Post-proliferative immature radial glial cells female-specifically express aromatase in the medaka optic tectum

Abstract

Aromatase, the key enzyme responsible for estrogen biosynthesis, is present in the brain of all vertebrates. Much evidence has accumulated that aromatase is highly and exclusively expressed in proliferating mature radial glial cells in the brain of teleost fish even in adulthood, unlike in other vertebrates. However, the physiological significance of this expression remains unknown. We recently found that aromatase is female-specifically expressed in the optic tectum of adult medaka fish. In the present study, we demonstrated that, contrary to the accepted view of the teleost brain, female-specific aromatase-expressing cells in the medaka optic tectum represent a transient subset of post-proliferative immature radial glial cells in the neural stem cell lineage. This finding led us to hypothesize that female-specific aromatase expression and consequent estrogen production causes some sex difference in the life cycle of tectal cells. As expected, the female tectum exhibited higher expression of genes indicative of cell proliferation and radial glial maturation and lower expression of an anti-apoptotic gene than did the male tectum, suggesting a female-biased acceleration of the cell life cycle. Complicating the interpretation of this result, however, is the additional observation that estrogen administration masculinized the expression of these genes in the optic tectum, while simultaneously stimulating aromatase expression. Taken together, these results provide evidence that a unique subpopulation of neural stem cells female-specifically express aromatase in the optic tectum and suggest that this aromatase expression and resultant estrogen synthesis have an impact on the life cycle of tectal cells, whether stimulatory or inhibitory.

Figure 1. Phylogenetic analyses of genes related to the cell life cycle in vertebrates.

Phylogenetic analyses of Pcna, Mki67, Sox2, Msi1, Gfap, Bcl2, Bcl2l1, and Baxa/Baxb to verify that the EST clones and predicted transcript identified in the present study encode the medaka orthologs of these proteins in other vertebrates. The number at each node indicates bootstrap values for 1000 replicates. Scale bars represent 0.1 substitutions per site.

Figure 2. Majority of the tectal aromatase-expressing cells are neither mature radial glial cells nor proliferating cells.

Absence of expression of the mature radial glial marker, Gfap (A–J) and proliferation markers, Pcna (K–N) and mki67 (O–R), in the majority of female-specific cyp19a1b-expressing cells in the medaka optic tectum. (A) Gfap-positive cells exhibiting a radial glial morphology with long cytoplasmic processes (indicated by arrows). (B) Gfap expression in the whole optic tectum. Arrows indicate the central portion of the optic tectum, where Gfap expression was observed. (C) cyp19a1b expression in the same field as panel A. (D) Merged image of panels B and C along with DAPI staining. (E) Gfap expression in the peripheral portion of the optic tectum. (F) cyp19a1b expression in the same field as panel E. (G) Merged image of panels E and F along with DAPI staining. (H) Gfap expression in the central portion of the optic tectum. (I) cyp19a1b expression in the same field as panel H. (J) Merged image of panels H and I along with DAPI staining. (K) Expression of Pcna and cyp19a1b in the whole optic tectum. Arrows indicate the peripheral growth zone, where Pcna expression was detected. Nuclei were stained with DAPI. (L) Pcna expression in the peripheral growth zone (arrows). (M) cyp19a1b expression in the same field as panel L. (N) Merged image of panels L and M along with DAPI staining. (O) Expression of mki67 and cyp19a1b in the whole optic tectum. Arrows indicate the peripheral growth zone. Nuclei were stained with DAPI. (P) mki67 expression in the peripheral growth zone (arrows). Note that the signals indicated by asterisks lie on the dorsal surface of the cerebellum, but not in the optic tectum. (Q) cyp19a1b expression in the same field as panel P. (R) Merged image of panels P and Q along with DAPI staining. Small boxes in panels E–J and L–N indicate regions shown at higher magnification in the insets in the respective panels. Scale bars, 100 µm (B–D, K, O), 50 µm (E–J, L–N, P–R), and 10 µm (A, all insets). OT, optic tectum; VGZ, ventricular gray zone of the optic tectum.

Figure 3. The tectal aromatase-expressing cells express genes indicative of multipotency and exhibit cell polarity.

Coexpression of cyp19a1b and the genes indicative of multipotency, sox2 (A–D) and msi1 (E–H), and epithelial/radial glial cell polarity marker, Zo1 (I–L), in female medaka optic tectum. (A) Expression of sox2 and cyp19a1b in the optic tectum overall. Arrows indicate the peripheral growth zone. Nuclei were stained with DAPI. (B) sox2 expression in the VGZ. (C) cyp19a1b expression in the same field as panel B. (D) Merged image of panels B and C along with DAPI staining. (E) Expression of msi1 and cyp19a1b in the whole optic tectum. Arrows indicate the peripheral growth zone. Nuclei were stained with DAPI. (F) msi1 expression in the VGZ. (G) cyp19a1b expression in the same field as panel F. (H) Merged image of panels F and G along with DAPI staining. (I) Expression of Zo1 and cyp19a1b in the whole optic tectum. Nuclei were stained with DAPI. (J) Zo1 expression in the VGZ. (K) cyp19a1b expression in the same field as panel J. (L) Merged image of panels J and K along with DAPI staining. Small boxes in panels B–D, F–H, and J–L indicate regions shown at higher magnification in the insets in the lower right-hand corner of the respective panels. Scale bars, 100 µm (A, E, I), 50 µm (B–D, F–H, J–L), and 10 µm (all insets). OT, optic tectum; VGZ, ventricular gray zone of the optic tectum.

Figure 4. The tectal aromatase-expressing cells are immature radial glial cells.

Morphology of female-specific cyp19a1b-expressing cells in the medaka optic tectum as revealed by imaging of cyp19a1b-GFP transgenic medaka (A–C) and expression of Blbp in these cells (D–M). (A) GFP fluorescence in the whole optic tectum in cyp19a1b-GFP transgenic medaka. Nuclei were stained with DAPI. (B) GFP fluorescence in the VGZ shown at higher magnification than panel A. (C) Highly polarized morphology of GFP-positive cells with long cytoplasmic processes (indicated by arrows). (D) Blbp-positive cells exhibiting a radial morphology with long cytoplasmic processes (indicated by arrows). (E) Blbp expression in the whole optic tectum. (F) cyp19a1b expression in the same field as panel E. (G) Merged image of panels E and F along with DAPI staining. (H) Blbp expression in the peripheral portion of the optic tectum. (I) cyp19a1b expression in the same field as panel H. (J) Merged image of panels H and I along with DAPI staining. (K) Blbp expression in the central portion of the optic tectum. (L) cyp19a1b expression in the same field as panel K. (M) Merged image of panels K and L along with DAPI staining. Small boxes in panels H–M indicate regions shown at higher magnification in the insets in respective panels. Scale bars, 100 µm (A, E–G), 50 µm (B, H–M), and 10 µm (C, D, all insets). OT, optic tectum; VGZ, ventricular gray zone of the optic tectum.

Figure 5. Sex differences in the tectal expression of cell life cycle-related genes and the tectal size.

Examination of sex differences in the expression of cell life cycle-related genes as well as cyp19a1b in the medaka optic tectum (A) and its size (B). (A) Examination of sex differences in the expression of cyp19a1b, proliferation markers, pcna and mki67, genes indicative of multipotency, sox2 and msi1, mature radial glial marker, gfap, anti-apoptotic genes, bcl2 and bcl2l1, and pro-apoptotic genes, baxa and baxb, in the medaka optic tectum. Values are given relative to the level of expression in the whole brain of males, which was arbitrarily set to 1. *, p < 0.05; **, p < 0.01; ***, p < 0.001. (B) Examination of sex differences in the optic tectum size in medaka. No significant difference was detected between males and females (p = 0.12).

Figure 6. Effects of estrogens on the tectal expression of cell life cycle-related genes.

Effects of ovariectomy (OVX) and E2 treatment on the expression of cyp19a1b and the cell life cycle-related genes differentially expressed between the sexes (pcna, mki67, gfap, and bcl2) in the optic tectum. Values are given relative to the level of expression in the whole brain of males, which was arbitrarily set to 1. Groups with different alphabetical characters are significantly different (p < 0.05).