Uncoupling protein 1 (UCP1) is a unique mitochondrial membranous protein expressed in brown adipose tissue (BAT) in mammals. While its expression in response to cold temperatures and adipogenic inducers is well-characterized in mammals and human infants, the molecular characterization and expression of UCP1 in fish remain unexplored. To address this gap, we analyzed UCP1 expression in response to adipogenic inducers in a fish cell line, rainbow trout gonadal cells (RTG-2), and compared it with UCP1 expression in three mammalian preadipocytes, 3T3-L1, T37i, and WT1 exposed to the Peroxisome proliferator-activated receptor gamma (PPARγ) agonists, rosiglitazone (Rosi). In mammalian preadipocytes, UCP1 protein was highly expressed by Rosi, with an induction of adipogenesis observed in a time-dependent manner. This suggests that UCP1 plays a significant role in adipogenesis in mammals. However, RTG-2 cells showed no response to adipogenic inducers and exhibited only marginal expressions of UCP1. These results imply that RTG-2 cells may lack crucial responsive mechanisms to adipogenic signals or that the adipogenic response is regulated by other mechanisms. Further studies are needed to confirm these phenomena in fish preadipocytes when an appropriate cell line is established in future research.

Mitochondrial and mitochondrial DNA (mtDNA) is maternally inherited in humans and most animals. The degradation of sperm-borne mitochondria after fertilization assures normal preimplantation embryo development and may prevent mitochondrial diseases derived from heteroplasmy. Although it has been known that ubiquitin-proteasome system (UPS) is the major degradation pathway of post-fertilization sperm mitochondria in mammals, it is unclear how the UPS, which is able to get rid of single protein molecule at a time, can eliminate whole sperm mitochondrial organelle. We considered that the autophagy receptors [sequestosome 1(SQSTM1), microtubule-associated protein 1 light chain 3 (LC3), and gamma-aminobutyric acid receptor-associated protein (GABARAP)] and the non-traditional mitophagy pathways involving UPS and the ubiquitin-binding protein dislocase, valosin-containing protein (VCP) may act independently or in concert during post-fertilization sperm mitophagy. We found that the association of SQSTM1 with sperm mitochondria was displayed in both pig and rhesus monkey zygotes after fertilization. Sperm mitochondrial proteins [mitochondrial trifunctional enzyme subunit alpha (HADHA), mitochondrial aconitase 2 (ACO2), and mitochondrial ATP synthase H+ transporting F1 complex β-subunit (ATP5B)] co-purified with the synthetic, SQSTM1-derived, ubiquitin-binding UBA domain were identified. Also, the accumulation of GABARAP-positive protein aggregates was observed around sperm mitochondrial sheaths in fertilized oocytes, which reflects autophagosome formation. Furthermore, the inhibition of VCP delayed the process of sperm mitophagy and completely blocked it when embryos were co-injected with autophagy-targeting antibodies, such as anti-SQSTM1 and/or anti-GABARAP. Thus, both SQSTM1-dependent autophagy pathway and VCP-mediated proteasomal proteolysis facilitate post-fertilization sperm mitophagy in mammals. This explains how the proteolytic pathway can coordinate autophagy pathway to degrade the sperm mitochondrial sheath inside the fertilized oocyte.

Until today, success in germline cells and tissue cryopreservation is limited mainly due to the poor understanding of the complex physiological processes can lead to cell damage during cryopreservation. Germline cells, from both male and female, have unique ability to differentiate into one or more cell lines and thus it becomes a crucial point to store them in subzero temperature with the minimal damage of their functional properties and maximum recovery of unchanged and viable cells when thawed. In the past three decades, a vast research has been performed using various different animal models which in fact have led to development of new methodologies and optimization of older one. However, successful use of animal model has provided the opportunity in research with human germline cells and tissues preservation, but not in all the cases. Therefore, the use of new cryo-protective chemicals and modified protocols have been often found in different groups of researchers based on the types, physical structures, utility and animal species of the specimens to be cryopreserved. This review discusses about the basics of different types of cryopreservation methodologies and commonly used optimized protocols and cryoprotectants for germline cells and tissues preservation.

국내에서 포유류는 축산학, 수의학, 실험동물학, 생태학, 유전학 등 다양한 분야에서 활발하게 연구되고 있으며, 최근 생물다양성의 중요성이 강조되면서 이들의 보전 및 관리는 국민적으로도 많은 관심을 받고 있다. 하지만 전문 연구와 국민 관심의 증가에도 불구하고 국내 포유류 연구동향을 파악한 사례는 아직까지 찾아보기 힘들다. 이 연구는 국내 포유류의 연구동향을 파악하여 향후 세부연구영역의 계획과 관련 정책 제시를 위한 기초자료 제공을 목표로 하였다. 2015년까지 국내에서 발행된 포유류 학술논문 392편을 분석 대상으로 하였으며, 최근 각광받는 연구영역을 파악하기 위해 텍스트마이닝과 동시출현단어 분석을 이용하였다. 그 결과, 국내 포유류 연구논문 발행 수는 점차 증가하 였으며, 연구대상 종 역시 점차 다양해진 것으로 나타났다. 텍스트마이닝과 동시출현단어 분석을 통해 파악된 주된 포유류 연구영역은 (1)진화/계통/유전학, (2)환경/생태학, (3)발생/생식/세포생물학, (4)기생충/수의학, (5)설치류/기생충 학, (6)세균/바이러스학, (7)해부/세포생물/실험동물학, (8)형태/해부수의학, (9)축산학, (10)해양포유류학, (11)익수목 연구 등 11개로 구분되었다. 환경/생태학 연구는 11개 연구영역 중에서 최근 가장 활발하였으며, 과거에 비해 연구 비율이 급격히 증가한 분야로 나타났다. 환경/생태학 연구분야는 생물다양성 보전의 핵심으로, 최근 생물다양성의 중요성이 강조됨에 따라 국내 서식 포유류의 생태연구에 대한 연구자들의 관심 역시 더욱 증가한 것으로 보인다. 이 연구결과가 미래 국내 포유류 연구의 계획과 관련 정책 수립을 위한 기초자료로 유용하게 활용되기를 희망한다

Sperm adhesion molecule 1 (SPAM1) and Hyaluronidase 5 (HYAL5) has been well-known as assistants for sperm penetrate through the cumulus mass surrounding the ovulated eggs. However, so far their role in mammalian fertilization remain elusive, because mouse sperm lacking SPAM1 or HYAL5 were still capable of penetrating the cumulus mass despite a delayed dispersal of cumulus mass. Those data collectively demonstrated that SPAM1 or HYAL5 deficiency alone was not sufficient to cause male infertility in mice. In the present study, SPAM1 and HYAL5-simultaneous deficient male mice model was generated. Because of inhibition in sperm hyaluronidases, SPAM1 and HYAL5-deficient male mice produced significantly smaller numbers of offspring than hetero type and wild type mice. Hyaluronic acid degradation assay and cumulus oocyte complex dispersal assay as well as sperm motility assay using double knock out sperm and extracts had severe adverse effects on the dispersal of cumulus oocyte complex, which was the main reason for the impaired fertility of double knock out male sperm. Moreover, hyaluronic acid degradation assay using human sperm extracts revealed that sperm hyaluronidase has a principal role in sperm penetration through the cumulus oocyte complex. In conclusion, our results suggest that sperm hyaluronidase deficiency may be sufficient to cause male sterility in mammal because SPAM1 and HYAL5 deficiency sperm not impaired the sperm motility in hyaluronic acid but also cumulus oocyte complex penetration.

Heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) have a important role in influence of pre-messenger RNA (pre-mRNA) processing and mRNA metabolism and transportation in cells. Recently, hnRNP A2/B1 can recognize m6A modifications on pre-mRNA or pre-miRNA and affect alternative splicing and miRNA processing in HeLa Cells. However, roles of hnRNP A2/B1 in various cells and tissues, especially in elary embryo development, are unclear. Here, we investigated the temporal and spatial expression patterns of hnRNPA2B13 during mammalian early embryo development. In mouse, hnRNPA2B1 was localized at the nucleus after 1-cell stage, however, hnRNPA2B1 was expressed after 2-cell stage in pig. Then, knockdown of hnRNP A2/B1 induced by RNA interference (RNAi) was used to analyze the effect of hnRNP A2/B1 in preimplantation develop in pigs. Knockdown of hnRNP A2/B1 delayed embryo development. Interestingly, ICM marker OCT4 and Sox2 was significantly decreased in blastocyst stage. mRNA expression show that transcription factors which is Pou5f1, Sox2, Nanog, Cdx2 and AP2γwas decreased the transcription levels without the changing of junction protein, ZO-1, occludin, and CXADR. Outgrowth results indicated that knock-down of hnRNPA2B1 embryos cannot format the colony. Knock-down of Methyltransferase like 3(METTL3) embryos mislocalized the hnRNPA2/B1 at the nucleus. In summary, the expression patterns of hnRNPA2/B1 differ between mouse and porcine embryos, and these differences may reflect species-specific functions during preimplantation embryo development. Our results suggested that hnRNPA2/B1 is necessary for newly synthesis of mRNA related with transcription factor, and early embryo development by the RNA epigenetic modification.

Actin nucleation factors, which promote the formation of new actin filaments, have emerged in the last decade as key regulatory factors controlling asymmetric division in mammalian oocytes. Actin nucleators such as formin-2, spire, and the ARP2/3 complex have been found to be important regulators of actin remodeling during oocyte maturation. We found that actin nucleation promoting factor called WASP homolog-associated protein with actin, membranes and microtubules (WHAMM) play crucial roles in mouse oocyte maturation by generation of ER-associated actin filaments during meiotic spindle migrations. We also investigate regulatory mechanism of actin nucleator spire and discovered the novel roles of Zinc in regulating spire localization and cytoplasmic actin mesh formation. Another class of actin-binding proteins including cofilin, tropomyosin, capping proteins and tropomodulin, are thought to control actin cytoskeleton dynamics at various steps of oocyte maturation. The heterodimeric actin-capping protein (CP) binds to the fast-growing (barbed) ends of actin filaments and plays essential roles in various actin-mediated cellular processes. When CP is knockdowned or inhibitory component was overexpressed, asymmetric divisions of oocytes have been compromised. It turns out that knockdown or inhibition of CP deplete cytoplasmic actin mesh level, which have been known to be essential for maintain cytoplasmic actin mesh. Another actin binding proteins, tropomodulin 3 (Tmod3), binds to the slow-growing end of actin filaments and knockdown or expression deletion mutant of Tmod3 also decrease actin mesh level in maturing oocyte and it severely ablated asymmetric division of oocyte. Finally, tropomyosin 3, actin filament binding proteins protect actin filament from depolymerization, is also important to maintain cortex integrity in maturing oocyte, therefore showed the importance maintenance of actin filaments during oocyte maturation. Taken together, our study on various actin nucleator and actin binding study showed the importance of actin dynamics in mammalian oocyte maturation and early embryonic developments.

The belief that honey bee venom (BV) can be used to treat certain immune-related diseases, such as arthritis and rheumatic conditions, goes back to antiquity. A growing number of reports have demonstrated that BV contains at least 18 pharmacologically active components, including phospholipase A2 (PLA2). Recent research has shown that bee venom PLA2 (bvPLA2) induces protective immune responses against several diseases including asthma, Parkinson’s disease, and drug-induced organ inflammation. However, the antiviral properties of bvPLA2 have not been well investigated. Hence, we examined the potential inhibitory effects of bvPLA2 and its possible mechanism of action against a broad panel of pathogenic viruses in vitro. Pre-treatment with bvPLA2 significantly inhibited the replication of vesicular stomatitis virus (VSV), coxsackie virus (H3), enterovirus-71 (EV-71), herpes simplex virus (HSV) and Adenovirus (AdV) dramatically. However, bvPLA2 did not show antiviral activity against Influenza A virus (PR8) and Newcastle disease virus (NDV). Such inhibitory effects were explained by blocking of the attachment of the virus to cells upon bvPLA2 treatment. Additionally, we observed that Heparan sulfate (HS) has an inhibitory effect on the attachment of HSV to the cell surface dose dependently, which was inconsistent with bvPLA2 treatment. These findings suggest that bvPLA2 has an inhibitory effect on the replication of diverse viruses by blocking their attachment to the cell surface and could be a promising source of natural antiviral agents.

The objective of this study was to evaluate the toxicities of permeable cryoprotectants and finally to establish the cryopreservation method of surplus embryos obtained during assisted reproductive technology (ART). Toxicities of permeable cryoprotectants, dimethyl sulfoxide (DMSO), ethylene glycol (EG), Glycerol, and 1,2-PROH were investigated using a murine embryo model. Female F-1 mice were stimulated with gonadotropin, induced ovulation with hCG and mated. Two cell embryos were collected and cultured after exposure to among DMSO, EG, Glycerol, and 1,2-PROH. Embryo development was evaluated up to the blastocyst stage. The total cell count of blastocysts that were treated with DMSO and Glycerol at the 2-cell stage was significantly lower than that were treated with EG (81.1±15.1), 1,2-PROH (88.0±21.1) or the control (99.9±21.3) (p<0.001). On comparison of four cryoprotectant treated groups, the DMSO and Glycerol treated group showed a decreased cell count compared with the EG and 1,2-PROH treated group (p<0.05). Both DMSO (14.7±1.3), EG (12.1±1.1), Glycerol (15.2±1.8), and 1,2-PROH (11.5±1.3) treated groups showed higher apoptosis rates of cells in the blastocyst compared with the control (6.5±0.7, p<0.0001). In addition, the DMSO or Glycerol treated group showed more apoptotic cells than the EG or 1,2-PROH treated group (p<0.001). The potential toxicity of cryoprotectants was uncovered by prolonged exposure of murine embryos to among DMSO, EG, Glycerol, and 1,2-PROH at room temperature. When comparing four permeable cryoprotective agents, EG and 1,2-PROH appeared to be less toxic than DMSO and Glycerol at least in a murine embryo model.

본 연구에서 배아의 생식세포 동결에 가장 흔히 쓰이고 있는 두 가지 동결 보호제, 즉 DMSO와 EG의 독성을 비교하고자 생쥐 수정란 모델을 이용한 실험을 하였다. 생후 6주령의 암컷 생쥐 F1 hybrid mice에 10 IU의 PMSG를 복강 주사하여 과배란을 유도하고, 2-세포기 배아를 획득하고 DMSO와 EG 각각 노출시킨 후, 배양을 하였다. 배반포의 전체 세포수는 2-세포기 단계에서 DMSO(68.1±24.1)로 EG(81.2±27.0) 혹은 control(99.0±18.3)(p<0.001) 처리구에 비해서 유의적으로 낮았다. DMSO 처리구가 EG 처리구에 비해 세포수가 적었다. DMSO(15.4±1.5)와 EG(10.2±1.4) 두 처리구는 대조구(6.1±0.9, p<0.0001)와 비교해서 배반포에서 세포사 비율이 더 높음을 확인했다. 또한, DMSO 처리구는 EG 처리구(p<0.001)보다 더 많은 세포사멸된 세포가 확인되었다. DMSO 또는 EG 처리군과 대조군 사이에는 배아 부화율에 있어서 차이가 있었으며, 이는 배아에 대한 동결 보호제의 잠재적인 독성을 확인한 결과였다. 이번 연구에서 장기간 처리했을 때 EG 처리군보다 DMSO 처리군에서 배아발달과 세포수가 저하된 것은 DMSO의 독성이 더 높을 것으로 사료된다.

본 연구에서 생쥐 초기 배아를 이용하여 동결 보존된 배아의 생존율 또는 발달율에 영향을 미치는 요인들의 상관관계를 알아본 결과, 배아의 발달 단계에 따른 동결 - 해동 후 생존율에 있어서는 2세포기 배아가 4~8세포기 배아보다 유의하게 높았으나(p＜0.01), 배 발달율에 있어서는 4~8세포기 배아가 유의하게(p＜0.01), 높아 생존율과 배 발달율과는 상관관계가 없는 것으로 사료된다. 또한, 동결 보호제에 따른 배아의 생존율에 있어서는 2, 4~8세포기 배아 모두 DMSO에서 유의하게 높았으나(p＜0.01), 배 발달율에 있어서는 EG가 DMSO에 비해 더 유의하게 높은 성적을 나타내어(p＜0.01, p＜0.05) 동해로 인한 상해가 적은 것으로 생각되어 2, 4~8세포기 배아에서 EG가 더 효과적인 동결 보호제로 사료되며, 동결 프로그램으로는 완만 동결 - 급속 해동법이 더 우수한 프로그램으로 보인다.

생쥐 2세포기, 4세포기, 8세포기를 각 발생 단계에서 채취하여 동결 보호제를 첨가한 서로 다른 배양액에서 배양하고, 배아의 동결 보존 및 융해 시 급속 처리와 저속 처리 단계로 비교하여 이들 조건이 배아의 생존과 발현에 미치는 영향을 조사하여 다음의 결과를 얻었다. 동결 보호제로 처리하여 배양액을 달리한 경우, 급속 단계에서는 모든 배양액에서 비슷한 발생율을 보였고, 저속단계의 4세포기와 8세포기는 D-PBS에서 높은 발생율을 보였다(P＜0.05, P＜0.01). 배아의 발생 시기에 따른 동결 보존 후, 발생율은 2, 4, 8세포기로 넘어갈수록 발생율의 증가를 보여 8세포기에서 발생율이 가장 높았다(P＜0.01). 동결 보호제의 처리단계에 따른 발생율은 2세포기의 급속 단계에서는 유사하였으나, 4세포기와 8세포기는 저속단계에서 높은 발생율을 보였으며(P＜0.05), 특히 8세포기에서 가장 높았다(P＜0.01).

Precise, rapid and simple methods for species identification in animals are among the most important techniques in the livestock industry and research fields including meat classification. In this study, polymerase chain reaction (PCR) based molecular identification using inter species polymorphisms were examined by PCR-restriction fragment length polymorphism (RFLP) analysis for mitochondrial DNA (mtDNA) cytochrome b (CYTB) gene sequences among four mammalian livestock animals (cattle, horse, goat and pig). The results from PCR-RFLP analysis using the AluI restriction enzyme were also provided for the species-specific band patterns among CYTB gene sequences in these four species. The AluI-digestion for CYTB genes provided interesting migration patterns differentially displayed according to each species. Cattle and horse had one AluI-recognition site at different nucleotide positions and their AluI-digested fragments showed different band patterns on the gels. Pig had two AluI-recognition sites within the amplified CYTB sequences and produced three bands on the gels. Goat had no AluI-recognition site and was located at the same position as the uncut PCR product. The results showed the species-specific band patterns on a single gel among the four livestock animal species by AluI-RFLP. In addition, the results from blind tests for the meat samples collected from providers without any records showed the identical information on the species recorded by observing their phenotypes before slaughter. The application of this PCR-RFLP method can be useful and provide rapid, simple, and clear information regarding species identification for various tissue samples originating from tested livestock species.

Insects and animals can recognize surrounding environments by detecting thousands of chemical odorants. Olfaction is a complicated process that begins in the olfactory epithelium with the specific binding of volatile odorant molecules to dedicated olfactory receptors (ORs). OR proteins are encoded by the largest gene superfamily in the mammalian genome. We report here the whole genome analysis of the olfactory receptor genes of S. scrofa using conserved OR gene specific motifs and known OR protein sequences from diverse species. We identified 1,301 OR related sequences from the S. scrofa genome including 1,113 functional OR genes and 188 pseudogenes. OR genes were located in 46 different regions on 16 pig chromosomes. We classified the ORs into 17 families, three Class I and 14 Class II families, and further grouped them into 349 subfamilies. We also identified inter- and intra-chromosomal duplications of OR genes residing on 11 chromosomes. A significant number of pig OR genes (n=212) showed less than 60% amino acid sequence similarity to known OR genes of other species. We also performed a similar analysis on the cattle OR subgenome and identified 1,071 OR related sequences. We show that S. scrofa has one of the largest OR repertoires, suggesting an expansion of OR genes in the swine genome. Considering available information from literature, it seems that OR systems between mammals and insects possess high similarity in their action mechanisms and rapid evolutionary changes due to differences in living environments.

In the first part of this study, a novel culture device the named oil-free micro tube culture (MTC) system for in vitro culture (IVC) of murine and porcine embryos was introduced. Parthenogenetic mouse and porcine embryos were placed into 0.2-mL thinwall flat cap PCR tubes and cultured to the blastocyst stage. Conventional drop culture was used as the control. Murine embryos in MTC had a higher blastocyst formation rate and larger population of cells in the blastocysts. This was due to higher numbers of trophectoderm (TE) cells rather than inner cell mass cells. On the other hand, the 'MTC' system in the pig showed similar (in 20 μl medium volume) or lower (in 10 μl medium volume) blastocyst formation rate when compared with drop culture system. In the second part of this study, dexamethasone (DEX) and leukemia inhibitory factor (LIF), which suppress PGF2α, were directly supplemented into ET media, and transfer of the embryos to surrogate was followed. In the cattle industry, embryo transfer technology has been used to produce the most valuable cows or bulls. Numerous factors such as heat stress, mastitis, manipulating female reproductive tract may contribute to early embryonic loss through premature increases of uterine luminal concentrations of PGF2α in cows. Furthermore, addition of PGF2α to culture medium has been shown to inhibit the development and hatching of mammalian embryos. When DEX and LIF were supplemented, the pregnancy rate (6 month post-ET) was increased from 56.0% to 68.3%. In IVC experiment, DEX and LIF supplementation supported hatching of bovine embryos in the presence of PGF2α in the medium (from 16.9% to 40.6%). Additional ET experiments using alternative drugs are currently under investigation. The present work was supported by the Technology Development Program for Agriculture and Forestry, Ministry for Food, Agriculture, Forestry and Fisheries (MIFAFF; 109020-3).

In this study we present a mammalian cell culture model that allows to study the effect of endocrine disruptors (EDCs) on aromatase activity of aquatic amphibian, Bombina orientalis. Bombina orientalis aromatase gene was subcloned into a mammalian express

Two-pore domain 칼륨() 통로는 흥분세포 및 비흥분세포의 안정막 전압을 일정하게 유지하는데 관여한다. 그러나 생식세포 및 생식기관에서 발현되는 통로의 분포영역 및 그 기능에 대해서는 연구자들에 의해 아직 정리되지 못하였다. 본 종설에서는 통로의 생식세포 및 생식기관에서 발현, 분포 및 생리학적 의의를 논하였다. 통로는 인간 영양막세포, 자궁근층, 태반혈관계, 자궁평활근조직, 태반융모조직 및 임신자궁조직에서 발현되어 임신에 있어서 관련성을 제