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Fig. 13. To identify factors that are regulated by DHP, we carried out gene expression cloning using eel testicular fragments that were cultured with (DHP) or without (Cont) 100 ng/mL DHP for 6 days. After cultivation, poly (A)+ RNAs were extracted from these testicular fragments and a subtracted cDNA library was then constructed from +DHP and –DHP cDNA enriched via the RDA procedure (Niwa et al. 1997). One thousand clones from each of these libraries were subsequently screened by differential hybridization, using each enriched cDNA preparation as a probe. We thereby screened 25 non-cross-hybridizing and up-regulated cDNA fragments after DHP treatment. Southern blotting analysis showed 12 typical cDNA fragments, whose names were eSRS36, 37, 38, 39, 40, 41, 42, 43, 44, 56, 29 and 33. Among these, an eSRS56 cDNA fragment corresponding to trypsinogen was identified (GenBank Acc. No. AB519643). Trypsinogen expression in the Japanese eel testis was determined by northern blot analysis of cultured testicular fragments. Pooled testicular fragments from 10 eels were cultured without (C) or with 10 ng/mL of 11-KT, E2 or DHP for 6 days. Lane IC shows the initial control before culturing. Northern blot analysis of EF1, which serves as reference, is also shown. Reprinted with permission from PNAS, 106, Miura et al., Trypsin is a multifunctional factor in spermatogenesis, 20972–20977, Fig. 1, © 2009, National Academy of Sciences.

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