• Asian-Australasian Journal of Animal Sciences
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• 제23권12호
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• pp.1557-1565
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• 2010

Methyl paraben (MP), which is used as a preservative in pharmaceutical and cosmetic (shampoo) products, foods and beverages, enters into the aquatic environment and can pose a potential fish health hazard. In this experiment, effects of MP were evaluated in adult male common carp (Cyprinus carpio) by exposing them to fractions (1/$143^{rd}$ to 1/29th) of the $LC_{50}$ dose with every change of water for 28 days. Vitellogenin induction, metabolic enzymes, somatic indices and bioaccumulation were studied at weekly intervals. The $96^{th}$ h $LC_{50}$ of MP in fingerlings was 120 mg/L. Compared to the control, except for increases (p<0.01) in alkaline phosphatase (EC 3.1.3.1), alanine aminotransferase (EC 2.6.1.2) and liver size, there were decreases (p<0.01) in activity of acid phosphatase (EC 3.1.3.2), aspartate aminotransferase (EC 2.6.1.1), and testiculosomatic index following exposure to any dose of MP. Vitellogenin induction was significantly higher (p<0.01) in exposed than unexposed (control) fish. The bioaccumulation of MP in testis, liver, brain, gills and muscle tissues of fish increased significantly (p<0.01) with increase of dose from 0.84 ppm to 1.68 ppm. Dose and duration of exposure (p<0.01) indicated that an exposure period of 1 to 2 weeks was sufficient to cause changes in the quantifiable parameters studied. Fish exposed to 4.2 ppm MP became lethargic after the $26^{th}$ d. Histologically, degeneration, vacuolization and focal necrotic changes in liver and fibrosis-like changes in testicular tissue were noted.

• 대한수의학회지
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• 제46권1호
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• pp.35-41
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• 2006

Vitellogenin (Vtg), a phospholipoglycoprotein precursor of egg yolk is synthesized and secreted from the liver in response to estrogens in female fish. Vtg is normally undetectable in the blood of male fish, but can be induced by exposure to chemicals possessing estrogenic activity. Thus, the presence of Vtg in blood of male fish can serve as a useful biomarker for assessing previous exposure to estrogenic compounds. In the present study, Vtg was abnormally expressed in Rhynchocypris oxycephalus using estradiol benzoate ($E_2$). As the result, it was found that the level of Vtg in blood from R. oxycephalus was increased by treated quantity of $E_2$ with dose-effect manner. Monoclonal antibodies were generated against Vtg of R. oxycephalus. The hybridoma were screened with an enzyme immunoassay for the production of specific anti-Vtg antibodies. Five positive cell lines with a high specificity were selected. Monoclonal antibodies against vtg of R. oxycephalus that was developed in this study, may be a useful bio-indicator for the detection of estrogenic contamination in the aquatic ecosystem.

• 한국어병학회지
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• 제26권3호
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• pp.241-254
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• 2013

본 연구는 환경내의 내분비교란화학물질 조사를 위한 바이오마커 시스템을 개발하기 위하여 쏨뱅이의 Vitellogenin(Vtg)을 바이오마커로서 활용하기 위하여 실험을 실시하였다. Vtg는 에스트로겐을 이용하여 유도하였으며, 어류의 혈청으로부터 겔 여과 및 이온교환크로마토그래피를 이용하여 정제하였다. 그 후 BALB/C 마우스를 이용하여 정제된 Vtg의 하이브리도마를 생산하여 항-Vtg의 항체를 생산하였다. 쏨뱅이로부터 유되된 Vtg의 크기는 Sepharose CL-6B을 이용한 겔 여과를 통하여 약 440 kDa 인 것으로 나타났으며 main-band는 175 kDa이었으며 몇 개의 sub-band가 발견되어졌다. 8개의 단클론 항체가 개발한 하이브리도마로부터 얻을 수 있었으며, 이 항체는 본 실험에서 조사한 어류 중 우럭볼락 Sebastes hubbsi을 제외한 다른 종에서는 교차반응이 이루어지지 않았다. 그 결과, 클론 S28과 S15의 단클론항체를 ELISA와 ICG assay의 추적 인자로 사용할 수 있을 것으로 판단된다. 본 연구에서 배발된 검출시스템은 다양한 내분비교란 활동의 확인뿐 만이 아니라 내분비교란화학물질로 알려진 물질을 검출하는 바이오 마커 분석에 활용될 수 있을 것이다.

• Fisheries and Aquatic Sciences
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• 제12권4호
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• pp.276-285
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• 2009

Despite great commercial interest, relatively little has been described about molecular mechanism of bivalve reproduction. We investigated genes involved in ovarian maturation of the Yesso scallop, Patinopecten yessoensis. GSI index and histological analysis revealed that maturation of ovary begin in February and spawning period is from April to June which is similar to the previous study in the East Sea. As result of combination analysis of differential display RTPCR (DDRT-PCR) and histological examination, vitellogenin (Vg), ferritin (Ft) and ADT/ATP carrier protein (ACC) were identified as differently expressed genes in maturating ovary. Endpoint RT-PCR results showed that Vg is ovary-specific genes whereas Ft and ACC are expressed ubiquitously suggesting that Vg can be good molecular markers for ovarian development and sex determination in bivalves. Quantitative PCR results revealed that Vg were expressed highest during growth stage and appears to play a major role in oocyte maturation. On the contrary, expression of Ft was highest after spawning stage, which suggests that up-regulation may be involved in spawning and inactive stages in which the scallops recover from spawning. In addition, high level of the mitochondrial gene, ACC, may play a role in energy metabolism in maturating oocytes. Isolation and molecular studies of these key genes will expand our knowledge of the physiological changes from various exogenous factors including temperature, salinity, pH, even or numerous endocrine disrupting chemicals (EDCs) during reproductive cycle. In addition, further study of these genes implicates various industrial applications including the stable seed production, increased food quality, or economic aquaculture system.

• 한국가축번식학회지
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• 제25권3호
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• pp.277-286
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• 2001

기존의 미세주입 및 정자 electroporation에 의한 보다 효율적인 유전자 도입방법을 개선하여 간단하고 고효율성의 유전자 변환기술을 위한 유전자 도입장치의 시제품 개발로 다수의 난자를 전기적으로 단순화할 수 있는 상업화의 가능성을 보여주었다. 도입된 유전자는 모든 초기배에서 발현됨을 보여 주었다. 특히 난황내의 합포체세포(syncytium)에서의 transient성의 강한 발현은 전기자극에 의해 많은 수의 난자에 유전자를 도입하고 100% 발현되는 체계를 이용하여 transient 시기에서 인간 유용단백질 생산의 가능성을 타진할 수 있는 결과를 보여 주었다. 어류유전자 발현의 작동되는가를 검색하기 위해 신경세로조직특이 tubulin promoter 를 이용한 결과 gfp의 발현이 뇌주변과 척추를 중심으로 체내 전반의 신경세포내에 발현이 강하게 나타남을 보여 주었다. 한편 reporter 유전자 이외에 간세포로부터 전체 RNA를 분리시켜 vitellogenin의 분해산물인 phosvitin cDNA의 길이와 promoter지역인 1.6 kb에 대한 primer쌍들을 선정한 상태에서 PCR에 의해 각각 cDNA와 gDNA로부터 cloning 중에 있으며 human factor Ⅶ과 epidermal growth factor, vitellogenin의 3종의 target 단백 질유전자를 구축 및 검정 화인 중에 있다.

• Animal cells and systems
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• 제7권3호
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• pp.227-235
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• 2003

Bisphenol A (BPA), nonylphenol (NP), and 4-tert-octylphenol (OP) are known endocrine disrupting chemicals (EDCs) with estrogenic activity in fish. This study compared the effects of BPA, NP and OP on in vitro vitellogenin (VTG) synthesis in primary cultures of hepatocytes of the Chinese minnow Phoxinus oxycephalus. The VTG secreted into the culture medium was measured using enzyme-linked immunosorbent assay (ELISA), which we developed in this study using an antibody prepared from homogenates of Chinese minnow egg. VTG synthesis was induced by estradiol-17$\beta$ ($E_2$) and phenols (BPA, NP and OP) treatment. $E_2$ at concentrations of 10$^{-6}$ M or higher increased VTG levels significantly (P < 0.05). Exposure to 10^5\;M\;BPA\;or\;10^-4$M NP and OPinduced in vitro VTG synthesis (P < 0.01). However, $10^-3$ M BPA, NP or OP did not induce VTG synthesis. These results suggest that SPA has the highest estrogenic potential in Chinese minnow hepatocytes. Tamoxifen, an anti-estrogen, drastically blocked the production of VTG by phenols (BPA, NP and OP) suggesting that phenols (BPA, NP and OP) may act via binding to estrogen receptor (ER) in Chinese minnow hepatocytes.

• Applied Microscopy
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• 제31권1호
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• pp.19-35
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• 2001

계절성 번식기 동안에 자성무지개송어 (Oncorhynchus mykiss)의 혈장내 $estradiol-17\beta$, 혈장성분, 전기영동상 그리고 미세구조 변화의 특징을 살펴보았다. 정상적인 자연조건에서 혈장내 $estradiol-17\beta$는 난황형성기(vitellogenic season)말인 9월에 peak를 나타내었고, 산란이 이루어진 시기 이후인 12월에는 서서히 감소하였으며, 난소의 재형성 초기인 2월과 3월에는 급격하게 감소하였다. 이러한 $estradiol-17\beta$의 변화 추이를 볼 때 이 호르몬은 자성 무지개송어에서 혈장내 calcium, phosphorus, glucose, albumin및 total protein수준의 증가에 의해서 증명된 것과 같이 난황전구물질(vitellogenin)의 생성을 자극하는 것으로 나타났다. Latematuring단계 혹은 산란중인 난모세포의 전기영동상(분자량이 각각 약 70,000과 200,000 Da)은 late perinucleolus단계의 난모세포의 전기명동상보다 훨씬 두껍고 진한 경향을 나타내었다. 이 단백질 2개의 전기영동상은 $estradiol-17\beta$ 호르몬 peak와 일치하는 추이를 나타내면서 SDS-PAGE에서 확인되었다. 생식소 숙도지수 일명 성성숙지수, [gonadosomatic indices(GSI)]는 10월로부터 그 이듬해 1월까지 유의성 있게 증가하는 경향을 나타내었고, 성숙난자의 급격한 수적인 증가와 일치하면서 1월에 가장 높은 peak를 나타내었다. 산란 전단계에서 estradiol-17 수준과 CSI간에는 정 (+)의 상관관계 (r=0.701, p<0.01)를 나타내었고, 간숙도지수 [hepatosomatic index (HSI)]는 번식시기인 12월에 가장 높은 수치를 나타내었다. 번식시기 (12월)에 생식소 자극호르몬 분비세포(gonadotropes)는 granules와 globules와 같은 생식소자극호르몬을 포함한 봉입체로 가득찬 상태를 나타내었다. 난황형성기에는 late perinucleolus 난모세포가 난황의 축적을 통해서 early maturing난모세포로 변형되는 과정이며, 이 시기의 난모세포는 late maturing단계로 되면서 난황으로 완전히 가득찬 상태로 확인되었다. 핵이주 단계에 있는 난모세포의 과립막세포의 미세구조를 살펴보면 팽창된 조면 내형질세망과 tubular cristae가 있는 간상의 미토콘드리아로 구성되어 있었다. 방사대 (zona radials)와 난황막 사이에 있는 미세융모(finger-like projections)는 각각 성장하면서 난황형성기에 pore canals을 이루면서 서로 연결되나, 난성숙기에 방사대가 더욱더 치밀하게 되면 수축되어 봉쇄되면서 위축되는 것으로 확인되었다. 방사대는 외측에 않은 homogeneous layer와 내측에 2개의 두꺼운 helicoidal layers (zona radiata interna and zona radials externa)총 3개의 구조물로 구성되어 있었다. 정상적인 환경조건에서 볼 때 난포는 난형성과 생식소에서 분비되는 스테로이드 흐르몬의 생성에 충분할 만큼 조직의 발달과 호르몬의 주기적인 분비에 영향을 주는 것으로 나타났다.

• Asian-Australasian Journal of Animal Sciences
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• 제26권8호
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• pp.1080-1088
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• 2013

Our previous results showed that kisspeptin-10 (Kp-10) injections via intraperitoneal (i.p.) once daily for three weeks notably promoted the egg laying rate in quails. In order to investigate the mechanism behind the effects of Kp-10 on enhancing the egg laying rate in birds, this study focused on the alternations of lipids synthesis in liver after Kp-10 injections. 75 female quails (22 d of age) were allocated to three groups randomly, and subjected to 0 (control, Con), 10 nmol (low dosage, L) and 100 nmol (high dosage, H) Kp-10 injections via i.p. once daily for three weeks, respectively. At d 52, quails were sacrificed and sampled for further analyses. Serum $E_2$ concentration was increased by Kp-10 injections, and reached statistical significance in H group. Serum triglyceride (TG) concentrations were increased by 46.7% in L group and 36.8% in H group, respectively, but did not reach statistical significance, and TG contents in liver were significantly elevated by Kp-10 injections in a dose-dependent manner. Serum total cholesterol (Tch) concentrations significantly decreased in H group, while in H group the hepatic Tch content was markedly increased. The level of non-esterified fatty acid (NEFA), apolipoprotein A1 and B (apoA1 and apoB) were not altered by Kp-10 injections. The genes expression of sterol regulatory element binding protein-1 (SREBP-1), fatty acid synthetase (FAS), apolipoprotein VLDL-II (apoVLDL-II), cholesterol $7{\alpha}$-hydroxylase (CYP7A1) and vitellogenin II (VTG-II) were significantly up-regulated by high but not low dosage of Kp-10 injection compared to the control group. However, the expression of SREBP-2, acetyl-CoA carboxylase ($ACC_{\alpha}$), malic enzyme (ME), stearoyl-CoA (${\Delta}9$) desaturase 1 (SCD1), apolipoprotein A1 (apoA1), fatty acid binding protein 2 (FABP2), 3-hydroxyl-3-methyl glutaryl-coenzyme A reductases (HMGCR), estrogen receptor ${\alpha}$, ${\beta}$($ER{\alpha}$ and ${\beta}$) mRNA were not affected by Kp-10 treatment. In line with hepatic mRNA abundance, hepatic SREBP1 protein content was significantly higher in H group. Although the mRNA expression was not altered, the content of $ER{\alpha}$ protein in liver was also significantly increased in H group. However, SREBP-2 protein content in liver was not changed by Kp-10 treatment. In conclusion, exogenous Kp-10 consecutive injections during juvenile stage significantly advanced the tempo of egg laying in quails, which was associated with the significant elevation in hepatic lipids synthesis and transport.

• Asian-Australasian Journal of Animal Sciences
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• 제20권10호
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• pp.1606-1611
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• 2007

The experiment was conducted to investigate the effects of dihydropyridine on laying performance and fat metabolism of laying hens. Five hundred and forty laying hens, 40 weeks old, were randomly allotted to three groups, each of which included four replicates of 45 hens. The groups were given a basal corn-soybean meal diet supplemented with 0, 150 mg/kg and 300 mg/kg dihydropyridine. Results showed that compared with the control group (0 mg/kg dihydropyridine), supplements of 150 and 300 mg/kg dihydropyridine increased egg production rate by 9.39% (p<0.01) and 12.97% (p<0.01), increased mean egg weight by 3% (p>0.05) and 4.8% (p>0.05), and improved feed efficiency by 9.54% (p<0.05) and 7.25% (p<0.05), respectively; The addition of 150 and 300 mg/kg dihydropyridine decreased percentage of abdominal fat by 35.4% (p<0.05) and 46.9% (p<0.05), decreased liver fat content by 32.4% (p<0.05) and 10.5% (p<0.05), increased HSL activity of abdominal fat by 39.64% (p<0.05) and 48.48% (p<0.05), increased HSL activity of liver by 9.4% (p>0.05) and 47.34% (p<0.05) and increased the content of cAMP in adenohypophysis by 14.67% (p<0.05) and 10.91% (p<0.05), respectively; The inclusion of 150 mg/kg dihydropyridine increased liver superoxide dismutase activity by 69.61% (p<0.05), and increased hepatic apoB concentration by 53.96% (p<0.05); The supplementation of 150 or 300 mg/kg dihydropyridine decreased malondialdehyde concentration of hepatic mitochondria by 30.90% (p<0.01) and 10.39% (p<0.05), respectively; Supplemented dihydropyridine had no significant effects on TG, Ch HDL-C and VLDL-C concentrations in serum; addition of 150 or 300 mg/kg dihydropyridine increased T3 levels in serum by 15.34% (p<0.05) and 11.88% (p<0.05) and decreased insulin concentration by 40.44% (p<0.05) and 54.37% (p<0.05), respectively. The results demonstrated that adding dihydropyridine had the tendency of improving very low density lipoprotein receptor (VLDLR) content in the ovary. It was concluded that dihydropyridine could improve laying performance and regulate the fat metabolism of laying hens and that 150 mg/kg dihydropyridine is the optimum dose for laying birds in practical conditions.

• Asian-Australasian Journal of Animal Sciences
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• 제14권2호
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• pp.174-183
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• 2001

During the rapid phase of gonadal development of the freshwater teleost, the catfish (Silurus asotus), the influence of hCG upon the inducement of final oocyte maturation and spawning was investigated electrophoretically and ultrastructurally. The electrophoretic patterns obtained were different in the presence and absence of some of the major or minor zones, because of the hormone level in catfish. The vitellogenin of hormone-treated fish was stained more intensively than that of sham-treated fish. These proteins showed some minor or main bands of egg extracts which migrated at positions corresponding to molecular weights of approximately 90,000. However, the thickness of electrophoretic band in molecular weight for hCG-treated fish was slightly lower than that for saline control. It seemed the plasma protein with molecular weight of approximately 45,000 in hCG-treated fish disappeared. In contrast to the control fish, the ovaries in the catfish treated with hCG shows a marked ultrastructural change under the electron microscope. No dilated profiles were seen in the granulosa cells of the mature oocyte before ovulation. After germinal vesicle breakdown (GVBD), the zona radiata interna (ZRI) becomes more compact, and there is a loss of all the processes from the pore canals. There is a wide space between the vitelline membrane and zona radiata. Also, during final maturation, the microvillar processes from the oocyte are seen no longer to penetrate deeply into the extracellular spaces of the overlying granulosa cells, and the reticulate patterns of the zona radiata interna becomes occluded, giving the zona radiata a more solid appearance. It has been possible to initiate 100% oocyte maturation in yolk granules and follicles in vivo by treatment with hCG and a high water temperature ($27^{\circ}C$). In hCG-treated fish, the percentages of successful artificial fertilization and hatching were maximal at 15 h after a single injection. It seems clear that a long acting preparation containing hCG can be successfully used in prespawning fish to advance the final events of gonadal maturation and initiate spawning. Further studies are necessary to evaluate the potential of hCG to either stimulate or inhibit the reproductive development of fish at other stages of the seasonal reproductive cycle.