Expression of aromatase in radial glial cells in the brain of the Japanese eel provides insight into the evolution of the cyp191a gene in Actinopterygians

Abstract

The cyp19a1 gene that encodes aromatase, the only enzyme permitting conversion of C19 aromatizable androgens into estrogens, is present as a single copy in the genome of most vertebrate species, except in teleosts in which it has been duplicated. This study aimed at investigating the brain expression of a cyp19a1 gene expressed in both gonad and brain of Japanese eel, a basal teleost. By means of immunohistochemistry and in situ hybridization, we show that cyp19a1 is expressed only in radial glial cells of the brain and in pituitary cells. Treatments with salmon pituitary homogenates (female) or human chorionic gonadotrophin (male), known to turn on steroid production in immature eels, strongly stimulated cyp19a1 messenger and protein expression in radial glial cells and pituitary cells. Using double staining studies, we also showed that aromatase-expressing radial glial cells exhibit proliferative activity in both the brain and the pituitary. Altogether, these data indicate that brain and pituitary expression of Japanese eel cyp19a1 exhibits characteristics similar to those reported for the brain specific cyp19a1b gene in teleosts having duplicated cyp19a1 genes. This supports the hypothesis that, despite the fact that eels also underwent the teleost specific genome duplication, they have a single cyp19a1 expressed in both brain and gonad. Such data also suggest that the intriguing features of brain aromatase expression in teleost fishes were not gained after the whole genome duplication and may reflect properties of the cyp19a1 gene of ancestral Actinopterygians.

Figure 1. Western blotting analysis of aromatase expression in male pituitary extracts.

(A) Incubation with the aromatase antibody diluted (1∶1000) yielded a single band at the expected size of 56 Kd. (B) Pre-absorption of the antiserum diluted 1∶1000 with the peptide NH2-EKDSE LTMMF TPRRR Q- COOH (25 μM) caused disappearance of the band.

Figure 2. Hormonal treatment strongly increases aromatase immunoreactivity in the brain of Japanese eel.

Transverse sections in the preoptic area of males (A–F) and females (G–L) showing aromatase-immunoreactivity in control animals (males: A–C; females G–I) and animals treated with either human chorionic gonadotrophin (HCG, males: D–F) or salmon pituitary homogenates (SPH, females: J–L). One can see that hormonal treatment strongly increases the aromatase immunoreactivity in cells bordering the preoptic recess (rpo). Pictures in A and D (or G and J) were taken with the same exposure time to allow comparison. PP: parvocellular preoptic nucleus; PM: magnocellular preoptic nucleus. A–F: Bar  = 75 μm; G–L: Bar  = 50 μm.

Figure 3. Distribution of cyp19a1b mRNA and aromatase protein in the brain of the Japanese eel.

A, D, G: Cyp19a1b mRNA in the brain of the Japanese eel as revealed by in situ hybridization in the supracommissural nucleus of the subpallium (Vs), the parvocellular preoptic nucleus (PP) and the magnocellular preoptic nucleus (PM). Note that the signal is consistently restricted to the regions adjacent to the ventricles. B, E, H: Aromatase protein in the brain of the Japanese eel as revealed by immunohistochemistry on the same sections than in A, D and G. One can see that immunoreactive cells have their nuclei along the ventricles and long lateral processes C, F, I: Merges showing overlapping (yellow color) of cyp19a1b mRNA and aromatase protein in the brain of the Japanese eel as revealed by immunohistochemistry on the same sections than in A, D and G. Only the radial processes do not show co-expression. All bars  = 20 μm.

Figure 4. Distribution of cyp19a1b mRNA and aromatase protein in the brain of the Japanese eel.

A, D, G: Cyp19a1b mRNA in the brain of the Japanese eel as revealed by in situ hybridization in the supracommissural nucleus of the subpallium (Vs), the dorsal central thalamic nucleus (CP) and around the 4^th ventricle (4V) between the valvula of the cerebellum (VC) and the midbrain tegmentum (MT). Note that the signal is consistently restricted to the regions adjacent to the ventricles. B, E, H: Aromatase protein in the brain of the Japanese eel as revealed by immunohistochemistry on the same sections than in A, D and G. One can see that immunoreactive cells have their nuclei along the ventricles and long lateral processes C, F, I: Merges showing overlapping (yellow color) of cyp19a1b mRNA and aromatase protein in the brain of the Japanese eel as revealed by immunohistochemistry on the same sections than in A, D and G. Only the radial processes do not show co-expression. All bars  = 20 μm.

Figure 5. Details of the aromatase-positive cells characterizing them as radial glial cells.

(A) Proximal processes (arrows) reaching the ventricle at the level of the anterior periventricular preoptic nucleus. Bar  = 15 μm (B) Long radial processes (arrowheads) some of which end at the ventral surface of the brain (arrow) while others project more laterally. Bar  = 75 μm (C) End-feet of the distal processes at the periphery of the lateral hypothalamus (L Hyp) close to the meninx. Bar  = 8 μm.

Figure 6. In situ hybridization of cyp19a1b mRNA combined to BLBP (Brain Lipid Binding Protein) immunohistochemistry confirms the radial glial nature of the aromatase-positive cells.

A, D, G: Cyp19a1b mRNA in the brain of the Japanese eel as revealed by in situ hybridization in the post-commissural nucleus of the subpallium (Vs), the magnocellular preoptic nucleus (PM) and the parvocellular preoptic nucleus (PP). Note that the signal is consistently restricted to the regions adjacent to the ventricles. Bar  = 15 μm B, E, H: BLBP protein in the brain of the Japanese eel as revealed by immunohistochemistry on the same sections than in A, D and G. One can see that immunoreactive cells have their nuclei along the ventricles and long processes running laterally. Bar  = 25 μm C, F, I: Merges showing overlapping (yellow color) of cyp19a1b mRNA and BLBP protein in the brain of the Japanese eel as revealed by immunohistochemistry on the same sections than in A, D and G. Only the radial processes do not show co-expression. Bar  = 20 μm.

Figure 7. Expression of aromatase in the pituitary gland of the Japanese eel.

A–C: High power view of aromatase-positive cells in the rostral pars distalis (RPD). One can see that positive cells are located in the outer region of the follicles and have a long process running to the follicle center (fc). Negative cells have their nuclei (stars) closer to the base of the follicles. Bar  = 7 μm D–F: Low power view of aromatase-positive cells in the pars intermedia (PI). Digitations of the neurohypophysis (nh) penetrate deeply within the proximal PI in which numerous positive cells are located. bc: blood cells showing endogenous fluorescence. Bar  = 75 μm G–H: High power view of aromatase-positive cells in the pars intermedia (PI) showing that positive cells have their nuclei located in the center of the PI cell population. However, they also have cytoplasmic processes ending onto the basal membrane that separates the neurohypophysis from the PI. bc: blood cells showing endogenous fluorescence. Bar  = 15 μm.

Figure 8. Aromatase-positive radial glial cells and pituitary cells exhibit proliferative activity.

A–C: Aromatase-positive cell in the ventral periventricular preoptic nucleus (PP: arrow in A) exhibits a nucleus positive for the proliferation marker PCNA (arrows in B and C). rpo: preoptic recess. D–F: Aromatase-positive cell in the nucleus of the lateral recess (NRL; arrow in D) exhibits a nucleus positive for the proliferation marker PCNA (arrows in E and F). G–L: Two examples of aromatase/PCNA-positive cells the proximal pars distalis (PPD) and pars intermedia; arrows in G and J). Figures I and L show that the PCNA-positive nuclei (arrows in H and K) correspond to aromatase positive cells in G and J, respectively. All bars  = 5 μm.

Figure 9. Hypothesis regarding evolution of the cyp19a1 gene in the Actinopterygian lineage (ray-finned fish).

From an ancestral gene having brain and gonad functions, the teleost specific genome duplication gave birth to two copies that evolved differently in Elopomorphs (Eels) and other teleosts. Soon after the duplication, eels probably lost one copy of the cyp19a1 gene and this remaining copy retained brain and gonad functions. In other teleost fishes, a sufunctionalization process occurred that led to partition of functions between the two copies, cyp19a1a (gonad) and cyp19a1b (brain).