Aromatase is expressed in the trigeminal ganglion in a sex-dependent manner and aging modifies its expression, inverting sex differences

Aromatase immunoreactivity was identified in primary sensory neurons within the ophthalmic, maxillary, and mandibular subdivisions of the trigeminal ganglion. Immunofluorescence employing the aromatase antibody was exclusively observed in the soma of primary sensory neurons, with satellite glial cells displaying no immunoreactivity (Fig. 1). The absence of immunoreactivity was noted when the primary antibody was omitted (data not shown). Immunoreactivity was exhibited in small, medium, and large primary sensory neurons with a similar staining intensity. This immunoreactivity was confined to the cytoplasm, while cell nuclei remained negative.

Fig. 1

Aromatase expression in the mouse trigeminal ganglion. Representative examples of aromatase immunofluorescence in the mouse trigeminal ganglion and quantification of gene expression in young adult and aged male and female mice. A, aromatase marker in the three portions of the trigeminal ganglion. B, Trigeminal ganglion cell nuclei labeled with DAPI. C, Merge. D, Magnification showing aromatase labeling exclusively in the soma of primary sensory neurons, with satellite glial cells not immunoreactive. E, Expression of aromatase. Male animals (n=4) and female animals (n=4). Data are represented as individual values ± SEM. The red line shows mean values. **, *** Significant differences (**p<0.01; ***p<0.001). Scale bar, 100 μm

Quantification of the Cyp19a1 gene expression was performed in the trigeminal ganglion of young adult and aged male and female mice. Two-way ANOVA revealed a significant effect of age [F(1,12)=117.4; P<0.0001], of gonadal sex [F(1,12)=7.829; P=0.0161], and the interaction between age and gonadal sex [F(1,12)=22.02; P=0.0005]. Fisher’s multiple comparison tests are presented in Fig. 1.E. Specifically, older animals (27 months of age) exhibited increased Cyp19a1 expression compared to young adult animals (P<0.0001), with older females demonstrating higher expression than older males (P<0.01). Cyp19a1 expression was higher in older females compared to young adult females (P<0.0001) and older males (P=0.0011), and higher in older males compared to young adult males (P<0.0001) (Fig. 1.E).

As a result of the changes in Cyp19a1 expression with aging, sex differences were inverted in older animals compared to younger ones. Consequently, Cyp19a1 expression was higher in young males than in young females (Fig. 2A); however, in older animals, Cyp19a1 expression was elevated in females (Fig. 1.E).

Fig. 2

Aging modifies the expression of Cyp19a1 in the trigeminal ganglion. Effect of aging on the aromatase expression in the trigeminal ganglion of aged male and female mice. Comparisons of Cyp19a1 expression levels in young adult and aged male and female animals. Young adult males (n=4), young adult females (n=4), older males (n=4), and older females (n=4). Data are represented as individual values ± SEM. The red line shows mean values. *, *** Significant differences (**p<0.01; ***p<0.001)

Aging modifies the expression of Esr1, Esr2, Gper1 and Ar in the trigeminal ganglion

Older mice exhibited a significantly higher expression of genes encoding for ERs in the trigeminal ganglion compared to young animals (Fig. 2A-C and E-G). Male animals displayed increased expression of Esr1 [t=12.07, df=6; P<0.0001], Esr2 [t=19.31, df=6; P<0.0001], and Gper1 [t=20.30, df=6; P<0.0001]. Similar results were obtained for female animals, with higher expression levels of Esr1 [t=15.93, df=6; P<0.0001], Esr2 [t=19.82, df=6; P<0.0001], and Gper1 [t=29.14, df=6; P<0.0001] higher in aged animals compared to adult animals.

In contrast to the effects on genes encoding for ERs, aging resulted in a significant decrease in the expression of Ar (Fig. 2D and H). Both male [t=3.469, df=6; P<0.0133] and female [t=9.585, df=6; P<0.0001] animals exhibited reduced expression of Ar.

Aging reverses sex differences in the expression of Esr1, Esr2, Gper1, and Ar in the trigeminal ganglion

Due to the aforementioned changes with aging, the sex differences in the expression levels of Esr1, Esr2, Gper1, and Ar were assessed in the trigeminal ganglion of young adults and aged male and female mice. Two-way ANOVA revealed a significant effect of age on the expression of Esr1 [F(1,12)=373.410; P<0.0001], Esr2 [F(1,12)=745.06; P<0.0001], Gper1 [F(1,12)=1462.41; P<0.0001] and Ar [F(1,12)=75.13; P<0.0001]. Furthermore, gonadal sex displayed a significant effect on Esr1 [F(1,12)=15.263; P=0.002], Esr2 [F(1,12)=15.33; P=0.002], Gper1 [F(1,12)=47.05; P<0.0001] and Ar [F(1,12)=107.07; P<0.0001]. Interaction effects between aging and gonadal sex were detected for Esr2 [F(1,12)=26.00; p=0.0002], Gper1 [F(1,12)=68.19; P<0.0001] and Ar [F(1,12)=10.12; P= 0.007], whereas no interaction was found for Esr1 [F(1,12)=0.163; P= 0.693]. Fisher’s multiple comparison tests are shown in Fig. 3.

Fig. 3

Aging modifies the sex differences in the expression of Esr1, Esr2, Gper1, and Ar in the trigeminal ganglion. Sex differences in estrogen and androgen receptors’ expression in the trigeminal ganglion of young adult and aged male and female mice. A, Esr1 (ERα) mRNA levels. B, Esr2 (ERβ) mRNA levels. C, Gper1 (GPER) mRNA levels. D, Ar (AR) mRNA levels in young adult aged male and female animals. Aged male animals (n=4) and aged female animals (n=4). Data are represented as values distribution ± SEM, the red line shows mean values. *, **, *** Significant differences (*p<0.05; **p<0.01; ***p<0.001)

In aged animals, the expression levels of Esr1, Esr2, and Gper1 were markedly increased, with females demonstrating the highest values. This pattern reverses the trend observed in younger subjects, where males exhibited elevated levels. Conversely, Ar expression levels experienced a substantial decline in aging mice, a trend more consistently apparent in female subjects.

FCG model reveals that sex differences depend on gonadal sex and are not determined by sex chromosome complement

The FCG model enables the determination of whether the origin of sex differences in a specific trait depends on either gonadal sex, sex chromosome complement, or a combination of both factors. This model produces four types of animals: XX and XY mice with testes, and XX and XY mice with ovaries. Comparisons between animals possessing the same type of gonad allow the assessment of sex chromosome influence, while evaluations involving animals with the same sex chromosome complement facilitate the assessment of the influence of gonadal endowment.

Analysis of androgen and estrogen receptor expression and aromatase in FGC animals revealed a significant effect of gonadal endowment (Fig. 4). Animals with testes demonstrated higher expression of Cyp19a1, Esr2, and Gper1 than those with ovaries (Fig. 4A, C and D), while animals with ovaries exhibited higher expression levels of Esr1 and Ar than those with testes (Fig. 4B and E), regardless of sex chromosome complement.

Fig. 4

Sex differences in the expression of Cyp19a1, Esr1, Esr2, Gper1, and Ar depend on gonadal sex and not on sex chromosome complement. Analysis of the influence of gonadal sex and chromosomal complement in the expression of estrogen and androgen receptors in the trigeminal ganglion using the FCG model. A, Cyp19a1 (Aromatase). B, Esr1 (ERα) mRNA levels. C, Esr2 (ERβ) mRNA levels. D, Gper1 (GPER) mRNA levels. E, Ar (AR) mRNA levels in FCG animals. XY males (n=4), XY females (n=4), XX males (n=4), and XX females (n=4). Data are represented as individual values ± SEM. The red line shows mean values. *, **, *** Significant differences (*p<0.05; **p<0.01; ***p<0.001)

Two-way ANOVA revealed a significant effect of gonads on the expression of Cyp19a1 [F(1,12)=40.80; P<0.0001], Esr1 [F(1,12)=116.2; P<0.0001], Esr2 [F(1,12)=86.99; P<0.0001], Gper1 [F(1,12)=34.89; P=0.0001] and Ar [F(1,12)=79.92; P<0.0001]. Additionally, sex chromosome complement displayed a significant effect on Ar expression [F(1,12)=16.53; P=0.0019], but not on Cyp19a1 [F(1,12)=1.310; P=0.2791], Esr1 [F(1,12)=0.3385; P=0.5714], Esr2 [F(1,12)=0.03360; P=0.8582], or Gper1 [F(1,12)=0.6596; P=0.4356] expression.

No interaction effects between gonadal sex and sex chromosome complement were detected for Cyp19a1 [F(1,12)=0.06679; P=0.8013], Esr1 [F(1,12)=1.276; P=0.2808], Esr2 [F(1,12)=4.543; P=0.0589], Gper1 [F(1,12)=1.023; P=0.3356] and Ar [F(1,12)=1.205; P=0.9973]. Fisher’s multiple comparison tests are shown in Fig. 4.

Significant positive correlation found between Cyp19a1 expression and Esr1, Esr2, and Gper1 expression in trigeminal ganglion

To investigate whether individual variations in the expression of the gene encoding the enzyme involved in estradiol synthesis (Cyp19a1 were associated with variations in the expression of genes encoding proteins participating in estradiol signaling, we examined the correlation between individual values of Cyp19a1 expression and those of Esr1, Esr2, and Gper1 in animals from all experimental groups (see Fig. 5).

Fig. 5

Cyp19a1 expression significantly and positively correlated with the expression of Esr1, Esr2, and Gper1 in the trigeminal ganglion. Correlation of the levels of aromatase and estrogen and androgen receptors’ expression. A, Correlation with Ers1 (ERα); B, Correlation with Ers2 (ERβ); C, Correlation with Gper1 (GPER); D, Correlation with Ar (AR)

As illustrated in Fig. 5, significant positive correlations were observed between Cyp19a1 expression values and Esr1 [Fig. 5A; r=0.95; R2=0.970; p=0.0001], Esr2 [Fig. 5B; r=0.98; R2= 0.9085; p=0.0007] and Gper1 [Fig. 5C; r=0.98; R2=0.8875; p<0.0004] expression values. Conversely, a non-significant correlation was demonstrated between Cyp19a1 expression values and Ar [Fig. 5D; r=-0.73; R2=0.1962; p=0.0995] expression values.

Significant positive correlation found between Age and Esr1, Esr2, Gper1, and Cyp19a1 expression in the trigeminal ganglion of both male and female animals

To investigate whether individual variations in the expression of the gene encoding the enzyme involved in estradiol synthesis (Cyp19a1) and estradiol and androgen receptor signaling were associated with variations in age, we examined with Spearman's rank correlation coefficient (ρ) the correlation between individual values of Cyp19a1, Esr1, Esr2, Gper1 and Ar expression at different age in male (Fig. 6A) and female (Fig. 6B) animals, respectively.

Fig. 6

Age significantly and positively correlated with the expression of Esr1, Esr2, and Gper1 in the trigeminal ganglion. Correlation of individual values of Cyp19a1, Esr1, Esr2, Gper1, and Ar expression at different ages. A, Correlation in male animals; B, Correlation in female animals

As illustrated in Fig. 6A, male animals showed significant positive correlations between age and Cyp19a1 [ρ=0.84, P=0.001], Esr1 [ρ=0.84, P=0.001], Esr2 [ρ=0.84, P=0.001], and Gper1 [ρ=0.84, P=0.001] expression and no correlation with Ar values [ρ=-0,12, P=0.72]. Similarly, female animals showed significant positive correlations between age and Cyp19a1 [ρ=0.84, P=0.001], Esr1 [ρ=0.84, P=0.001], Esr2 [ρ=0.84, P=0.001], and Gper1 [ρ=0.84, P=0.001] but significant negative correlation with Ar values [ρ=-0.84, P=0.001] (Fig. 6B).