Autophagic activation in vitrified–warmed mouse oocytes

Bang, Soyoung,Shin, Hyejin,Song, Haengseok,Suh, Chang Suk,Lim, Hyunjung Jade BioScientifica 2014 Reproduction Vol.148 No.1

<P>Vitrification involves the use of cryoprotectants (CPAs) and liquid nitrogen (LN<SUB>2</SUB>), which may cause osmotic damage and cryoinjury to oocytes. Autophagy is widely recognized as a survival or response mechanism elicited by various environmental and cellular stressors. However, the induction of autophagy in vitrified–warmed oocytes has not been examined. In this work, we investigated whether the vitrification–warming process induces autophagy in mouse oocytes. Metaphase II (MII) oocytes that were vitrified and stored in LN<SUB>2</SUB> for at least 2 weeks were used in the study. In RT-PCR analyses, we observed that several <I>Atg</I> genes such as <I>Atg5</I>, <I>Atg7</I>, <I>Atg12</I>, <I>LC3a</I> (<I>Map1lc3a</I>), <I>LC3b</I> (<I>Map1lc3b</I>), and <I>Beclin1</I> were expressed in MII mouse oocytes. Slight reduction in mRNA levels of <I>Atg7</I> and <I>Atg12</I> in vitrified–warmed oocytes was noted, and expression of these genes was not significantly influenced. Confocal live imaging analysis using oocytes from GFP-LC3 transgenic mice revealed that vitrified–warmed oocytes had a significantly higher number of GFP-LC3 puncta in comparison to fresh oocytes. The expression of BECLIN1 protein was also increased in vitrified–warmed oocytes. Treatment with 3-methyladenine, an inhibitor of autophagy, did not significantly affect the rates of oocyte survival, IVF, and embryonic development after warming and IVF. The results suggest that the observed autophagic activation in vitrified–warmed oocytes is a natural adaptive response to cold stress. Collectively, we show for the first time that vitrified–warmed mouse oocytes exhibit autophagic activation during warming and that this response is not induced by CPA-containing solutions. The induction of autophagy by cold temperature is first reported herein.</P>

Induction of autophagy in the vitrified-warmed mouse oocytes

Soyoung Bang,Hyejin Shin,Hyunjung Jade Lim 한국발생생물학회 2013 한국발생생물학회 학술발표대회 Vol.2013 No.8

Vitrification uses cryoprotectants and liquid nitrogen, which may cause osmotic stress and cryodamage to oocytes. Autophagy is widely considered as a survival or responsive mechanism to various environmental and cellular stresses. However, the status of autophagy in vitrified-warmed oocytes has not been studied. In this work, we investigated if vitrification-warming process induces autophagy in mouse oocytes. Four-week-old female ICR mice and GFP-LC3 transgenic mice were used. The mice were superovulated with 5IU PMSG and 5IU hCG and ovulated MII oocytes were collected from oviducts. Oocytes obtained from several mice were pooled and divided into three groups. Group1: fresh oocytes. Group2: oocytes treated with vitification solutions (1.3 M EG+1.1 M DMSO and 2.7 M EG+2.1 M DMSO+0.5 M sucrose for 2.5 min) and warming solutions (0.5 M, 0.25, 0,125, and 0 M sucrose at intervals 2.5 min). Group3: vitrified-warmed oocytes (loaded onto an EM copper grid, and were stored in LN2 for 2 weeks). RT-PCR and confocal live imaging of GFP-LC3 were performed to examine the effects of vitrification-warming process on autophagy in oocytes. In RT-PCR analyses, expression of autophagy related (Atg) genes, such as Atg5, Atg7, Atg12, LC3a, LC3b, and Beclin1 was examined. Expression of Atg7 and Atg12 was slightly reduced in Group 3 (vitrified-warmed oocytes). The expression levels of other Atg genes did not change. Confocal live imaging analysis using oocytes from GFP-LC3 transgenic mice revealed that some vitrified-warmed oocytes showed green puncta which indicate autophagic activation. All oocytes of Group 1 and Group 2 show no puncta formation. Our results suggest that induction of autophagy may serve as an indicator of conditions of vitrification-warming process. Moreover, it offers the possibility that development of methods to modulate autophagic response during cryopreservation could improve efficacy of oocyte cryopreservation.

Vitrification, in vitro fertilization, and development of Atg7 deficient mouse oocytes

Bang, Soyoung,Lee, Geun-Kyung,Shin, Hyejin,Suh, Chang Suk,Lim, Hyunjung Jade The Korean Society for Reproductive Medicine 2016 Clinical and Experimental Reproductive Medicine Vol.43 No.1

Objective: Autophagy contributes to the clearance and recycling of macromolecules and organelles in response to stress. We previously reported that vitrified mouse oocytes show acute increases in autophagy during warming. Herein, we investigate the potential role of Atg7 in oocyte vitrification by using an oocyte-specific deletion model of the Atg7 gene, a crucial upstream gene in the autophagic pathway. Methods: Oocyte-specific Atg7 deficient mice were generated by crossing Atg7 floxed mice and Zp3-Cre transgenic mice. The oocytes were vitrified-warmed and then subjected to in vitro fertilization and development. The rates of survival, fertilization, and development were assessed in the Atg7 deficient oocytes in comparison with the wildtype oocytes. Light chain 3 (LC3) immunofluorescence staining was performed to determine whether this method effectively evaluates the autophagy status of oocytes. Results: The survival rate of vitrified-warmed $Atg7^{f/f}$;Zp3-Cre ($Atg7^{d/d}$) metaphase II (MII) oocytes was not significantly different from that of the wildtype ($Atg7^{f/f}$) oocytes. Fertilization and development in the $Atg7^{d/d}$ oocytes were significantly lower than the $Atg7^{f/f}$ oocytes, comparable to the $Atg5^{d/d}$ oocytes previously described. Notably, the developmental rate improved slightly in vitrified-warmed $Atg7^{d/d}$ MII oocytes when compared to fresh $Atg7^{d/d}$ oocytes. LC3 immunofluorescence staining showed that this method can be reliably used to assess autophagic activation in oocytes. Conclusion: We confirmed that the LC3-positive signal is nearly absent in $Atg7^{d/d}$ oocytes. While autophagy is induced during the warming process after vitrification of MII oocytes, the Atg7 gene is not essential for survival of vitrified-warmed oocytes. Thus, induction of autophagy during warming of vitrified MII oocytes seems to be a natural response to manage cold or other cellular stresses.

재생에너지 수용성 확대를 위한 송전혼잡 예측에 관한 연구

박소영(Soyoung Park),문승필(Seungpil Moon),방형필(Hyeongpil Bang),허진(Jin Hur) 대한전기학회 2023 대한전기학회 학술대회 논문집 Vol.2023 No.7

재생에너지 수용성 확대를 위한 송전혼잡 예측에 관한 연구

박소영(Soyoung Park),문승필(Seungpil Moon),방형필(Hyeongpil Bang),허진(Jin Hur) 대한전기학회 2023 대한전기학회 학술대회 논문집 Vol.2023 No.7

The formation of multivesicular bodies in the trophectoderm is associated with autophagic activation in dormant and activated blastocysts in mice

Hyejin Shin,Soyoung Bang,Hyunjung Jade Lim 한국발생생물학회 2014 한국발생생물학회 학술발표대회 Vol.2014 No.9

Dormant blastocysts during delayed implantation exhibit heightened autophagic activation. Activation of autophagy, the self-eating process within cells, was suggested as an adaptive response to unfavorable environment of prolonged survival in utero. During the course of this study, we observed by transmission electron microscopy that multivesicular bodies (MVBs) accumulate in the trophectoderm of dormant blastocysts upon activation of implantation by estrogen. MVBs are the late endosomes which are characterized by the presence of diverse internal vesicles within a large vesicle. Autophagosomes fuse with MVBs during autophagic activation, and efficient autophagic degradation requires functional MVBs. Biogenesis of MVBs depends on a dynamic network of ESCRT complexes 0, I, II, and III. Tsg101 (a component of the ESCRT-I complex) and CD63 are often used as a marker of MVBs. Lysobisphosphatidic acid (LBPA) is an abundant lipid in MVBs and required for the formation of MVBs. In this study, we performed immunofluorescence staining for detection of MVB makers in dormant and activated embryo. In dormant blastocysts, expression of Tsg101 and LBPA exhibited a uniform pattern throughout the trophectoderm. In contrast, expression of both markers prominently increased in the mural trophectoderm of activated blastocysts. To investigate the relationship with MVB formation and autophagy activation in activated blastocyst, 3-MA, a widely used inhibitor of autophagy, was daily injected intraperitoneally to ovx mice. Interestingly, 3-MA injection to block autophagy during delayed implantation led to a reduction of the signal of MVB markers, suggesting that prolonged activation of autophagy in dormant blastocysts is associated with MVB formation upon activation of implantation. Collectively, these results show that expression of MVB makers increase in the trophectoderm of blastocysts upon activation of implantation and that the formation of MVB is associated with heightened autophagy during delayed implantation.

공단지역 및 청정지역 식물 잎권의 잎표면세균 및 내산성세균의 분포

안종훈,방숙진,한남정,송왕영,황소영,이인수,박성주 대전대학교 이과대학 기초과학연구소 1997 自然科學 Vol.- No.-

산성강하물의 영향을 받는 대천공단지역과 영향을 받지 않는 청정지역인 대전 계족산 자연휴양림에서 자라는 밤나무(Castanea crenat)의 잎표면에서 서식하는 총세균수, 생존세균수, 종속영양세균수, 내산성세균수를 1996년 8월부터 1997년 8월까지 5회에 걸쳐 조사하였다. 공단지역 잎표면 평균 총세균수, 생존세균수 및 종속영양세균수는 각각 9.9×10^(5) cell/㎠, 1.6×10⁴cell/㎠, 7.1×10³cell/㎠,로서 청정지역에 비하여 각각 1.5배, 2배, 2.6배 정도로 관찰되었다. MPN법으로 측정한 pH 5.6애서의 잎표면 내산성세를수는 공단지역 3.3×10⁴, 청정지역 3.4×10⁴MON/㎠로 거의 같았고, pH 4.0에서의 내산성세균수는 공단지역애서 1.9×10^-(-1)MPN/㎠인 반면 청정지역에서는 전혀 검출되지 않았다. pH 3.0에서의 내산성 잎표면세균수는 공단지역과 청정지역의 잎권 어느 곳에서도 검출되지 않았다. 한편 계절별 잎표면세균수의 분포는 대체로 잎이 나기 시작하여 크기가 가장 작은 5월에 최대를, 그리고 낙엽이 지는 11월에 최소를 나타내었다. 이런 결과는 공단지역의 대기오염물질의 침적이 주변의 식물 잎표면 세균수를 감소시키지는 않으며, 특히 산성강하물의 영향으로 내산성세균수가 증가함을 보여주고 있다. Total, direct viable count, and acid-tolerant epiphytic bacterial population sizes were quantified on leaves of chestnut tree (Castanea crenata S. et Z.) near Taejon Industrial Estate affected by acid precipitation and deposition as well as in the clean natural forest area, Mt. Kyejok, in Taejon city from August 1996 to August 1997. Geometric mean numbers of total, direct viable count, and acid-tolerant epiphytic bacteria were 9.9×10^(5) cell/㎠, 1.6×10^(6)cell/㎠, and 7.1×10³cfu/㎠ respecfvely, being 1.5, 2, and 2.6 times those in the clean area. Acid-to-lerant epiphytic bfcterial numbers at pH 5.6 by MPN method were 3.3×10" in the industrial area, about the same as the number,3.4×104 MPNicni, of the clean area. Acid-tolerant bacterial number at pH 4.0 was 1.9×10^(-1)MPN/㎠ in the industrial area, whereas none was detected in the clean area. Acid-tolerant bacteria at pH 3.0 were not detected at all in the industrial area as well as in the clean area. Epiphytic bacterial population sizes were generally the greatest in May when leaves are emerged and grew but the lowest in November when defoliation occurs. These results showed that air pollutant deposition on leaves did not cause a deuease of epiphytic bacteria at least and acid deposition on leaves did cause an increase of acid-tolerant bacteria.

알로에 베라 젤 및 껍질 추출물의 생리활성 평가

조은혜(Eunhye Cho),김소영(Soyoung Kim),방순일(Soonil Bang),김동청(Dong Chung Kim),인만진(Man-Jin In),채희정(Hee Jeong Chae) 한국생물공학회 2014 KSBB Journal Vol.29 No.6

In vitro biological activities of Aloe vera gel and skin extracts were evaluated. Total polyphenol contents of Aloe vera skin were measured 41.12 mg/g. DPPH radical scavenging activity of Aloe vera skin-70% EtOH extract, Aloe vera skin-water extract, Aloe vera gel-70% EtOH extract and Aloe vera gel-water extract were 55%, 38%, 11% and 10%, respectively. In addition, 70% EtOH extract and water extract were compared with respect to SOD-like antioxidant activity of Aloe vera-70% EtOH extract has higher activity than Aloe vera water extract. Tyrosinase inhibition rate of Aloe vera gel extract was higher than Aloe vera skin extract. Alcohol dehydrogenase (ADH) and Acetaldehyde dehydrogenase (ALDH) relative percentage activity of Aloe vera gel extract were 126% and 216%, respectively. It was suggested that Aloe vera gel and skin extracts could be used as a functional biomaterial for functional food and cosmetics.

Poster Session : PS 0734 ; Rheumatology ; Bone Morphogenetic Protein 6 Polymorphisms are Associated with Radiographic Progression in Ankylosing Spondylitis

( Young Bin Joo ),( Soyoung Bang ),( Seung Hun Lee ),( Kyung Bin Joo ),( Tae Hwan Kim ) 대한내과학회 2014 대한내과학회 추계학술대회 Vol.2014 No.1

Background: Nearly 25 genetic loci associated with susceptibility to ankylosing spondylitis (AS) have been identified by several large studies. However, there have been limited studies to identify the genes associated with radiographic severity of thedisease. Thus we investigated which genes involved in bone formation pathways might be associated with radiographic severity in AS. Methods: A total of 417 Korean AS patients were classifi ed into two groups based on the radiographic severity as defi ned by the modifi ed Stoke` Ankylosing Spondylitis Spinal Score (mSASSS) system. Severe AS was defi ned by the presence of syndesmophytes and/or fusion in the lumbar or cervical spine (n=195). Mild AS was defi ned by the absence of any syndesmophyte or fusion (n=170). A total of 251 single nucleotide polymorphisms (SNPs) within 52 genes related to bone formation were selected and genotyped. Odds ratios (OR) and 95% confi dence interval (95% CI) were analysed by multivariate logistic regression controlling for age at onset of symptoms, sex, disease duration, and smoking status as covariates. Results: We identifi ed new loci of bone morphogenetic protein 6 (BMP6) associated with radiographic severity in patients with AS that passed false discovery rate threshold. Two SNPs in BMP6 were signifi cantly associated with radiologic severity [rs270378 (OR 1. 97, p = 6. 74 x 10-4) and rs1235192 [OR 1. 92, p = 1. 17 x 10-3]) adjusted by covariates. Conclusions: This is the first study to demonstrate that BMP6 is associated with radiographic severity in AS, supporting the role wingless-type like/BMP pathway on radiographic progression in AS.

Exosomal Proteins in the Aqueous Humor as Novel Biomarkers in Patients with Neovascular Age-related Macular Degeneration

Kang, Gum-Yong,Bang, Joo Young,Choi, Ae Jin,Yoon, Jeehyun,Lee, Won-Chul,Choi, Soyoung,Yoon, Soojin,Kim, Hyung Chan,Baek, Je-Hyun,Park, Hyung Soon,Lim, Hyunjung Jade,Chung, Hyewon American Chemical Society 2014 JOURNAL OF PROTEOME RESEARCH Vol.13 No.2

<P>Age-related macular degeneration (AMD) describes the progressive degeneration of the retinal pigment epithelium (RPE), retina, and choriocapillaris and is the leading cause of blindness in people over 50. The molecular mechanisms underlying this multifactorial disease remain largely unknown. To uncover novel secretory biomarkers related to the pathogenesis of AMD, we adopted an integrated approach to compare the proteins identified in the conditioned medium (CM) of cultured RPE cells and the exosomes derived from CM and from the aqueous humor (AH) of AMD patients by LC–ESI–MS/MS. Finally, LC–MRM was performed on the AH from patients and controls, which revealed that cathepsin D, cytokeratin 8, and four other proteins increased in the AH of AMD patients. The present study has identified potential biomarkers and therapeutic targets for AMD treatment, such as proteins related to the autophagy–lysosomal pathway and epithelial–mesenchymal transition, and demonstrated a novel and effective approach to identifying AMD-associated proteins that might be secreted by RPE in vivo in the form of exosomes. The proteomics-based characterization of this multifactorial disease could help to match a particular marker to particular target-based therapy in AMD patients with various phenotypes.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/jprobs/2014/jprobs.2014.13.issue-2/pr400751k/production/images/medium/pr-2013-00751k_0007.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/pr400751k'>ACS Electronic Supporting Info</A></P>