Fig 1: Correlation between plasma 11-KT and preoptic crh mRNA levels in wild female Japanese eels (A. japonica) (n = 100). Blood plasma was extracted with diethyl ether as following de Jesus et al. [112]. Analyses were made in duplicate on each sample using the 11-Keto-Testosterone ELISA Kit (Item No. 582751, Cayman Chemical Inc., ANN Arbor, MI, USA) according to the manufacturer’s instructions. Brains of each fish were dissected out under microscopy, mixed with ISOGEN (Nippon Gene), and total RNA was isolated according to the manufacturer instructions. Total RNA was treated with RNase-free DNase I (Takara). After inactivation of DNase, reverse transcription was carried out using the SuperScript II First-Strand Synthesis System (Invitrogen). QPCR for crh was performed with LightCycler 480 Instrument (Roche Diagnostics) as described by in Yada et al. [113]. The sequences of primers in the assays to amplify crh were 5'-TCACGCAGCGTCTTTTGC-3', forward; 5'-GCTGGCTAGCGTAGCTGCTT-3', reverse, which were designed based on the Gene Bank accession number LC01940.