Ganglioside GT1b, glycosphigolipids with three sialic acid, is known to play an important role in signal transduction such as epidermal growth factor receptor (EGFR). EGF is also known to induce resumption of meiosis and cumulus cells expansion during porcine oocyte maturation. Therefore, this study was conducted to evaluate the effects of ganglioside GT1b on resumption of meiosis and cumulus cells expansion in porcine oocyte maturation. First, porcine cumulus-oocyte complexes were cultured in NCSU-23 medium supplemented with GT1b (0, 1, 2 and 4 μM) at 44 h. We observed that the proportion of the metaphase II (M II) stage was significantly increased in the 2 μM GT1b (78.0 ± 2.3) treated group than in the other groups. Furthermore, expression of cumulus cells expansion factor genes (Has2, TNFAIP6, Ptx3) were significantly increased in the 2 μM GT1b treated group than in the other groups. Next, we investigated the meiotic maturation and the expressions of cumulus cells expansion factor genes after GT1b and/or EGF treatment. The proportion of the M II stage was significantly higher in the GT1b+EGF (90.1 ± 2.3) treated group than in the other groups. Moreover, expressions of cumulus cells expansion factor genes were significantly increased in the GT1b+EGF treated group than in the control group. After in vitro fertilization, fertilization rate, preimplantation development competence and quality of blastocyst were improved in oocytes derived from GT1b+EGF treated group. Taken together, these results suggest that exogenous ganglioside GT1b improving the developmental competence of porcine embryos via increase of resumption of meiosis and cumulus cells expansion during in vitro maturation of porcine oocytes.

Ganglioside GD1a is specifically formed by the addition of sialic acid to ganglioside GM1a by ST3 β- galactoside α -2,3-sialyltransferase 2 (ST3GAL2). Above all, GD1a are known to be related with the functional regulation of several growth factor receptors, including activation and dimerization of epidermal growth factor receptor (EGFR) in tumor cells. The activity of EGF and EGFR is known to be a very important factor for meiotic and cytoplasmic maturation during in vitro maturation (IVM) of mammalian oocytes. However, the role of gangliosides GD1a for EGFR-related signaling pathways in porcine oocyte is not yet clearly understood. Here, we investigated that the effect of ST3GAL2 as synthesizing enzyme GD1a for EGFR activation and phosphorylation during meiotic maturation. To investigate the expression of ST3GAL2 according to the EGF treatment (0, 10 and 50 ng/ml), we observed the patterns of ST3GAL2 genes expression by immunofluorescence staining in denuded oocyte (DO) and cumulus cell-oocyte-complex (COC) during IVM process (22 and 44 h), respectively. Expression levels of ST3GAL2 significantly decreased (p<0.01) in an EGF concentration (10 and 50 ng/ml) dependent manner. And fluorescence expression of ST3GAL2 increased (p<0.01) in the matured COCs for 44 h. Under high EGF concentration (50 ng/ml), ST3GAL2 protein levels was decreased (p<0.01), and their shown opposite expression pattern of phosphorylation-EGFR in COCs of 44 h. Phosphorylation of EGFR significantly increased (p<0.01) in matured COCs treated with GD1a for 44 h. In addition, ST3GAL2 protein levels significantly decreased (p<0.01) in GD1a (10 μM) treated COCs without reference to EGF pre-treatment. These results suggest that treatment of exogenous ganglioside GD1a may play an important role such as EGF in EGFR-related activation and phosphorylation in porcine oocyte maturation of in vitro.

Mitochondria are well known to regulate the mammalian embryo development. Recent studies showed that the mitochondrial dynamics responses are mainly generated through mitochondrial membrane potential (MMP) and cellular ATP production, which is dependent on mitochondrial reactive oxygen species (ROS). However, these mechanisms are unclear on development process of preimplantation porcine embryos. The aim of this study was to evaluate that difference of the mitochondrial dynamics-derived various functions on the embryo development according to lipid composition of zygote. First, zygote was classify two groups (high lipid, grade 1: G1 and low lipid, grade 2: G2) by lipid composition of cytoplasm. And, we performed the in vitro culture (IVC) using zygote of divided groups. The nuclei numbers and developmental rates of blastocysts were lower in G2 than those of G1 embryos. Next, we investigated the intracellular ROS and mitochondrial derived superoxide production in porcine embryos by using DCF-DA and Mito-SOX staining. As expected, both intracellular ROS and mitochondrial derived superoxide were significantly increased (p<0.05) in the preimplantation stage embryos of G2 group compared with G1 group. In addition, to observe difference of the mitochondrial functions, we investigated the mitochondrial membrane potential (MMP, ΔΨ) and contents of ATP in the preimplantation stage embryos by using JC-1 kit and ATP determination kit. These functions of mitochondria were dramatically reduced in cleavage stage embryos or blastocysts of G2 group. Finally, to verify the difference of the mitochondrial dynamics-derived various functions, we investigated the expressions of mitochondrial fission (Drp1, pDrp1-616) and fusion (Mfn1, Mfn2) factors by Western blotting analysis. Interestingly, the protein levels of pDrp1-616 in embryos of G1 group were continuously increased until blastocyst stage. Whereas, the expression patterns of Mfn1/2 in embryos of G2 group were significantly reduced during IVC progression. The expression patterns of mitochondria dynamic between the two groups were shown opposite. These results demonstrated that the lipid contents of zygote were related the positive-correlation with mitochondrial dynamics-derived functions in porcine embryos. Moreover, we suggest that lipid of zygote is play a important role on mitochondrial functions and dynamics during preimplantation embryos development in pigs.

The plastic monomer bisphenol A (BPA) is well known as a representative environmental hormones. Recent studies showed that the BPA exposure induced mitochondrial dysfunction and mitochondrial derived reactive oxygen species (mito-ROS). However, changes of antioxidant enzymes expression and ROS production from mitochondria according to the BPA exposure on in vitro maturation (IVM) of porcine oocytes have not been studied. We hypothesized that regulation of ROS production from mitochondria by BPA may play a critical role in meiotic maturation or expansion of cumulus cells in cumulus-oocyte complexes (COCs). To investigate the negative effects of BPA exposure on oocyte maturation, immature pig oocytes were matured in NCSU-23 medium supplemented with BPA (50, 75 and 100 μM) for 44 h. Expectedly, the rates of meiotic maturation and cumulus cell expansion of COCs in the BPA (75 μM) treated group was significantly lower than those of control group (p<0.01). Most of secretion factors expressions from COCs were significantly decreased (p<0.05) in the BPA treated COCs. Next, we investigated the intracellular ROS and mitochondrial specific superoxide production according to the BPA exposure using DCF-DA and mito-SOX staining, respectively. BPA exposure were showed that increasing of both intracellular ROS and mito-ROS, as well as mitochondrial related antioxidant enzymes (sod2, prdx3, prdx5) mRNA expression significantly increased (p<0.01) in COCs. And then, mitochondria membrane potential (MMP) dramatically reduced, and mitochondrial-derived apoptotic factors (bax, bcl-xl, caspase 3) mRNA expressions were increased (p<0.01) in BPA treated COCs. In additon, protein levels of mitochondrial-derived apoptosis genes (AIF, cleaved parp1 and caspase 3) were significantly increased (p<0.05) by BPA exposure. To confirm the reduction of BPA-induced mito-ROS, we used to the mitochondrial-targeted ROS scavenger, mito-TEMPO. Interestingly, addition of mito-TEMPO (0.1 μM) to the BPA pre-treated COCs recovered in meiotic maturation of porcine oocytes. These results demonstrated that BPA exposure was induced increasing of mitochondrial dysfunction, mito-ROS and mitochondrial-mediated apoptosis on pig oocyte maturation. Therefore, we suggest that controlling of mito-ROS plays a critical role in pig oocyte maturation in vitro. These findings will be helpful to solve causes of mitochondrial-related infertility.

Melatonin has an important role as anti-oxidative effect and reducing of endoplasmic reticulum(ER)-stress on oocyte maturation and embryo development. Under ER-stress condition, unfolding protein response (UPR) is a defence mechanism in mammalian cells. Recently, regulation of UPR signaling genes are involved in oocyte maturation, embryo development and female reproduction. However, there is no report on the role of melatonin for UPR signaling and ER-stress mediated apoptosis during pig oocyte maturation progression. Moreover, the changes of UPR genes expression according to the porcine oocyte maturation is not yet fully understood. Here, we investigated the changes of UPR signal (BIP/GRP78, ATF4, p90/p50ATF6, and XBP1) and ER-stress apoptotic factor CHOP genes expressions in porcine oocyte maturation by Western blot and RT-PCR analysis. During oocyte maturation, UPR marker and CHOP genes expressions were significantly increased in matured oocytes or cumulus-oocyte complexes (COCs). UPR markers expressions were significantly increased by ER-stress inducer, tunicamycin (Tm), treated (1, 5, 10 μg/ml) groups in a dose-dependent manner compared with control group. To confirm the reducing of ER-stress by melatonin (0.1 μM), we were compared to the effects of ER-stress inhibitor, TUDCA (200 μM), after pre-treated Tm (5 μg/ml) for 22 h maturation. Expressions of UPR markers and meiotic maturation were recovered by melatonin (0.1 μM) in COCs. And, we observed the role of Grp78/Bip as UPR signaling beginning marker using siRNA. In result, reduction of Grp78/Bip gene expression by siRNA was induced the inhibition of oocyte maturation (32.5±10.1 vs control; 77.8±5.3), and p50ATF6 protein level was significantly decreased (p<0.001) in cultured COCs for 44 h. In addition, these results were recovered through the addition of melatonin (0.1 μM) or TUDCA (200 μM) in maturation medium. These results demonstrated that the regulation of UPR signaling via Grp78/Bip gene induction plays a critical role in porcine oocyte maturation in vitro. Furthermore, this present study first confirmed a functional link between inhibition effect of ER-stress by melatonin and regulating of UPR signaling in porcine oocyte maturation. In conclusion, melatonin improves the oocyte maturation and cumulus cells expansion of COCs through the regulation of UPR signal pathway by BIP/GRP78 against the ER-stress during porcine oocyte maturation periods.

Gangliosides exist in glycosphingolipid-enriched domains on the cell membrane and regulate various functions such as adhesion, differentiation, and receptor signaling. Ganglioside GM3 by ST3GAL5 enzyme provides an essential function in the biosynthesis of more complex ganglio-series gangliosides. However, the role of gangliosides GM3 in porcine oocytes during in vitro maturation and early embryo development stage has not yet understood clear. Therefore, we examined ganglioside GM3 expression patterns under apoptosis stress during maturation and preimplantation development of porcine oocytes and embryos. First, porcine oocytes cultured in the NCSU-23 medium for 44 h after H2O2 treated groups (0.01, 0.1, 1 mM). After completion of meiotic maturation, the proportion MII (44 h) was significantly different among control and the H2O2 treated groups (76.8±0.3 vs 69.1±0.4; 0.01 mM, 55.7±1.0; 0.1 mM, 38.2±1.6%; 1 mM, P<0.05). The expressions of ST3GAL5 in H2O2 treated groups were gradually decreased compared with control group. Next, changes of ST3GAL5 expression patterns were detected by using immunofluorescene (IF) staining during preimplantation development until blastocyst. As a result, we confirmed that the expressions of ST3GAL5 in cleaving embryos were gradually decreased (P<0.05) according to the early embryo development progress. Based on these results, we suggest that the ganglioside GM3 was used to the marker as pro-apoptotic factor in porcine oocyte of maturation and early embryo production in vitro, respectively. Furthermore, our findings will be helpful for better understanding the basic mechanism of gangliosides GM3 regulating in oocyte maturation and early embryonic development of porcine in vitro.

Edaravone (Eda) is a potent scavenger of inhibiting free radicals including hydroxyl radicals (H2O2). Reactive oxygen species (ROS) such as H2O2 can alter most kinds of cellular molecules such as lipids, proteins and nucleic acids, cellular apoptosis. In addition, oxidative stress from over-production of ROS is involved in the defective embryo development of porcine. Previous study reported that Eda has protective effects against oxidative stress-like cellular damage. However, the effect of Eda on the preimplantation porcine embryos development under oxidative stress is unclear. Therefore, in this study, the effects of Eda on blastocyst development, expression levels of ROS, and apoptotic index were first investigated in preimplantation porcine embryos. After in vitro fertilization, porcine embryos were cultured for 6 days in PZM medium with Eda (10 μM), H2O2 (200 μM), and Eda+H2O2 treated group, respectively. Rate of blastocyst development was significantly increased (P<0.05) in the Eda treated group compared with only H2O2 treated group. And, we measured intracellular levels of ROS by DCF-DA staining methods and investigated numbers of apoptotic nuclei by TUNEL assay analysis is in porcine blastocyst, respectively. Both intracellular ROS levels and the numbers of apoptotic nucleic were significantly decreased (P<0.05) in porcine blastocysts cultured with Eda (10 μM). More over, the total cell number of blastocysts were significantly increased (P<0.05) in the Eda-treated group compared with untreated group and the only H2O2 treated group. Based on the results, Eda was related to regulate as antioxidant-like function according to the reducing ROS levels during preimplantation periods. Also, Eda is beneficial for developmental competence and preimplantation quality of porcine embryos. Therefore, we concluded that Eda has protective effect to ROS derived apoptotic stress in preimplantation porcine embryos.

녹색두부용으로 개발된 청두 1호의 녹색도를 증진시키고, 밥밑용으로 개발된 청자 3호의 소비촉진을 위하여 두부용으로 이용 가능성을 검정한 결과는 다음과 같다. 녹색두부의 녹색도를 증진시키기 위해 미나리, 부추, 솔잎, 시금치와 쑥을 첨가하여 두부를 제조한 결과 모든 두부에서 명도를 나타내는 L값과 적색도를 나타내는 a값이 유의하게 낮아졌다. 첨가물 중녹색도의 증진 정도가 가장 좋은 시금치를 녹색두부 제조에 이용하였다. 청두 1호와 청자 3호에 시금치를 첨가하여 제조한 두부는 무첨가 두부에 비해 L과 a값이 낮았으며 클로로필, 카로티노이드와 조섬유 함량이 높았다. 시금치를 첨가한 두부의 물성검사 결과 무첨가구에 비하여 청두 1호는 경도, 검성, 응집성과 씹힘성이 유의하게 변하였고, 청자 3호는 탄력성에 차이를 보였다. 관능검사 결과 두부 색에 대한 기호도는 청두 1호로 만든 녹색두부가 청자 3호 보다 높았으나, 시금치 첨가가 식미에 좋지 않은 영향을 끼쳤을 것으로 판단되므로 녹자엽 콩을 이용한 녹색두부의 녹색도를 증진시키고 소비자들의 기호도를 높이기 위해서는 추후 이를 고려한 첨가량 설정 연구가 필요할 것이다.

In the present study, we investigated the role of binding immunoglobulin protein/glucose-regulated protein, 78-kDa (BIP/GRP78)-regulated endoplasmic reticulum (ER)-stress on meiotic maturation and cumulus cells expansion in porcine cumulus-oocyte complexes (COCs). Previously, it has been demonstrated that unfolded protein response (UPR)- related genes, such as molecules involved in ER-stress defense mechanisms, were expressed in matured oocytes and cumulus cells during in vitro maturation (IVM) of porcine oocytes. However, BIP/GRP78-mediated regulation of ER stress in porcine oocytes has not been reported. Firstly, we observed the effects of knockdown of BIP/GRP78 (an UPR initiation marker) using porcine-specific siRNAs (#909, #693, and #1570) on oocyte maturation. Among all siRNAs, siRNA #693 significantly reduced the protein levels of UPR marker proteins (BIP/GRP78, ATF4, and P90ATF6) in porcine COCs observed by Western blotting and immunofluorescence analysis. We also observed that the reduction of BIP/GRP78 levels by siRNA#693 significantly inhibited the meiotic maturation of oocytes (siRNA #693: 32.5±10.1% vs control: 77.8±5.3%). In addition, we also checked the effect of ER-stress inhibitors, tauroursodeoxycholic acid (TUDCA, 200 μM) and melatonin (0.1 μM), in BIP/ GRP78-knockdown oocytes. TUDCA and melatonin treatment could restore the expression levels of ER-stress marker proteins (BIP/GRP78, p-eIF2α, eIF2α, ATF4, and P90ATF6) in siRNA #693-transfected matured COCs. In conclusion, these results demonstrated that BIP/GRP78-mediated regulation of UPR signaling and ER stress plays an important role in in vitro maturation of porcine oocytes.

We recently reported rice promoters that are active in late stages of pollen development. However, rice promoters that allow manipulation of gene expression at earlier stages of pollen development are still very limited to date. In this study, we have chosen 10 putative microspore promoters, OsMSP1 through OsMSP10, based on publicly available transcriptomic datasets in rice (Oryza sativa L.). Sequence analysis of these promoter regions revealed some cis regulatory elements involved in pollen-specific expression. We also examined promoter activities using the promoter-GUS reporter constructs in both transgenic rice and Arabidopsis. In rice, all of the 10 promoters directed GUS signals from the microspore stage throughout the all stages of pollen development. In addition, while GUS signals from 4 promoters, OsMSP2, OsMSP7, OsMSP9 and OsMSP10, seem to be expressed preferentially during pollen development, those from other six promoters were observed in vegetative tissues such as leaves, stems, and roots of seedlings. Similarly, in Arabidopsis, all of the 10 promoters directed GUS signals during pollen development. In detail, 8 promoters, OsMSP1 ~ OsMSP8 directed GUS signals from the microspore stage, whereas 2 promoters, OsMSP9 and OsMSP10, exhibited GUS signals from tricellular stage. Furthermore, seven promoters, except for OsMSP1, OsMSP2 and OsMSP10, showed GUS signals in shoot apical region or root tissues of seedlings. Furthermore, we verified microspore activity of four promoters, OsMSP1, OsMSP2, OsMSP3 and OsMSP6, by complementation analysis of the sidecar pollen (scp) mutant which displays microspore-specific defects. Currently, further analyses are underway for GUS expression of T2 generation in transgenic rice and scp complementation with remaining promoters.

In the course of map-based cloning, mutant genes are identified through linkage to specific region on genetic map. Here, we demonstrated gametophytic mutant line, named as AP-28-23, in which mutant gene was mapped on chromosome 2. Based on phenotypic analysis of mature pollen, mutant phenotype of AP-28-23 was classified into three classes, wild-type showing 2-4%, moderate 35-53% and severe type 97-100% on aberrant pollen frequencies, respectively. The severe type is completely sterilized with 100% unfertilized ovules. We also revealed that the transmission was reduced through male gametophyte in the AP-28-23 line. The transmission efficiency (TE) through the male gametophyte is only 0.67%, whereas in the female gametophyte is 89.87%.

본 연구에서는 단옥수수 주요 품종인 고당옥과 구슬옥을 재배하여 고품질 단옥수수 생산 재배법을 보급하기 위한 기 초자료를 제공하고자 종실의 등숙 중 이화학적 특성 변화를 조사하였다. 파종시기와 관계없이 등숙이 진행됨에 따라 수 분 함량은 감소하고, 종실 100립의 생중과 건중은 증가하였 고(p<0.05), 파종시기가 늦어질수록 100립중의 증가 속도는 느려졌다. 종실의 조단백과 조회분 함량은 출사 후 일수가 경과함에 따라 감소하는 경향을 보였고, 조지방 함량은 유 의하게 증가하였다(p<0.05). 단옥수수 종실의 총 유리당 함 량은 파종시기와 관계없이 출사 후 일수가 경과함에 따라 유의하게 감소하였으나(p<0.05), 유리당을 구성하는 fructose, glucose, sucrose와 maltose의 조성은 파종시기에 따라 차이를 보였으며 그 중 sucrose가 대부분을 차지하고 있었다. 종 실의 전분 함량과 경도는 출사 후 일수가 경과함에 따라 유 의하게 증가하는 경향을 보였으며(p<0.05), 전분 함량의 증 가 속도 역시 100립중과 마찬가지로 파종이 늦어질수록 느 려지는 경향을 보였다. 수확 적기의 종실 수분함량은 68~ 69% 정도였으며, 전분 함량은 7월 10일 파종이 가장 높았 고 7월 30일 파종이 가장 낮았다. 적기에 수확한 단옥수수 의 단맛을 비교한 결과 고당옥은 4월 10일 파종한 것에 비 해 7월 20일, 7월 30일 파종이 약 1.8배와 1.4배 단맛이 높 은 반면 구슬옥은 파종시기와 상관없이 단맛의 정도가 비슷 하였다.

The correct development of male gametophytes (pollen grains) in flowering plants is essential for proliferate in gamete production. Here we have taken a map-based cloning approach using Arabidopsis male gametophytic mutant, named gemini pollen3 (gem3) to identify and characterize key gene that is expressed gametophytically for the completion of microgametogenesis focusing on genes which control cell division and cell fate determination. Previously reported gem1 and gem2 mutants with similar characteristics to gem3 that are disturbed at asymmetric division and cytokinesis at pollen mitosis I (PMI) in Arabidopsis. However, gem3 was mapped to a different genetic locus, and pollen developmental analysis revealed that gem3 exert an effect at meiosis and mitosis causing complete sterility. We also discovered that gem3 homozygous lines produce aberrant pollen grains, arising from incomplete cytokinesis during male meiosis with sporophytic phenotypes of twisted-shape leaves, large flowers. This mutation shows reduced genetic transmission of gem3 allele through male gametophyte. In previous results, the gem3 locus was confirmed by mapping to the region located on chromosome 5. To further confirm strong candidate gene, we performed sequencing and genetic complementation analysis. Currently, we are performing functional studies of the gem3 gene for the better understanding of molecular mechanisms that control asymmetric division at meiosis and mitosis during pollen development.

OsLPS is pollen specific gene that express at late stage of pollen development in rice. Based on microarray database, promoter region of two genes Os03g0106900 and Os03g0106500 were identified. The sequence of 2287bp and 2468bp upstream region of these genes were amplified and designated as OsLPS10 and OsLPS11. These promoters were fused with GUS-GFP reporter gene in a destination vector, pKGWFS7 and introduced into rice (Dongjin cultivar) and Arabidopsis (Col-0). The results of GUS assay showed different pattern of gene expression in pollen of rice and Arabidopsis. In Arabidopsis, the OsLPS10 gene strongly activated in young anther and not expressed in mature pollen. Pollen development analysis revealed GUS expression was detected at unicellular stage and strongest at the bicellular pollen developmental stage. No GUS signal was recorded in mature pollen. In case of OsLPS11, no GUS signal was detected in during pollen development of inflorescent. By contrast, in rice, the GUS expression pattern of OsLPS10 and OsLPS11 exhibited similar. GUS expression was first detectable in the anthers of spikelets at the bicellular stage and intensity increased in tricellular and mature pollen. The GUS signal was not detected in the anthers in unicellular microspores in both genes, OsLPS10 and OsLPS11. The results suggested that these genes were different activity in heterologous plant system, monocot and dicot. Complementation analysis and Cis-regulatory elements will be examined to illuminate the characteristic of these genes

Based on the results of microarray analysis we selected ten candidate genes that express in pollen at the early pollen developmental stage. By PCR amplification, the promoter region of these genes were amplified from rice genomic DNA (Nipponbare) and cloned into the destination pKGWFS7 vector via an entry vector, pDONR201. The characteristic of promoters were evaluated in Arabidopsis thaliana (Col-0) through GUS expression analysis. Fifty T2 plants respectively from each promoter were tested. Whole inflorescence of individual plant was stained with 1mM X-Gluc solution to observe tissue-specific GUS expression patterns. The results showed that all 10 promoters activated in pollen tissues. Among them six promoters expressed at the early developmental stage (unicellular) of pollen and the others expressed at both early (unicellular) and late pollen developmental stage (mature pollen). The results indicated that these promoters would be potential applicable for the studies of pollen function. Currently, we are performing these promoters analysis in rice transgenic plants as well as molecular characterization.