Histology of corpuscles of Stannius in Lake Van fish (Alburnus tarichi Güldenstädt, 1814) (Cyprinidae).

In the present study, the location, histology and number of corpuscles of Stannius (Sc), which are endocrine glands associated with the kidneys of teleost fish, were investigated for the first time in Lake Van fish (Alburnus tarichi), an anadromous and endemic inhabiting Turkey's Lake Van Basin. The Sc, which were ovoid or spheroid and white or cream in colour, were found to vary in number between three and five among the examined fish. The glands were located in the caudal part of the kidney, and either partially or completely embedded, and found to be present on both the ventral and dorsal surface of either side of the caudal part of the kidney. The Sc were surrounded by a connective tissue capsule that penetrated and divided the gland into incomplete lobules. Two types of cells were determined in the parenchyma of the gland. Type-I cells were predominant throughout the parenchyma and larger than the second (type-II). In the type-I cells, the cytoplasm was observed as weakly or moderately eosinophilic with haematoxylin and eosin staining and weakly or moderately acidophilic with Mallory's triple staining. In the type-I cells, the cytoplasm exhibited weak to moderate periodic acid-Schiff staining and slight or uniform staining with aldehyde fuchsin. The type-II cells were round, had a darkly stained spherical nucleus and were dispersed among the type-I cells. They displayed no cytoplasmic staining with the abovementioned stains.

Polyclonal antibody-based immunoassay of vitellogenin in Van fish (Alburnus tarichi).

Van Lake is the third largest closed lake in the world and the biggest lake in Turkey. An ELISA method has developed with the aim of determining the pollution caused by estrogens and estrogen-like chemicals that have come to the lake Van in recent years. First, the vitellogenin in estrogen-treated male fish plasma was purified by ion exchange chromatography, injected into rats, and the obtained polyclonal antibodies were tested for specificity by Western blot and immunohistochemical methods. Immunohistochemical labeling of the vitellogenin-synthesized liver resulted in the intense marking of the liver of the animals injected with estrogen, while no markings were observed in the control group. The limit of detection of the developed enzyme-linked immunosorbent assay was 4.6 µg L-1, and the working range was 7.8 to 2000 µg L-1. Intra- and inter-assay variations were 13.0 % and 13.3%. The highest level of vitellogenin in male fishes measured was 23.56 µg mL-1.

Vitellogenin in the male Lake Van fish (Chalcalburnus tarichi Pallas, 1811).

Chalcalburnus tarichi is an endemic cyprinid species living in Lake Van, Turkey. In the present study, we investigated vitellogenin (Vtg) in the plasma of adult male C. tarichi, sampled monthly from Edremit, Campus and Çarpanak sites of Van Edremit Gulf (VEG) of lake, between October 2004 and May 2005. The fish living around VEG winters in deep water around Edremit site. University and Çarpanak sites are also on fish migration way from VEG to fresh water (Karasu River). The levels of measured plasma Vtg changed from trace levels to 26.35 µg/mL. The highest level of plasma Vtg was detected in April, with a concentration of 21.60 ± 1.29 µg/mL. We also performed immunohistochemical Vtg staining in the liver sections of male fish and determined Vtg positive hepatocytes in the liver of 1/10 sampled fish. Our results indicated that C. tarichi living in Lake Van might be exposed to endocrine-disrupting chemicals.

Isolation of estrogen receptor subtypes and vitellogenin genes: expression in female Chalcalburnus tarichi.

Reproductively arrested gonadal development has been previously described in the teleost pearl mullet (Chalcalburnus tarichi, Cyprinidae) from Van Edremit Gulf of Lake Van, Turkey. Oocyte development in some females was arrested at the previtellogenic stage, while gonadosomatic index (GSI) and plasma 17ß-estradiol (E2) level were low. A subset of the females was found to have normal ovaries and relatively higher plasma E2 and GSI. These two groups were termed reproductively arrested (RA) and reproductively non-arrested (RN) females. In this study, we cloned estrogen receptor (ER) isoforms (ERα, ERß1 and ERß2) and vitellogenin (Vtg), and their mRNA levels were measured in RA and RN fish tissues. C. tarichi ERs fell in the same clade with other fish ERs and ERα and ERß1 had 97% and 98% identity with the roach (Rutilus rutilus) ERs, respectively. Both Vtg and ER isoforms' mRNA abundance were higher in the liver than in the ovary and hypothalamus (liver>ovary>hypothalamus). The level of ERα mRNA was significantly lower in the liver, ovary and brain of RA fish than in the RN fish tissues. ERß1 mRNA levels were not different in the liver and ovary from RA and RN fish while ERß2 expression significantly increased in the liver and ovary from RA fish. All ER subtype expression was found to be lower in the brain from RA fish than RN fish. The level of Vtg mRNA was significantly lower in the liver and ovary from RA fish than RN fish tissue. These results suggest that ER subtypes are differentially regulated by E2, and their functions are also different in vitellogenesis. Analysis of organic contaminants in sediments revealed that C. tarichi living in Van Edremit Gulf of Lake Van are exposed to the contaminants bis(2-ethylhexyl) phthalate and 4,4(') DDT. We suggest that the RA fish represent a segment of the population that is more sensitive to exposure to endocrine disrupting compounds.

The effects of 17alpha-ethynylestradiol, 4-nonylphenol and phenol red on vitellogenin synthesis in juvenile Chalcalburnus tarichi primary hepatocyte culture.

In this study, the effects of the endocrine-disrupting chemicals known as 17α-ethynylestradiol (EE2), 4-nonylphenol (NP) and phenol red on vitellogenin (Vtg) synthesis in juvenile Chalcalburnus tarichi primary hepatocyte culture were investigated depending on dose and time. Hepatocytes were isolated by collagenase perfusion method and the cells were kept alive in cell culture for more than 2 weeks without using serum. As a result, it was determined that EE2 increased the Vtg synthesis depending on dose and time (p < 0.05), while NP and phenol red did not have any effect on the Vtg synthesis in the Chalcalburnus tarichi primary hepatocyte culture. Besides that, it was observed that high doses of NP (10( -3) and 10(-4)M) had toxic effects to the hepatocyte culture, too.

Effects of 17α-ethynylestradiol and nonylphenol on liver and gonadal apoptosis and histopathology in Chalcalburnus tarichi.

Chalcalburnus tarichi is an endemic cyprinid fish living in the Lake Van basin located in the Eastern Turkey. Fish (3+ ages) were exposed to 17α-ethynylestradiol (EE(2); 1, 10, 100 ng L(-1); nominal concentrations) and nonylphenol (NP; 10, 60, 200 µg L(-1) ; nominal concentrations) for 32 days under semistatic daily renewal conditions. The exposure period was followed by an evaluation of liver and gonadal apoptosis and gonad histopathology in males and females. Exposure to the highest concentrations of EE(2) (100 ng L(-1)) and NP (200 µg L(-1) ) caused significant increases in the extent of apoptosis in liver and gonads. Treatment with 100 ng L(-1) of EE(2) and 200 µg L(-1) NP increased the number of TUNEL positive hepatocytes significantly in both sexes compared to controls. The rates of apoptosis in testicular germ cells and ovarian follicular cells were significantly greater at the same concentrations. Exposure to EE(2) (100 ng L(-1)) and NP (60 and 200 µg L(-1)) caused thickening of interstitial connective tissue (fibrosis) in the seminiferous tubule wall and testis-ova formation in males. In females treated with 100 ng L(-1) EE(2) , increased percentage of atretic ooctytes and fibrotic areas in the ovarian somatic stromal tissue were found in the ovaries. Increase in atresia, without a statistical significance, and fibrotic stromal tissue were also noted in 60 and 200 µg L(-1) NP treatments. Results suggest that EE(2) - and NP-dependent hepatotoxicity and gonadotoxicity are causally related to the increase in apoptosis in C. tarichi.

An investigation on endoparasites of Rana ridibunda complex (s. l.) with dermal parasite (metacercariae) in Dalaman (Turkey).

In this study, endoparasites of the Rana ridibunda specimens collected from Dalaman area, southwestern Turkey were investigated. In the intestine of 17 specimens of R. ridibunda, five different parasites, represented by three species of the trematoda (Diplodiscus sp, Pleurogenoides sp, Plagiorchis sp) and two genera of round worms (Cosmocerca sp; Foleyella sp) were recognized. The trematoda and one of the round worms were found in the rectal region whereas the other round worm species are observed in the abdominal cavity.