Absorbance was read at 450 nm (measured in a Plate reader, Bioteck, USA), using 100 μl of TMB solution and 100 μl 2 N HCl as a blank control. Manufacturer’s information shows that the kit anti-bradykinin

anti-body reacts with bradykinin, kallidin, [Des-Arg1]-bradykinin and biotinyl-bradykinin and this peptides detection limit is of approximately 0.004 ng/ml. The follicular fluid was assayed using enzyme-linked immunosorbent assay (ELISA), to determine estradiol concentration, following the manufacturer’s instructions (Cayman Biochemical). The differences on continuous data between hours during the ovulation process were accessed BMN 673 by analysis of variance (ANOVA) and multi-comparison between hours was performed by least square means. Data were tested for normal distribution using the Shapiro–Wilk test and normalized when necessary. All analyses were performed using the JMP software (SAS Institute Inc., Cary, USA) and a P < 0.05

was considered statistically check details significant. Data are presented as mean ± sem. There were no differences regarding follicular diameter in different time-points before the ovariectomy (evaluated through ultrasound; data not shown). The concentration of estradiol increased 3 h after treatment with GnRH, the expected endogenous LH surge time, and gradually decreased thereafter, up to 24 h (data not shown). The KKS precursor expression, or KNG, was similar for both follicular cell types, granulosa and theca, during the ovulation (P > 0.05, Fig. 1A and B). The mRNA expression of the B2R receptor was constant during the ovulation process in granulosa cells, with no difference (P > 0.05) at different times after the LH surge induction ( Fig. 1C). However, in theca cells, the mRNA B2R receptor expression showed an increase (P < 0.05) after the GnRH (hour zero) injection up until 6 h and gradual decrease at 12 h, remaining constant until 24 h ( Fig. 1D). The B1R receptor mRNA expression was different in both follicular cells types during the assessed times. In granulosa cells ( Fig. 1E), the

expression increased only at 6 h and decreased after that. In theca cells ( Fig. 1F), the B1R Phosphatidylinositol diacylglycerol-lyase expression increased at 3 and 6 h, decreased at 12 h and then remained constant until 24 h. Results for kallikrein-like activity in the follicular fluid showed a decrease (P < 0.05) between the LH ovulatory surge induction (hour zero) and 24 h ( Fig. 2A). There was, however, no difference between zero and 12 h. The bradykinin presented differences (P < 0.05) during the ovulation. The BK increased after zero hour until 6 h, decreased until 12 h and remained constant up until 24 h ( Fig. 2B). This study demonstrated for the first time that components of the KKS system are produced in the ovary during ovulation in monovular species, using a sensitive semi-quantitative RT-PCR and enzymatic assay for the KKS components.