28 Expression Analysis of Sex-Specific and Endocrine-Disruptors 369
OLGIs analyzed, we identified OLGIs corresponding to genes preferentially
expressed in either male or female. In the comparisons of gene expression between
male and female, OLGIs that gave the ratios of averaged expression levels more
than two-fold were considered as markers for male- or female-specific genes. By
this criterion, 1,243 OLGIs were estimated to represent genes specifically expressed
in the female, and 2,276 represented male-specific genes. The female-specific
OLGIs represented sequences of previously characterized female-specific genes
such as vitellogenin I/II, choriogenin H/L, estrogen receptor, ZP family genes
(ZPA, ZPB, and ZPC), cyclin Bs, FIGα, and 42Sp50 (Kanamori 2000). Unlike the
female- specific OLGIs, most of the male-specific OLGIs have not yet been assigned
or characterized. Nonetheless, these OLGIs are good candidates for male-specific
biomarkers. These results suggest that medaka microarrays can be used to distinguish
physiological sex by analyzing the expression profiles of sex-specific OLGIs.
28.3.3 Gonadal Histology of Male Medaka Exposed
to Estrogenic Chemicals
Estrogenic chemicals such as synthetic estrogens and alkylphenols may change
the reproductive status of organisms by adversely affecting endocrine systems
(Colborn et al. 1996; Oberdorster and Cheek 2001; Lathers 2002). In medaka,
exposure to endogenous steroid hormones or exogenous endocrine disruptors
causes inter-sex (testis-ova) or sex reversal in gonad (Yamamoto and Matsuda
1963; Kang et al. 2002a,b; Balch et al. 2004). Particularly, E2, EE2, NP, OP and
BpA have been reported to induce vitellogenin and testis-ova in adult male
medaka (Gronen et al. 1999; Kang et al. 2002a,b, 2003; Seki et al. 2002). Based
on these previous studies, exposure concentrations of 100 ng/L E2 and EE2,
100 µg/L NP and OP, and 2 mg/L BpA were selected in this study. These expo-
sure concentrations were used to ensure histological impairment (e.g. testis-ova
formation) observed in the exposed male medaka, although some of the
concentrations selected (NP, OP, and BpA) were higher than environmentally-
relevant concentrations.
Histological analysis showed the formation of testis-ova in the testes of male
medaka exposed to the estrogenic chemicals. Fig. 28.2 shows typical sections of the
testis of the control male and the testis-ova gonad. No histologic abnormality was
observed in the gonad of the control male fish. High incidences (100%, 8/8) of testis-
ova were observed in 100 ng/L E2- and EE2-treated male. Four male of eight males
(50%) developed testis-ova by 2 mg/l BpA-treatment, whereas testis-ova were
observed in only one of 8 males (12.5%) exposed to 100 µg/l NP or OP. On the other
hand, spermatozoa were still observed in the testis of the EE2-treated male (Fig.
28.2), suggesting active spermatogenesis even in the testis-ova gonads. These results
suggest that the reproductive activity of the male medaka may be retained even
though the estrogenic chemical treatments significantly induce female structures
(oocytes) in testes of male medaka.