Earticle

Background: Mycotoxins which mainly consist of Aflatoxin (AF), Zearalenone (ZEN) and Deoxynivalenol (DON) are commonly found in many food commodities, each component has been shown to cause organ toxicity and oxidative stress in several species. Our previous study showed that mycotoxin-contaminated diet could cause oxidative stress in liver, kidney, spleen. Recently we examined its effects on oocyte quality. Materials and Methods: Mycotoxins-contaminated maize (AF 597μg/kg, ZEN 729μg/kg, DON 3.1mg/kg maize) was incorporated into the diet at three different doses (0, 5 and 20%) to feed the mice for 4 weeks. Results: Our results showed that the both the index of ovary and the number of good GV oocytes decreased in the mycotoxin-treated mice. The oocytes from mycotoxin- treated mouse displayed low developmental competence showing with lower GVBD and polar body extrusion rate; the embryo developmental competence also showed the similar pattern, most embryos could not develop to blastocyst stage. The cytoskeleton component actin expression in both oocyte cortex and cytoplasm decreased, and the expression of actin nucleation factor Profilin and mDia1 also decreased, indicating that mycotoxin may affect oocyte quality through the effects on actin. Moreover, a big proportion of oocytes with mycotoxin contaminated diet treatment showed disrupted cortical granule free domain, spindle morphology and mitochondria distribution, further confirmed the oocyte quality declination. We also used the in vitro model to confirm this, we cultured the oocytes in the medium with Zearalenone, a key component of mycotoxins, and the results were similar with the in vivo model. Conclusion: Our data indicated that the mycotoxins were toxic to mouse reproductive system and induced the oocyte quality declination.

과학기술의 빠른 발전으로 생명공학이 이루어낸 가장 큰 성공 중의 하나가 인체치료용으로 사용할 수 있는 재조합단백질(바이오신약)을 생산할 수 있게 되었다는 것이고, 이러한 바이오신약분야는 바이오산업의 핵심분야로서 세계시장의 선점을 위한 기술 강대국들의 경쟁력이 가장 치열한 분야이다. 일반적으로 재조합체 단백질 생산에는 적합한 합성 기작을 가지고 있는 박테리아 및 세포들을 이용한 배양으로 생산하고 있으나, 이러한 목적에 잘 맞는 또 하나의 선택이 형질전환동물을 이용한 생체반응기(Bioreactor)이고, 산업적으로 이용할 수 있는 양의 재조합단백질을 세포배양이 아닌 유즙, 계란, 혈액, 오줌, 혈청 등 형질전환동물의 생체에서 생산하는 것이다. 이러한 연구개발의 첫 번째 목표는 형질전환기술을 이용하여 형질전환동물을 생산하는 것이며, 두 번째로는 개발된 형질전환동물 생체로부터 생산된 치료용 재조합단백질을 분리 정제하여 약리효능을 검증하고, 신약으로서의 유효성 평가를 하는 것이다. 지금까지 국내에서도 많은 연구자들이 이러한 목표를 향해서 형질전환동물들은 개발하였으나, 두번째 목표인 생산된 재조합체단백질을 분리 정제하는 기술이 부족하여 실용화에 걸림돌이 되고 있다. 본 연구팀도 유즙에서 재조합단백질을 생산하는 형질전환동물을 개발하였으며, 두 번째 연구목표를 달성하기 위해서 분만돼지 유즙을 채취하고 가공처리하는 유즙전처리공정을 개발하여 이를 통해 유즙에 들어있는 불순물들을 효과적으로 제거할 수 있는 기술을 구축하였고, 재조합체 분리정제단계에서는 초기 재조합체 회수율을 높이고자 친화성 결합체를 이용한 정제방법을 선택하였다. 또한, 고전적인 이온결합 및 소수성결합 정제방법등도 활용하여 재조합체를 분리정제를 시도하였으며, 앞으로는 정제산물에 대한 유효성평가를 통하여 형질전환동물이 생산한 재조합체 단백질에 대한 약리 약효성을 분석할 계획이다. 특히, 본 연구를 통하여 형질전환동물을 활용한 바이오신약개발에 대한 연구기반을 확립하고자 한다.

Dimethyl sulfoxide (DMSO) is one of the most commonly used solvents in drug screening or for cryoprotection. In this study, we exposed mouse-derived zygote to blastocyst to three different concentrations of DMSO [0% (control), 0.5% (medium dose) and 1.0～2% (high dose)] to identify the safest dose that could effectively be used as solvent. We found that DMSO treatment substantially altered the developmental competency of mouse preimplantation embryos in concurrence with a significant reduction in embryo viability in a dose-dependent manner. Furthermore, Gene expression studies revealed a selective expression change of key markers associated with genome-wide DNA methylation reprogramming during preimplantation stages. Immunocytochemistry analysis demonstrated substantial alterations in the levels of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), increasing of apoptosis, and reduction of total cell numbers. Our study advocates for the first time that DMSO exposure induces significant alteration in gene expression, epigenetic reprogramming, and functionality of the preimplantation embryos. Also, the present study identified DMSO as a novel inducer of Tet1 and Tet2 expressions, and as a novel inhibitor for 5mC/5hmC conversion. Overall, our experiments warrant that mouse preimplantation-based assays can provide timely alerts about the outcome of widespread applications of DMSO as drug solvent and cryoprotectant agent.

Mx is regulated by type I interferons and contains a typical GTP-binding motif like other members of the GTPase dynamin family. However, the functions and working mechanisms of the Mx protein in chicken spermatogonial stem cells (SSCs) are not well documented. In the present study, Mx-overexpressing chicken SSCs (chMx-SSCs) were established and the antiviral activity of chMx-SSCs against Newcastle disease virus, avian influenza viruses was investigated in vitro. For chicken SSCs isolation, day 20 fetal males derived testes were initially subjected to digestion by collagenase IV followed by 0.25% trypsin–EDTA. After discarding the supernatant, the cells were cultured in SSC medium. SSC colonies expressed pluripotent markers such as stagespecific embryonic antigen-1, Oct-4, Nanog, and Sox-2. Chicken Mx gene was constructed in plasmid DNA vector (pcDNA3.1/V5-His A-chMX) and ChMx-SSCs lines were established with chMX constructs. The antiviral activity of ChMx-SSCs was determined by real-time RT-PCR, flow cytometry, and western blot analyses after infection with Newcastle disease virus-green fluorescent protein (GFP) and avian influenza viruses (H9N2 and H1N1). ChMx-SSCs inhibited recombinant Newcastle disease virus (rNDV)- GFP replication as determined by the calculation of the proportion GFP signal- positive cells by FACS analysis. When SSCs showed 100% GFP expression, chMx- SSCs had only 3.6% GFP expression. At 24 h after avian influenza virus infection, chMx-SSCs had a lower hemagglutinin protein level and a higher level of Mx protein. When the number of released virion particles was estimated by plaque-formation assay, chMx-SSCs had significantly fewer stained visible plaques in the MDCK layer than SSCs. Our results suggest that overexpression of chicken Mx directly stimulates antiviral activity resulting in downregulation of viral progeny release. Chicken Mx overexpression in chicken SSCs can be applied for the production of virus resistant transgenic chicken via direct transplantation of chMx-SSCs into the testis.

Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are members of the transforming growth factor β (TGF-β) family, and their roles in oocyte maturation and cumulus expansion are well known in the mouse and human, but not in the pig. We investigated GDF9 and BMP15 expression in porcine oocytes during in vitro maturation. A significant increase in the mRNA levels of GDF9 and BMP15 was observed at germinal vesicle breakdown, with expression levels peaking at metaphase I (MI) but decreasing at metaphase II (MII). GDF9 and BMP15 protein localized to the oocyte cytoplasm. While treatment with GDF9 and BMP15 increased cumulus expansion and the expression of genes involved in both oocyte maturation (c-mos, cyclinb1, and cdc2) and cumulus expansion (has2, ptgs2, ptx3, and tnfaip6). SB431542 (a TGFβ–GDF9 inhibitor) decreased meiotic maturation at MII. Following parthenogenetic activation, the percentage of blastocysts in SB431542 treatment was lower than in the control (74.4% and 41.3%, respectively). Treatment with GDF9 and BMP15 also increased the mRNA levels of maternal genes such as c-mos (a regulatory subunit of mitogen-activated protein kinase (MAPK)), and cyclinb1 and cdc2 (regulatory subunits of maturation/M phase promoting factor (MPF)); however, SB431-542 significantly decreased their mRNA levels. These data were supported by poly (A)-test PCR and protein activity analyses. Our results show that GDF9 and BMP15 function in cumulus expansion and that they stimulate MPF and MAPK activity in porcine oocytes during in vitro maturation.

Meiotic maturation in many species is initiated by the activation of maturation-promoting factor (MPF) with concomitant inactivation of counteracting phosphatases, most notably protein phosphatase 2A (PP2A). Recently, Greatwall (Gwl) has been identified as a cell cycle regulator that inhibits PP2A activity. In this study, we demonstrate that Gwl is required for meiotic maturation in porcine oocytes. Gwl is expressed from germinal vesicle (GV) to metaphase II (MII) stages of porcine oocytes and dramatically decreases with the progression of the meiotic cell cycle. Gwl is initially localized in the nucleus of GV oocytes and is associated with spindle fibers following GV breakdown (GVBD). Depletion of Gwl inhibits or delays meiotic maturation secondary to defects in chromosome congression and spindle formation. Conversely, overexpression of Gwl overcame meiotic arrest and initiated progression to the MII stage. However, these oocytes had severe spindle defects. Furthermore, MII oocytes depleted of Gwl progressed to pronuclear formation. Taken together, our data demonstrate that Gwl is required not only for meiotic maturation but also for maintenance of MII arrest in porcine oocytes.

G protein-coupled receptor kinase 5 (GRK5) is one of the seven GRK family members whose primary function is to desensitize G protein-coupled receptors (GPCRs). In recently, GRK5 deficiency has been linked to early Alzheimer disease (AD), but the mechanism by which GRK5 deficiency may accelerate to AD pathogenesis remains elusive. The GRK5 mRNA is expressed widely in brain and peripheral tissues, with highest expression evident heart, lung, and placenta. In cellular model systems, GRK5 can phosphorylate several neuronal GPCRs including ß2-adrenergic, M2-muscarinic, secretin, angiotensin AT1, and thyroid stimulating hormone receptors. Transcription activator-like effector nucleases (TALENs) are programmable nucleases that join FokI endonucleases with the modular DNA-binding domain of TALEs and highly effective in inducing mutations at specific genome loci. TALEN-mediated mutagenesis in zygotes is a potential alternative to conventional gene targeting in mice. In the presented study, we report the generation of mice with genetic knockout of the GRK gene using TALENs. We designed TALEN vectors for exon 1, 3 and 5 of mouse GRK5 gene and tested their ability to alter the each surrogate vector in 293T cells. We prepared of mRNAs for the linearized TALEN using the mMessage mMachine T7 Ultra kit. mRNAs (4ng/μl) was injected into cytoplasm of 180 one-cell embryos. After incubation for 24 hours, the selected two-cell embryos transferred into the oviduct of seven pseudopregnant C57BL/6 mice. We confirmed the genotype of Fo mice by sequencing and T7E1 assay. We found 6 mutant mice lines (11%) from 53 newborns. We also mated 3 Fo GRK5 mutant lines with wild type mice and confirmed the genotype of the F1 progenies. All the mutations observed in Fo mice were transmitted through the germline but not all progenies (8/3, 13/4, 7/4). Taken together, TALEN-mediated mutagenesis might accelerate the creation of genetically engineered mouse models and elucidate the mechanism of AD pathogenesis using GRK5 knock out mice.