Molecular isolation and characterization of the kisspeptin system, KISS and GPR54 genes in roach Rutilus rutilus.

The reproduction of vertebrates is regulated by endocrine and neuro-endocrine signaling molecules acting along the brain-pituitary-gonad (BPG) axis. The understanding of the neuroendocrine role played in reproductive function has been recently revolutionized since the KiSS1/GPR54 (KiSS1r) system was discovered in 2003 in human and mice. Kisspeptins, neuropeptides that are encoded by the KiSS genes, have been recognized as essential in the regulation of the gonadotropic axis. They have been shown to play key roles in puberty onset and reproduction by regulating the gonadotropin secretion in mammals while physiological roles in vertebrates are still poorly known. In order to provide new knowledge on basic reproductive physiology in fish as well as new tools to assess impacts of endocrine disrupting compounds (EDCs), the neurotransmitter system, i.e., gene/receptor, KISS/GPR54 might constitute an appropriate biomarker. This study provides new understandings on the neuroendocrine regulation of roach reproduction as well as new molecular tools to be used as biomarkers of endocrine disruption. This work completes the set of biomarkers already validated in this species.

Endocrine disruption effects in male and intersex roach (Rutilus rutilus, L.) from French rivers: An integrative approach based on subcellular to individual responses.

Among the chemicals found in the aquatic environment, some have been reported to interact with the endocrine system of organisms and have been identified as endocrine disrupting compounds. In this study, we have assessed the biological effects due to environmental estrogenic compounds in adult male roach (Rutilus rutilus). Wild fish were collected in the Seine River, and one of its affluent, the Epte River in Normandy, France and impacts on endocrine and reproductive functions have been assessed. This study provides innovative data on the intersex condition in wild fish that are crucial to understand estrogenic contamination impacts and maintenance of sustainable fish population. The biomarker responses found in fish from contaminated sites included high plasmatic vitellogenin concentrations, altered steroid levels, histopathological and up to 50% intersex fish among the male. However, the overall sex ratio of the roach population indicated sign of feminization with some sites showing only 20% of males. The chemical organic contamination at the different location sites has been characterized by the levels of PAHs, PCBs and phthalates and total estrogenic activity was measured by the YES in vitro assay.

Brain cytochrome P450 aromatase activity in roach (Rutilus rutilus): seasonal variations and impact of environmental contaminants.

P450 aromatase catalyses the conversion of C19 androgens to C18 estrogens which is thought to be essential for the regulation of the reproductive function. In this study, brain aromatase activity (AA) was measured monthly over a reproductive cycle in wild roach (Rutilus rutilus) sampled in a reference site in Normandy. AA peaked during the breeding season, reaching 35 fmol mg(-1)min(-1) in both male and female fish, and was low during the rest of the year except for a significant rise in October. AA was correlated with ovary maturation (measured either as gonado-somatic index or by histological analysis of the gonads) and plasma sex-steroid levels (11-ketotestosterone in males and 17-ß-estradiol in females). Measurements of AA in polluted sites showed that activity was significantly upregulated in sites with fish showing high levels of plasma vitellogenin and large proportion of intersexuality (20-50%) thus suggesting the occurrence of estrogenic compounds and their involvement in AA modulation.

A new in vitro screening bioassay for the ecotoxicological evaluation of the estrogenic responses of environmental chemicals using roach (Rutilus rutilus) liver explant culture.

There is growing evidence that many chemicals released in the environment are able to disturb the normal endocrinology of organisms affecting the structure and function of their reproductive system. This has prompted the scientific community to develop appropriate testing methods to identify active compounds and elucidate mechanisms of action. Of particular interest are in vitro screening methods that can document the effects of these endocrine disrupting compounds on fish. In this study, an in vitro bioassay was developed in the roach (Rutilus rutilus) for evaluating the estrogenicity or antiestrogenicity potency of environmental pollutants by measuring vitellogenin (VTG) induction in cultured liver explants. The cell viability was assessed by the measurement of nonspecific esterase activity using a fluorescein diacetate hydrolysis assay. Results showed that explants could be cultured for 72 h without any significant loss of activity. Dose-dependent responses have been measured with estrogenic model compounds such as 17-ß-estradiol (E2) and 17-α-ethynylestradiol (EE2) or antiestrogenic compounds such as tamoxifen. Lowest observable effective concentrations were 1 nM for E2, 1 nM for EE2, and 100 nM for tamoxifen, showing a good sensitivity of the test system. Estrogenicity of butyl 4-hydroxybenzoate, 4-nonylphenol, and bisphenol A was tested. bisphenol A (100 µM) or butylparaben induced a twofold increase in VTG production when compared with 100 nM E2, whereas this production was only 20% with 100 µM 4-nonylphenol. Overall, this study shows that the bioassay could provide valuable information on endocrine disrupting chemicals including metabolites and mixtures of compounds.

Seasonal variations and alterations of sex steroid levels during the reproductive cycle of male roach (Rutilus rutilus).

Seasonal variations of plasma steroid concentrations i.e. progesterone (P), 11-ketotestosterone (11-KT) and 17-ß-estradiol (E2) were determined immunoenzymatically during a whole reproductive cycle in male roach (Rutilus rutilus) caught in a reference site. Plasma 11-KT concentrations were significantly correlated with gonad growth, expressed as the gonado-somatic index (R² =0.60; p<0.05) and highest concentrations (757 ± 99 pg ml⁻¹ ) coincided with the final testis maturation in March. E2 and P concentrations peaked once during the reproductive cycle. E2 synthesis was significantly induced during the spawning period while P concentration peaked at the very start of the gametogenesis (June) thus suggesting specific roles of these steroids in the reproductive cycle. Comparison of reference levels were then made with plasma steroid concentrations from male roach sampled in polluted areas in the North of France. A significant decrease of E2 (50-60%) and 11-KT (76-84%) was measured, indicating that endocrine disrupting compounds may have interfered with the normal sex steroid synthesis. Contrary to the E2 and 11-KT sex steroids, plasma P concentration was not significantly affected in fish inhabiting impacted areas.

Roach (Rutilus rutilus) reproductive cycle: a study of biochemical and histological parameters in a low contaminated site.

Fish reproduction is subjected to worrying trends in many aquatic environments. In this study, we report the absence of histological and biochemical alterations in fish sampled in a low contaminated site (characterised by the absence of detectable oestrogenic activity and mutagenicity in sediment extracts). A total of 474 roach (Rutilus rutilus) were monthly sampled during 18 months, and no intersex fish were recorded after careful histological examination, thus indicating that the incidence of this phenomenon may be very low under natural conditions. Furthermore, mean male plasma vitellogenin concentration was 24 ng ml(-1) and was only slightly elevated during the spawning period (up to 120 ng ml(-1)) indicating that these low values may be characteristic of a low contaminated site. Of the male roach, 45.3% were sampled, a sex-ratio that did not significantly deviated from the expected 1:1 ratio between male and female. Results also showed that natural conditions can greatly affect the reproductive cycle of roach. Gametogenesis showed a biphasic pattern with first gonad maturation between September and December and a final maturation occurring at the end of winter/early spring. Under decreasing temperatures, particularly below 6 degrees C, gametogenesis was stopped or even regressed with secondary oocytes becoming rare under histological observation. Conversely, elevated temperatures during the winter lead to an earlier gonad maturation.