Figure 2. Induction of proteolysis by Lh, Tnfα and PGF_2α in brown trout preovulatory follicles.

(a) In vitro effects of sLh (25 ng/mL), in the absence or presence of TAPI-1 (50 μM), rTnfα (50 ng/mL) and PGF_2α (2 μg/mL) on trout gelatinase/collagenase activity in ovarian follicle homogenates. (b) Expression of proteolytic genes in ovarian follicles incubated with sLh (25 ng/mL), in the absence or presence of TAPI-1 (50 μM), or with rTnfα (50 ng/mL). The relative expression of mmp2, kt14, top2 and tsg6 was determined by qPCR and normalized to the abundance of 18s. (c) Inhibition of rTnfα-induced gelatinase/collagenase activity in ovarian follicle homogenates by prostaglandin synthesis inhibitors. Follicles were incubated with rTnfα (50 ng/mL) in the absence or presence of INDO (10 μg/mL), NS-398 (10 μg/mL) and SC-560 (10 μg/mL) for 16 h at 15 °C. (d,e) Determination of ovarian Mmp2 activity by gelatin zymography in follicles treated with sLh (25 ng/mL) (d) and rTnfα (50 ng/mL) in the absence or presence of INDO (10 μg/mL) (e). Clear bands in the zymogram indicated enzymatic digestion of gelatin. Densitometric analyses of Mmp2 gelatinase activity were quantified and expressed as fold increase of the total (pro-, intermediate and active Mmp2) or active Mmp2 content in control (set at 1 and represented by the dashed line) and sLh and rTnfα ± INDO-treated follicles. Representative zymograms are shown in the graph and the full-length gels are presented in Supplementary Figure S3. In all graphs, data are the mean ± SEM (n = 3–4). *P < 0.05, with respect to control; ^#P < 0.05, with respect to sLh (b) and with respect to rTnfα (50 ng/mL) (c).