Sex differences in the rapid control of aromatase activity in the quail preoptic area

Abstract

Adult male quail show high levels of aromatase activity in the preoptic area-hypothalamus (POA-HYP), which parallels the high number of aromatase-immunoreactive cells and elevated mRNA concentrations detected in this brain region by in situ hybridisation. Interestingly, females display considerably lower aromatase activity than males but have almost equal numbers of aromatase-immunoreactive cells and express similar levels of aromatase mRNA. Aromatase activity in the male POA-HYP can be rapidly regulated by calcium-dependent phosphorylations, in the absence of changes in enzyme concentration. In the present study, we investigated whether aromatase activity is differentially regulated by phosphorylations in males and females. A linear increase in accumulation of aromatisation products was observed in both sexes as a function of time but the rate of conversion was slower in females. Saturation analysis confirmed the lower maximum velocities (V(max) ) in females but indicated a similar affinity (K(m) ) in both sexes. Aromatase activity in females reacted differentially to manipulations of intracellular calcium. In particular, chelating calcium with ethylene glycol tetraacetic acid (EGTA) resulted in a larger increase of enzymatic activity in males than in females, especially in the presence of ATP. A differential reaction to kinase inhibitors was also observed between males and females (i.e. a larger increase in aromatase activity in females than in males after exposure to specific inhibitors). These findings suggest that the nature of aromatase is conserved between the sexes, although the control of its activity by calcium appears to be different. Additional characterizations of intracellular calcium in both sexes would therefore be appropriate to better understand aromatase regulation.

Figure 1

Schematic presentation of the experimental protocols for experiments 1 to 4. The sequential treatments applied to the tissue homogenates are indicated above the vertical lines and the incubation duration and temperature on the horizontal line. Δ4= androstenedione

Figure 2

Kinetic characterization of aromatase activity in the male and female quail POA-HYP. A) Measure of aromatase activity as a function of incubation time (Conditions: pH 7.2, 25 nM radioactive substrate, 1 mg wet weight per assay). B) Effect of substrate concentration on aromatase activity in the POA-HYP of the male and female quail (Conditions: pH 7.2, 20 min incubation time, 1 mg wet weight per assay). The time-course shown in Fig. 2A was studied in only one pool of brains for each sex and no measure of biological variation is therefore available for these data.

Figure 3

Calcium-dependent changes in aromatase activity in the male and female quail POA-HYP. Males and female tissue homogenates were exposed to 0 or 0.5 mM EGTA combined with the presence or absence of ATP/Mg²⁺/Ca²⁺, thus creating four different experimental conditions. The insert presents the same data after transformation of all individual enzymatic activities as percentage of the average sex-typical activity in the absence of EGTA and of ATP/Mg²⁺/Ca²⁺. The grey coding of the bars is the same as in the main part of the figure. The box summarizes the results of the three-way ANOVAs used to analyze these data with results on each line referring first to data expressed in fmol/h/POA-HYP and then in parentheses to data expressed as percentage. **= p<0.01, ***= p<0.001, NS= not significant; ATP = ATP/Mg²⁺/Ca²⁺.

Figure 4

Graph illustrating the interactions between the sex of the experimental subjects and the effects of ATP/Mg²⁺/Ca²⁺ (A, C) or of EGTA (B, D) on brain aromatase activity in the data presented in figure 3. The top two panels (A,B) represent interactions as detected in raw data (in fmol/h/POA-HYP) while the bottom panels (C, D) represent these same interactions with data expressed as percentage of the average sex-typical activity in the absence of EGTA and of ATP/Mg²⁺/Ca²⁺. No ATP/ATP = absence/presence of ATP/Mg²⁺/Ca²⁺; 0/0.5 = concentrations of EGTA in mM.

Figure 5

Effects of various kinase inhibitors on the ATP/Mg²⁺/Ca²⁺-induced reduction in aromatase activity in male and female quail POA-HYP. All groups except Controls were treated with ATP/Mg²⁺/Ca²⁺. In addition, they were exposed to buffer or to one of the following kinase inhibitors: Genistein (Gen), a tyrosine kinase inhibitor (Tyr K), Staurosporin (Stau), a general serine/threonine kinase inhibitor (Ser/Thr K), Bisindolylmaleimide (Bisin), a protein kinase C inhibitor (PKC) or H89, a protein kinase A inhibitor (PKA). The insert presents the same data after transformation of all individual enzymatic activities as percentage of the average sex-specific activity in control (KTH) conditions. The box summarizes the results of the two-way ANOVAs used to analyze these data with results on each line referring first to data expressed in fmol/h/POA-HYP and then in parentheses to data expressed as percentage. **= p<0.01, ***= p<0.001, NS= not significant. The asterisks above the female bars in the main part of the figure refer to results of the two-way ANOVA that were performed to compare specifically in males and females the effect of each condition compared to the ATP/Mg²⁺/Ca²⁺ with no kinase inhibitor condition (see text).