The glycoprotein hormone family consists of follicle-stimulating hormone (FSH; GTH1), luteinizing hormone (LH; GTH2), and thyroid-stimulating hormone (TSH), which are secreted by the pituitary gland in all mammalian species, and chorionic gonadotropin, which is secreted by placental trophoblast cells in primates and equids. These hormones consist of non-covalently associated α-, β- subunits. Within a species, the amino acid sequence of α-subunit is identical across all glycoprotein hormones and is encoded by a single gene. The αβ dimer is the active form of the hormone, and biological specificity is conferred by the β-subunit. Both of α and β subunit of eel FSH has two N-glycosylation sites (α-subunit: Asn56 and Asn79; β -subunit: Asn5 and Asn22, respectively). In the present study, we constructed deglycosylated mutants at single and double sites in each subunits of eel FSH for identification of Asn linked oligosaccharides' biological role. Mutant cDANs were cloned into pcDNA3 expression vector and transiently transfected into CHO suspension cells. The quantity of rec-eelFSHs were quantified by sandwich ELISA system, using monoclonal antibodies produced in our lab. The wild type rec-FSH protein was detected at the predicted molecular weight of 34 kDa by western blot. The molecular weight of deglycosylated mutants at single site decreased with about 4 kDa and of mutants at double sites decreased with 8 kDa. After PNGase treatment in the rec-eel FSH proteins, molecular weight also decreased to 7-8 kDa. We generated stably parental cell lines, engineered to express a β-arrestin 2EA fusion protein, expressing eel FSHR and C-terminal deleted mutant. 2 out of 5 receptor cells each were selected by G-418 and we tested these cell lines in a receptor functionality using PathHunter β arrestin assay (DiscoverX). Follicle stimulating hormone acts through binding to its specific receptor. Binding of ligand to the receptor activates the adenosine 3',5'-cyclic monophosphate (cAMP) pathway (McFarland et al., 1989; Ji and Ji, 1991a; Rose, 1998) and the inositol 1phosphate (IP1) the second messenger systems. After stimulation of eelFSH receptor stably transfected Parental CHO cells with FSH wild type and mutant hormones as a ligand, production of cAMP and IP-1 were evaluated (Cisbio). cAMP IC-50 values by eelFSHwt; αΔ56; αΔ79; αΔ56_79; and βΔ5 were 33.1; 1154.7; 22; 410 and 311.9 ng/ml, respectively. IP-1 IC-50 values by eelFSHwt; αΔ56; αΔ79; αΔ56_79 and βΔ5 were 6.8; 7.1; 4.4; 3.8 and 10.2 ng/ml, respectively too. The cAMP activation was greatly decreased in the αΔ56αmutant. Thus, the site of α56 oligosaccharide in the eel plays an pivotal role for the cAMP stimulation using eel FSH receptor cell lines. In the IP-one assay, the activity in the αΔ56 and βΔ5 mutants was a little decreased than the wt. The biological roles of N-linked oligosaccharides in GPCR internalization are going to be estimated by measuring β arrestin recruitment system.

소음 스트레스로 인한 뱀장어(Anguilla japonica)의 영향을 파악하기 위하여 스트레스 지표로 사용되는 코티졸, 포도당, 알부민 및 glucocorticoid receptor(GCR) 유전자의 발현 양을 측정, 분석하여 노출되지 않은 대조구와 비교하였다. 그 결과, 알부민은 노출 1시간 후에 낮은 값을 보인 반면 코티졸과 포도당은 대조구에 비해 매우 큰 차이를 보이며 높게 나타났다. GCR 유전자의 조직 발현 결과 간, 아가미, 근육 및 소장에서 많이 발현하였다. 소음 노출에 따른 시간의 변화에서 간과 아가미 근육과 소장에서 발현이 감소하는 양상을 나타내었다. 실험결과 뱀장어의 glucocorticoid receptor 유전자의 발현변화가 소음 스트레스로 인한 영향을 파악하는데 유용한 지표가 될 수 있음을 확인하였다.

Gene expressions of cytochrome P4501A (CYP1A), aryl hydrocarbon receptor (AhR) and vitellogenin (Vg) by endocrine disruptors, benzo[]pyrene (B[a]P) and tributyltin (TBT) were examined in cultured eel hepatocytes which were isolated from eels treated previously with B[a]P (10 mg/kg) or estradiol- (20 mg/kg) in vivo, and the relationship between CYP1A, AhR and Vg genes were studied. When the cultured eel hepatocytes were treated with B[a]P () the gene expressions of CYP1A and AhR were enhanced in a concentration-dependent manner. However, when treated with TBT () the gene expressions of CYP1A and AhR were suppressed at high concentrations (), while having no effects at low concentrations (). Gene expression of Vg was also suppressed by TBT in a concentration-dependent manner in cultured eel hepatocytes which was previously treated in vivo with estradiol-.

We investigated the effects of temperature changes on the oxygen consumption rhythm in Japanese eels, Anguilla japonica, using an automatic intermittent flow respirometer (AIFR). The endogenous rhythm of the oxygen consumption rate (OCR) in the eels (n = 18; 44-74 cm, 145-690 g), freshly collected by bag net from estuaries, was nearly synchronous with the tidal pattern of the estuarine collection site. The magnitude of mean OCR (mOCR) of eels showed variable range of 82.2 - 116.5 ml O2 kg-1 ww h-1 under constant conditions. In case of increasing temperature from 25 to 38℃, the OCR of eels exhibited a gradually increasing trend with a rhythmic pattern until 36℃. Above 36℃, the rhythms of the OCR dampened and the OCR decreased rapidly at around 36 - 37℃. The OCR of the eels exhibited the maximum value at 38℃, and then it sharply decreased. The results suggested that the critical thermal maximum (CTM) regarding the endogenous rhythms of the eels was at around 36 - 37℃ when water temperature increased at 0.5℃/14 h following the acclimation at 25℃. In case of decreasing temperature (0.5℃/14 h) from 25 to 0℃, the OCR of the eels displayed a abrupt decrease up to 23℃, and between at 23 and 20℃, there was an agitation which showed a slight increase in the OCR with a duration of 1-2 days. Below 9℃, the OCR rhythm of the eels showed a constant state regardless of temperature decreasing. These results suggest that the Japanese eel has an upper incipient lethal temperature at 36℃, with a lower thermal limit at 9℃. The biochemical aspects of the eels influenced by water temperature need to be further studied.

Vg은 난생 척추동물의 성숙한 암컷 혈청에 존재하는 성 특이 단백질로서 에 의해 합성이 유도된다. 본 연구는 뱀장어 Vg과 ER 유전자 발현에 대한 androgen과 성장호르몬(GH)의 영향을 조사하였다. 미성숙 뱀장어()에 , 뱀장어 recombinant GH(eGH, ) 또는 methyltestosterone(MT, )를 각각 단독 또는 eGH 또는 MT와 혼합하여 주사한 후 10일 후에 샘플을 채취하였다. 간 ER과 Vg mRNA는 RT-PCR을