Butanol

Diuron (3-(3,4-dichlorophenyl)-1,1 dimethylurea, CAS number 330-54-1, Pestanal, analytical standard), acetonitrile (anhydrous, 99.8 %), 1-chloro-2,4-dinitrobenzene (CDNB), disodium salt of reduced form of nicotinamide adenine dinucleotide phosphate (NADPH), ultrapure reduced glutathione (GSH), hydrogen peroxide, imidazole, glutamine, and hydroxylamine were purchased from Sigma Aldrich Chemicals (France). Formaldehyde 35 %, glacial acetic acid, and butanol 100 % were purchased from Carlo Erba (France), glycerin 86 % was purchased from Roth (France), ethanol 95 % was purchased from VWR, and paraffin (paraplast®) was purchased from Leica (France). Trizol Reagent and DNAse I were purchased from Invitrogen, M-MuLV Reverse Transcriptase was purchased from BioLabs, and Syber Green Mix was purchased from Roche. Ultrapure deionized water was prepared using a Milli-Q system (Millipore, Molsheim, France). All other reagents were of analytical grade.

Transverse sections were fixed for histology in Davidson's fixative at 4 °C (10% glycerol, 20% formaldehyde, 30% ethanol 95°, 30% sterile seawater, 10% acetic acid) for 48 h. The tissue samples were dehydrated in successive dilutions of ethanol, then transferred in butanol (Carlo Erba, France) and embedded in paraffin wax (Roth, France). Three micrometers sections were stained according to the Prenant-Gabe trichrome or Feulgen staining protocols (Gabe 1968 (link)). The stages of the gametogenetic cycle were individually determined according to Heude Berthelin et al. (2001) (link).