Heat shock proteins (HSPs) are highly conserved cellular proteins that contribute to adaptive responses of organisms to a variety of stressors. In response to stressors, cellular levels of HSPs are increased and play critical roles in protein stability, folding and molecular trafficking. The mRNA expression pattern of two well-known heat shock protein transcripts, HSP70 and HSP90 were studied in two tissues of nerve ganglia, cerebral ganglion and pleuropedal ganglion of Pacific abalone (Haliotis discus hannai). It was observed that both HSP70 and HSP90 transcripts were upregulated under heat stress in both ganglion tissues. Expression level of HSP70 was found higher than HSP90 in both ganglia whereas cerebral ganglion showed higher expression than pleuropedal ganglion. The HSP70 and HSP90 showed higher expression at Day-1 after exposed to heat stress, later decreased at Day-3 and Day-7 onwards. The present result suggested that HSP70 and HSP90 synthesize in nerve ganglion tissues and may provide efficient protection from stress.

수온의 변화는 어류의 거의 모든 생리학적 부분에 영향을 미친다. 기후 변화로 인한 수온의 상 승은 어류에게 물리적 피해를 줄 수 있다. 이 연구는 최적의 수온(15°C)보다 높은 수온(20°C)에 서의 대서양 연어의 건강상태를 평가하기 위해 수행하였다. 간 조직은 열 적응에 중요한 대사 기능을 발휘하기에 본 연구에 간 조직을 사용하였다. 생체지표유전자의 개발을 위한 분석 방법 으로는 NGS RNAseq 방법을 사용하였고, 생체지표유전자의 발현 양상을 관찰하기 위한 분석 방 법으로는 RT-qPCR을 사용하였다. NGS RNAseq 분석을 통해 1,366개의 차별적 발현 유전자를 확인하였으며, 그 중에서 880개의 증가하는 유전자와 486개의 감소하는 유전자를 확인하였다. 생체지표유전자로는 heat shock protein 90 alpha (Hsp90α), heat shock protein 90 beta (Hsp90β) 및 cytochrome P450 1A (CYP1A)을 선정하였는데 이들 유전자는 NGS RNAseq 분석에서 수온의 변화에 민감하게 반응하는 유전자들이었다. 이들 유전자의 RT-qPCR을 통한 발현 양상은 NGS RNAseq 분석과 유사하게 나타났다. 이 연구의 결과는 다른 어종에도 적용할 수 있으며, 산업 적으로도 유용하다고 생각된다.

The Apostichopus japonicus is an important species in some Asia countries including Korea, China and Japan. The purpose of the present study was to investigate the differential gene expression of heat shock protein90 (Hsp90) and ferritin as a biomarker for the thermal stress during water temperature rising in the sea cucumber, A. japonicus. The A. japonicus (1.4 g) was cultured in incubator of separate temperature (15°C, 20°C, 25°C and 30°C) for each 0, 3, 6, 12, 24, 48 hours. The mRNA expression levels of Hsp90 and ferritin were examined using RT-PCR assay. Results showed that, the expression of Hsp90 mRNA was not significantly changed at 15°C. The expression of Hsp90 mRNA was significantly increased at high temperature such as 20°C and 25°C. Furthermore, Hsp90 mRNA was early increased at 25°C than 20°C. The ferritin mRNA was similar expression pattern with Hsp90. But, Hsp90 mRNA was more sensitive than ferritin mRNA at high thermal stress. These results indicate that Hsp90 and ferritin mRNAs were involved in the temperature changes response and may be play an important role in mediating the thermal stress in A. japonicas.

The present study was performed to identify the relationship between plasminogen activator (PA) and Heat Shock Protein-90 (HSP-90) in porcine uterus tissues during the estrous cycle. Porcine uterus tissues were obtained from preovulatory (Pre-Ov), post-ovulatory (Post-Ov) and early to mid-luteal (Early-mid L) stages. The protein was extracted from uterus tissue by using M-PER Mammalian Protein Extraction Reagent. Proteins were refined by RIPA Buffer and quantified by BCA methods. As results, t-PA expression was significantly (p<0.05) higher from pre-ovulatory(Epithelium tissue: 29,067 μg/μl, Myometrium tissue: 30,797 μg/μl) compared to the post-ovulatory stage(Epithelium tissue: 54,357 μg/μl, Myometrium tissue: 53,270 μg/μl) and early to mid-luteal stage(Epithelium tissue: 42,380 μg/μl, Myometrium tissue: 43,139 μg/μl). On the other hand, the uPA expression indicated higher from early to mid-luteal stage (Epithelium tissue: 0.02198 μg/μl, Myometrium tissue: 0.02412 μg/μl) than pre-ovulatory stage (Epithelium tissue: 0.01577 μg/μl, Myometrium tissue: 0.01531 μg/μl) and post-ovulatory stage(Epithelium tissue: 0.01414 μg/μl, Myometrium tissue: 0.01429 μg/μl). However, expression of u-PA did not differ from each estrous cycle in the epithelium tissue and myometrium tissue(p<0.05). Expression of HSP-90 was differ t-PA and u-PA from pre-ovulatory in Epithelium tissue(25,423 μg/μl) and early to mid-luteal stage in epithelium tissue(177,922 μg/μl) and myometrium tissue(26,664 μg/μl). These results suggest that HSP-90 and u-PA were related with change of uterus cycle according to the reformation of the tissues in porcine uterus.

Heat shock protein 90 (Hsp90) is ATPase-directed molecular chaperon and affects survival of several cells. In our previous study, inhibitory effect of Hsp90 by inducing cell cycle arrest and apoptosis in the pig embryonic and primary cells was reported. However, its role during early bovine embryonic development is not sufficient. In this study, we traced the effects of Hsp90 inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), on early bovine embryonic development. We also investigated several indicators of developmental potential, including structural integrity, gene expression (apoptosis-related genes), and apoptosis, which are affected by 17-AAG. Bovine embryos were cultured in the CR1-aa medium with or without 17-AAG for 7 days. In result, significant differences in developmental potential were detected between the embryos that were cultured with or without 17-AAG (33.1±9.6 vs 21.7± 8.3%). The structural integrity of the blastocysts was examined by differential staining. Blastocysts from the dbcAMP- treated group had higher numbers of ICM, TE, and total cells than those from the untreated group. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) showed that the number of containing fragmented DNA at the blastocyst stage increased in the 17-AAG treated group compared with control (11.2 vs 3.9, respectively). Blastocysts that developed in the 17-AAG treated group had low structural integrity and high apoptotic nuclei than those of the untreated control, resulting in decrease the embryonic qualities of preimplantation bovine blastocysts. The m-RNA expression of the pro-apoptotic gene (Bax) increased in 17-AAG treated group, whereas expression of the antiapoptotic gene (Bcl-XL) decreased. In conclusion, Hsp90 also appears to play a direct role in bovine early embryo developmental competence including structural integrity of blastocysts. Also, these results indicate that Hsp90 is closely associated with apoptosis-related genes expression in developing bovine embryos.

Heat shock protein 90 (Hsp90) is ATPase-directed molecular chaperon and affects survival of cancer cell. Inhibitory effect of Hsp90 by inducing cell cycle arrest and apoptosis in the cancer cell was reported. However, its role during oocyte maturation and early embryo development is very insufficient. In this study, we traced the effects of Hsp90 inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), on meiotic maturation and early embryonic development in pigs. We also investigated several indicators of developmental potential, including structural integrity, gene expression (Hsp90-, cell cycle-, and apoptosis-related genes), and apoptosis, which are affected by 17-AAG. Then, we examined the roles of Hsp90 inhibitor on viability of primary cells in pigs. Porcine oocytes were cultured in the NCSU-23 medium with or without 17-AAG for 44 h. The proportion of GV arrested oocytes was significantly different between the 17-AAG treated and untreated group (78.2 vs 34.8%, p<0.05). After completion of meiotic maturation, the proportion of MII oocytes was lower in the 17-AAG treated group than in the control group (27.9 vs 71.0%, p<0.05). After IVF, the percentage of penetrated oocytes was significantly lower in the 17-AAG treated group (25.2%), resulting in lower normal pronucleus formation (2PN of 14.6%). Therefore, the inhibition of meiotic progression by Hsp90 inhibitor played a critical role in fertilization status. Porcine embryo were cultured in the PZM-3 medium with or without 17-AAG for 6 days. In result, significant differences in developmental potential were detected between the embryos that were cultured with or without 17-AAG. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) showed that the number of containing fragmented DNA at the blastocyst stage increased in the 17-AAG treated group compared with control (7.5 vs 4.4, respectively). Blastocysts that developed in the 17-AAG treated group had low structural integrity and high apoptotic nuclei than those of the untreated control, resulting in decrease the embryonic qualities of preimplantation porcine blastocysts. The mRNA expressions of cell cycle-related genes were down-regulated in the 17-AAG treated group compared with control. Also, the expression of the pro-apoptotic gene Bax increased in 17-AAG treated group, whereas expression of the anti-apoptotic gene Bcl-XL decreased. However, the expression of ER stress-related genes did not changed by 17-AAG. Cultured pESF cells were treated with or without 17-AAG and used for MTT assay. The results showed that viability of pESF cells were decreased by treatment of 17-AAG (2 μM) for 24 hr. These results indicated that 17-AAG decreased cell proliferation and increased cell death. Expression patterns Hsp90 complex genes (Hsp70 and p23), cell cycle-related genes (cdc2 and cdc25c) and apoptosis-related genes (Bax and Bcl-XL) were significantly changed by using RT-PCR analysis. The spliced form of pXbp-1 product (pXbp-1s) was detected in the tunicamycin (TM) treated cells, but it is not detected in 17-AAG treated cells. In conclusion, Hsp90 appears to play a direct role in porcine early embryo developmental competence including structural integrity of blastocysts. Also, these results indicate that Hsp90 is closely associated with cell cycle- and apoptosis-related genes expression in developing porcine embryos.