Gene expression profiles in the testis associated with testis–ova in adult Japanese medaka (Oryzias latipes) exposed to 17α-ethinylestradiol
Highlights
► We analyzed testis–ova marker genes in the testis of estrogen-exposed adult medaka. ► Testis–ova were induced by ethinylestradiol and estradiol benzoate. ► Microarray revealed up-regulation of zona pellucida genes in the testis with oocytes. ► These genes can be used as marker genes for detection of testis–ova in male medaka.
Introduction
Recently, there has been an increase in global concern for environmental pollution. Particular focus has been on estrogen mimicking chemicals, due to their possible effects on sexual development and reproduction in wildlife. Aquatic organisms, in particular, are easily exposed to environmental pollutants and therefore, fish have been used for studies on the effects of estrogenic pollutants on reproduction (Sumpter and Jobling, 1995, Tyler et al., 1998, Lange et al., 2009).
The occurrence of oocytes in the testes of male fish (testis–ova) is a well-documented phenomenon and has shown to be associated with environments contaminated with estrogenic chemicals. One key example of occurrence of testis–ova is wild roach (Rutilus rutilus) living downstream of sewage treatment plant discharges in the United Kingdom (Jobling et al., 1998). Exposure to sewage effluent causes altered sexual development resulting in reduced fertility in roach (Lange et al., 2011). Natural and synthetic estrogens, including the pharmaceutical estrogen, 17α-ethinylestradiol (EE2), are some of the major and potent estrogenic contaminants in effluents. EE2 has been measured in sewage effluents at concentrations up to 7.0 ng L−1 in the United Kingdom (Desbrow et al., 1998), 15 ng L−1 in Germany (Ternes et al., 1999), 42 ng L−1 in Canada (Ternes et al., 1999) and 2.8 ng L−1 in Switzerland (Johnson et al., 2005). Long-term exposure to environmentally relevant concentrations of EE2 can affect fish reproductive physiology by disturbing development and reproduction, hence possibly decreasing fertility in wild fish species, and risking a long-term reduction in population size (Kidd et al., 2007).
A recent study on roach collected from rivers contaminated with effluents has shown that intersex is an important determinant for reproductive success (Harris et al., 2011). The formation of testis–ova is clearly accompanied with the disturbance of spermatogenesis, resulting in reduced fertility (Jobling et al., 2002). Many investigators have focused on the occurrence of testis–ova in recent years. In laboratory experiments, testis–ova can be induced in various fish species by exposure to estrogenic chemicals (Urushitani et al., 2007). Testis–ova can be induced in Java-medaka (Oryzias javanicus) by exposure from the embryonic stage for six months to at least 159 ng L−1 17β-estradiol (E2) (Imai et al., 2005) and in adult Japanese medaka (Oryzias latipes) by exposure to at least 29.3 ng L−1 E2 for 21 days (Kang et al., 2002). Furthermore, early life exposure (until 90 days post hatch) of Japanese medaka to 10 μg L-1 bisphenol A, a weak estrogenic chemical, also induces testis–ova (Metcalfe et al., 2001). In roach, exposure to 4 ng L−1 EE2 during sexual differentiation induces testis–ova in 30% of males in later life and 720-day exposure from fertilization induces complete female type-gonads, suggesting sex reversal (Lange et al., 2009). Despite of a large amount of studies reporting the observation of testis–ova, induction mechanisms of testis–ova have not been well elucidated.
Testis–ova can be used as a valid endpoint when studying disruptive effects of estrogenic chemicals and currently, histological analysis is often employed to assess its development in sampled organisms. However, this method is laborious and unsuitable for a quantitative evaluation, and observation based upon a limited area of the testis could possibly lead to an overestimation or underestimation of testis–ova development. Thus, more rapid, easy, and reliable assay methods are required for detecting testis–ova and using marker genes that highly correlate with the presence of testis–ova would address the limitations of the histological approach. So far, however, gene expression profiles associated with testis–ova have not been reported.
Medaka is a small teleost fish, and a suitable test organism for investigating developmental abnormalities of gonads (Uchida et al., 2010), because this species displays gonadal abnormalities in response to estrogenic chemical exposure including the development of testis–ova even after sex differentiation is completed. In the present study, adult male medaka were exposed to two concentrations of EE2 to induce testis–ova; a higher nominal concentration of EE2 (100 ng L−1) was expected to cause a high incidence of testis–ova (experiment I), whereas a lower, environmentally relevant concentration of EE2 (20 ng L−1) would cause a minimum occurrence of testis–ova in adult male medaka (experiment II). Here we employ gene expression analyses using a microarray and quantitative RT-PCR to investigate marker genes related with the induction of testis–ova for ecotoxicological research.
Section snippets
Animals and chemicals
Japanese medaka (O. latipes, HdrR strain for experiment I and NIES strain for experiment II) were reared in dechlorinated, charcoal treated water, and maintained at laboratory conditions (exp. I: 26 ± 2 °C; 14-h light, 10-h dark cycle, exp. II: 24 ± 2 °C; 16-h light, 8-h dark cycle). Fish were fed twice per day with brine shrimp (Artemia nauplii).
17α-Ethinylestradiol (EE2) and estradiol benzoate (EB) were purchased from Sigma–Aldrich Corp. (St. Louis, MO, USA). For exp. I, EE2 and EB were dissolved
Experiment I-Induction of oocytes in the testis at high doses of EE2 and EB
The induction of testis–ova was histologically analyzed in sexually mature male medaka exposed to estrogens (EB and EE2) or vehicle alone. The testis from control medaka showed complete gonadal differentiation and maturation and contained all stages of germ cells, including the spermatogonia, spermatocytes, spermatids, and spermatozoa Fig. 1A. No testis–ova were found in the control testis. In 800 ng L−1 EB-exposed male medaka, a large number of testis–ova were observed after a 3-week exposure
Conclusion
Endocrine disrupting chemicals lead to the reduction of reproductive success, consequently affecting population growth and biodiversity. Current guidelines that focus exclusively on the expression of hepatic vitellogenin and choriogenin genes as biomarkers for exposure of estrogenic chemicals need to be reconsidered and multiple endpoints should be evaluated for more accurate, robust environmental monitoring. Induction of testis–ova has been evaluated as adverse effects of exposure to
Acknowledgements
We are grateful to Dr. Anke Lange, University of Exeter, for her critical readings of the manuscript. This work was supported by UK-Japan Research Collaboration Grants from the Ministry of the Environment, Japan, and Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan to S.M., Y.K. N.T., T.K. Y.O. and T.I.
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These authors contributed equally to this work.