supplemented with 3 mg/ml BSA without serum or growth factors. The mRNA levels of the several factors were investigated by quantitative real-time PCR analysis. Freshly isolated 0-, 4-, and 8-day-old ovaries were used as control. Results: The mRNA of AMH and FSHR as granulosa cell factors was highly increased according to the ovarian development in both of 4- and 8-day-old control. However, the mRNA expression was not induced in both of 4- and 8-day in vitro cultured ovaries. The mRNA expression of GDF-9 known to regulate follicle growth as an oocyte factor was different between in vivo and in vitro developed ovaries. In addition, the transcript of GDF-9 was expressed in the primordial follicles of mouse ovaries. The mRNA expression of c-kit was not significantly different during the early folliculogenesis in vitro. Conclusion: This is the first report regarding endogenous AMH and FSHR expression during the early folliculogenesis in vitro. In conclusion, it will be very valuable to evaluate cuboidal granulosa cell factors as functional marker(s) for normal early folliculogenesis in vitro.

• 한국수정란이식학회지
• /
• 제19권2호
• /
• pp.101-112
• /
• 2004

본 연구는 포유류인 $Cl^-$통로의 존재 여부를 확인하기 위하여 수행되었다. $Cl^-$통로는 비흥분성 세포에서 용적변화와 pH 조절, 이온운반계 등에 중요한 역할을 수행하므로 활발한 세포분화가 이루어지는 난자의 생존과 기능에 필수적 $Cl^-$통로 존재 여부를 확인하고자 하였다. 단일통로 전류를 기록하는 patch clamp 기법을 이용하였다. 세포외(pipette) 용액을 140 mM NaCl로 하고 세포내(bath) 용액을 70 mM, 140 mM, 280 mM NaCl로 바꾸어 주었을 때, 역전압($E_{rev}$) 은 $Cl^-$평형전압을 반영하는 -9.8${\pm}$0.5mV, 0mV, 11.5${\pm}$0.2mV로 변화하였다(n=4). 이런 결과는 이론치와 약간의 편차를 보이고 있으므로 이를 보정하기 위항 Goldman-Hodgkin-Katz(GHK) 식을 이용한 결과 $Cl^-$에 대한 $Na^+$의 투과성 ($P_{Na}$/$P_{Cl}$/)은 0.25로 추측할 수 있었다. Inside out patch mode에서 Pipette 용액과 bath 용액에 imperment인 NMDG-Cl로 구성하였을 경우 단일 통로가 기록되었다.(n=22). 막전압이 증가함에 따라 전도도(conductance)가 같이 증가하였으며 전류-전압 관계는 outward rectification을 보였다. 이런 특성을 나타내는 단일통로로 전류는 선택적 $Cl^-$통로 차단제인 IAA-94(indanyloxyacetic acid 94)에 의해 농도 의존적으로 차단되었으며 가역적으로 회복되었다. IAA의 $IC_{50}$은 32.6$\mu\textrm{M}$이었다. DIDS(4,4'-diisothiocyan ostillben-2-2'disulfonic acid)에 의해서는 전류크기가 감소 되는 빠른 억제기전을 나타내는 차단효과를 보였으며 시간경과에 따라 seblevel로 감소되는 subconductance block 이 나타났다. 이상의 결과는 햄스터 난자에서 chloride 통로가 존재하여, 물질이동과 pH 조절기능을 나타내는 외향성 전류-전압관계를 보이는 $Cl^-$ 통로의 성상으로 미루어 볼 때 난자의 pH 조절과 용적조절과 같은 생리적 환경 조성에 관여할 것으로 추정된다.

• 한국동물학회지
• /
• 제39권3호
• /
• pp.292-298
• /
• 1996

안점의 꽃갯지렁이(Pseudopotamilla occelata)의 난자형성중 난황단백질의 전구체인 체강액 단백질(CP)은 수용체에 의해 중개되는데 이러한 수용체 중개에 의한 난모세포내로의 유입은 GTP-binding protein에 의해 조절되는 것으로 확인되었다. 체강액 단백질(CP)을 가장 활발히 투과시키는 중기 난모세포내로의 125 I-CP의 유입은 GTP에 의해 촉지되었고, GTP 유사체인 GTPrS나 GTP$\beta$S의 효과를 금입자로 표지된 체강액 단백질을 이용하여 전자현미경으로 확인해 본 결과, gold-labeled CP는 난황립에 집중되었고, 이러한 현상도 GTP에 의해서는 촉진되었고 GTPrS에 의해서는 억제되었다.

• 한국발생생물학회지:발생과생식
• /
• 제14권1호
• /
• pp.27-34
• /
• 2010

A fertilized oocyte can get the competence for implantation through cleavage and stage-specific gene expression. These are under the control of autonomous and exogenous regulators including physiological culture condition. Endogenous and exogenous growth factors are considered as critical regulators of cleaving embryos during travel the oviduct and uterus. In this study, an effort was made to evaluate comprehensively the quality of embryos for implantation, grown in media enriched with EGF and PAF. The study evaluated developmental rates on given time, blastulation and hatching rates, and adhesion rates. Developmental rates of blastocyst to the hatching stage were significantly high in PAF treated group compared to the control in a dose-dependent manner but not in EGF group. Implantation rates were significantly high both PAF and EGF in a dose-dependent manner. H7, a PKC inhibitor, blocked the process of hatching of the blastocysts but combined treatment of EGF and PAF enhanced the hatching and implantation of blastocsyts. Based on these results it is suggested that EGF and PAF support acquirement of implantation competence at blastocyst stage through a PKC pathway.

• 한국동물번식학회:학술대회논문집
• /
• 한국동물번식학회 2001년도 춘계학술발표대회
• /
• pp.12-12
• /
• 2001

To get insight into the nature of foreign mitochondria and syngamy during mammalian fertilization we compared fertilization processes in porcine oocytes following microinjection of porcine or mouse spermatozoa. Pronuclear movement, sperm mitochondria, and DNA synthesis were imaged with propidium iodide, mitotracker, and BrdU under confocal laser scanning microscope. Intracytoplasmic injection of either porcine or mouse spermatzoon activated porcine oocytes without additional parthengenetic stimulation. Foreign mitochondria in either mouse or porcine sperm midpiece were introduced into porcine oocytes following sperm injection, but rapidly disappeared from the actively developing porcine oocytes. BrdU experiment showed new DNA synthesis in porcine oocytes following injection of mouse spermatozoon or sperm head. At 24 h after injection of mouse isolated sperm head or a spermatozoon, mitoic metaphase was seen in oocyte, but they did not go to normal cell division (Table). These results suggest that pronuclear formation, foreign mitochondria disruption, DNA synthesis and syngamy formation during fertilization are not species specific processes.(Table Omitted).

• Clinical and Experimental Reproductive Medicine
• /
• 제40권1호
• /
• pp.1-6
• /
• 2013

Objective: Oocyte-specific homeobox 4 (Obox4) is preferentially expressed in oocytes and plays an important role in the completion of meiosis of oocytes. However, the Obox4 expression pattern has not been reported yet. In this study, we investigated the subcellular localization of Obox4 using a green fluorescent protein (GFP) fusion expression system. Methods: Three regions of Obox4 were divided and fused to the GFP expression vector. The partly deleted homeodomain (HD) regions of Obox4 were also fused to the GFP expression vector. The recombinant vectors were transfected into HEK-293T cells plated onto coated glass coverslips. The transfected cells were stained with 4',6-diamidino-2-phenylindol and photographed using a fluorescence microscope. Results: Mutants containing the HD region as well as full-length Obox4 were clearly localized to the nucleus. In contrast, the other mutants of either the N-terminal or C-terminal region without HD had impaired nuclear localization. We also found that the N-terminal and C-terminal of the Obox HD contributed to nuclear localization and the entire HD was necessary for nuclear localization of Obox4. Conclusion: Based on the results of the present study, we demonstrated that the intact HD region of Obox4 is responsible for the nuclear localization of Obox4 protein in cells.

• Genomics & Informatics
• /
• 제3권1호
• /
• pp.24-29
• /
• 2005

TRPV2 is a non-specific cation channel expressed in sensory neurons, and activated by noxious heat. Particularly, TRPV2 has six transmembrane domains and three ankyrin repeats. TRPV2 has been cloned from various species such as human, rat, and mouse. Oocytes of Xenopus laevis - an African clawed frog ­have been widely used for decades in characterization of various receptors and ion channels. The functional property of rat TRPV2 was also identified by this oocyte expression system. However, no TRPV2 orthologue of Xenopus laevis has been reported so far. Hence, we have focused to clone a TRPV2 orthologue of Xenopus laevis with the aid of bioinformatic tools. Because the genome sequence of Xenopus laevis is not available until now, a genome sequence of Xenopus tropicalis - a close relative species of Xenopus laevis - was used. After a number of bioinformatic searches in silico, a predicted full-length sequence of TRPV2 orthologue of Xenopus tropicalis was found. Based on this predicted sequence, various approaches such as RT-PCR and 5' -RACE technique were applied to clone a full length of Xenopus laevis TRV2. Consequently, a full-length Xenopus laevis TRPV2 was cloned from heart cDNA.

• 한국동물생명공학회지
• /
• 제38권2호
• /
• pp.54-61
• /
• 2023

Background: Germ cells undergo towards male or female pathways to produce spermatozoa or oocyte respectively which is essential for sexual reproduction. Mesenchymal stem cells (MSCs) have the potential of trans-differentiation to the multiple cell lineages. Methods: Herein, rat MSCs were isolated from bone marrow and characterized by their morphological features, expression of MSC surface markers, and in vitro differentiation capability. Results: Thereafter, we induced these cells only by retinoic acid supplementation in MSC medium and, could able to show that bone marrow derived MSCs are capable to trans-differentiate into male germ cell-like cells in vitro. We characterized these cells by morphological changes, the expressions of germ cell specific markers by immunophenotyping and molecular biology tools. Further, we quantified these differentiated cells. Conclusions: This study suggests that only Retinoic acid in culture medium could induce bone marrow MSCs to differentiate germ cell-like cells in vitro. This basic method of germ cell generation might be helpful in the prospective applications of this technology.

• BMB Reports
• /
• 제37권2호
• /
• pp.153-158
• /
• 2004

The acid-labile subunit (ALS) is known to interact with the IGF binding protein (IGFBP) in the presence of insulin-like growth factors (IGFs). Studies, however, indicate that ALS forms a doublet with IGFBP3, independent of IGFs. To characterize the structural domain required for the IGF-free ALS-IGFBP3 interaction, seven recombinant human IGFBP3 mutants were generated: three deletion mutants and four site-specific mutants that had altering N-terminal regions of IGFBP3. ALS and IGFBP3 mRNAs were co-injected into Xenopus oocytes, and their products were cross-linked and immunoprecipitated using antisera against ALS or IGFBP3. Among the deletion mutants, the mutant of D40 (deleted in 11-40th amino acids) exerted no effect in the interaction with ALS, while D60 (${\Delta}11$-60) demonstrated a moderate reduction. D88 (${\Delta}11$-88), however, showed a significant decrease. In the case of site-specific mutants, the mutation that alterated the IGF binding site (codons 56 or 80) exerted a significant reduction in the interaction, whereas codons 72 or 87 showed no significant change in the interaction with ALS. The stability of the ALS-IGFBP3 interaction was analyzed according to a time-dependent mode. Consistent with the binding study, mutants on the IGF binding sites (56 or 80) consistently show a weakness in the ALS-IGFBP3 interaction when compared to the mutants that covered the non-IGF binding sites (72 or 87). This study suggests that the N-terminal of IGFBP3, especially the IGF binding site, plays an important role in interacting with ALS as well as in stabilizing the dual complex, independent of IGFs.