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. 2004 Jan 20;101(3):865-70.
doi: 10.1073/pnas.2630225100. Epub 2003 Dec 23.

Adult male rat hippocampus synthesizes estradiol from pregnenolone by cytochromes P45017alpha and P450 aromatase localized in neurons

Yasushi Hojo  1 , Taka-Aki Hattori, Taihei Enami, Aizo Furukawa, Kumiko Suzuki, Hiro-Taka Ishii, Hideo Mukai, John H Morrison, William G M Janssen, Shiro Kominami, Nobuhiro Harada, Tetsuya Kimoto, Suguru Kawato
Affiliations

Adult male rat hippocampus synthesizes estradiol from pregnenolone by cytochromes P45017alpha and P450 aromatase localized in neurons

Yasushi Hojo et al. Proc Natl Acad Sci U S A. .

Abstract

In adult mammalian brain, occurrence of the synthesis of estradiol from endogenous cholesterol has been doubted because of the inability to detect dehydroepiandrosterone synthase, P45017alpha. In adult male rat hippocampal formation, significant localization was demonstrated for both cytochromes P45017alpha and P450 aromatase, in pyramidal neurons in the CA1-CA3 regions, as well as in the granule cells in the dentate gyrus, by means of immunohistochemical staining of slices. Only a weak immunoreaction of these P450s was observed in astrocytes and oligodendrocytes. ImmunoGold electron microscopy revealed that P45017alpha and P450 aromatase were localized in pre- and postsynaptic compartments as well as in the endoplasmic reticulum in principal neurons. The expression of these cytochromes was further verified by using Western blot analysis and RT-PCR. Stimulation of hippocampal neurons with N-methyl-d-aspartate induced a significant net production of estradiol. Analysis of radioactive metabolites demonstrated the conversion from [(3)H]pregnenolone to [(3)H]estradiol through dehydroepiandrosterone and testosterone. This activity was abolished by the application of specific inhibitors of cytochrome P450s. Interestingly, estradiol was not significantly converted to other steroid metabolites. Taken together with our previous finding of a P450scc-containing neuronal system for pregnenolone synthesis, these results imply that 17beta-estradiol is synthesized by P45017alpha and P450 aromatase localized in hippocampal neurons from endogenous cholesterol. This synthesis may be regulated by a glutamate-mediated synaptic communication that evokes Ca(2+) signals.

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Figures

Fig. 1.
Fig. 1.
Immunohistochemical staining of P45017α in the hippocampal formation of an adult male rat. (A) The coronal section of the whole hippocampal formation. (B) The CA1 region. (C) The CA1 stained with anti-P45017α IgG preadsorbed with purified P45017α. (D) Fluorescence dual staining of P45017α (green) and neuronal nuclear antigen (red). (E) Fluorescence dual staining of P45017α (green) and glial fibrillary acidic protein (red). (F) Fluorescence dual staining of P45017α (green) and myelin basic protein (red). In D–F, superimposed regions of green and red fluorescence are represented by yellow. so, stratum oriens; pcl, pyramidal cell layer; sr, stratum radiatum. (Scale bar, 800 μm for A and 120 μm for B–F.)
Fig. 2.
Fig. 2.
Immunohistochemical staining of P450arom in the hippocampal formation of an adult male rat. (A) The coronal section of the whole hippocampal formation. (B) The CA1 region. (C) The CA1 stained with P450arom IgG preadsorbed with purified P450arom. (D) The CA3, where not only cell bodies but also processes of neurons are densely stained. so, stratum oriens; pcl, pyramidal cell layer; sr, stratum radiatum. (Scale bar, 800 μm for A and 120 μm for B–D.)
Fig. 3.
Fig. 3.
Immunoelectron microscopic analysis of the distribution of P45017α (A1–A3) and P450arom (B1–B3) within axospinous synapses, in the strata radiatum of the hippocampal CA1 region at the central region of the rostrocaudal level. Gold particles (indicated with arrowheads) were observed to be localized in the endoplasmic reticulum (A1 and B1), the presynaptic region (A2 and B2), and the postsynaptic region (A3 and B3) of pyramidal neurons. In the axon terminal (A2 and B2), gold particles were associated with small synaptic vesicles (A2 and B2). In dendritic spines, gold particles were found within the head of the spine (A3 and B3). Pre, presynaptic region; Post, postsynaptic region. (Scale bar, 200 nm.)
Fig. 4.
Fig. 4.
Western immunoblot analysis of P45017α (A) and P450arom (B) in microsomes from adult rat. (A) Lung (50 μg protein), testis (1 μg), and hippocampus (50 μg). (B) Lung (50 μg protein), ovary (1 μg), and hippocampus (50 μg). Lung was used as a negative control.
Fig. 5.
Fig. 5.
RT-PCR analysis of mRNAs for P45017α (A) and P450arom (B) in the adult rat. The RT-PCR products (50 ng each) were visualized with ethidium bromide in Top. (A) Lane 1, marker (100-bp ladder); lane 2, testis diluted at 1/100; lane 3, testis diluted at 1/1,000; lane 4, cerebral cortex; lane 5, hippocampus; lane 6, hypothalamus; lane 7, cerebellum; lane 8, peripheral blood leukocytes. (B) Lane 1, marker; lane 2, ovary diluted at 1/100; lane 3, ovary diluted at 1/1,000; lane 4, cerebral cortex; lane 5, hippocampus; lane 6, hypothalamus; lane 7, cerebellum; lane 8, liver. Peripheral blood leukocytes and liver were used as the negative controls. (Middle) Southern hybridizations. (Bottom) Ethidium bromide staining of GAPDH.
Fig. 6.
Fig. 6.
RIA analysis of estradiol concentrations in adult male rats. Estradiol concentration in the hippocampus before incubation (basal) (column 1), the hippocampus after a 30-min incubation without NMDA (column 2), the hippocampus after a 30-min incubation with 100 μM NMDA (column 3), the hippocampus after a 30-min incubation with 100 μM NMDA in the presence of MK-801 (column 4), and plasma (column 5). The vertical axis indicates the estradiol concentration in fmol/mg protein for the hippocampus (columns 1–4) and in fmol/μl for plasma (column 5). The significance of the NMDA-induced production of estradiol was confirmed by using the Student t test (*, P < 0.05). The data represent an average over three independent experiments.
Fig. 7.
Fig. 7.
HPLC analysis of steroid metabolism in adult rat hippocampal slices. A total of 106 cpm purified metabolites were applied to HPLC. (A) HPLC profiles of [3H]PREG metabolites using elution solvent A. Slices were incubated for 5 h in the absence (line a) or in the presence (line b) of SU-10603. (B) Reverse-phase HPLC (solvent C) of [3H]DHEA fractions from line a in A. (C) Profiles of [3H]DHEA metabolites (solvent B) in the absence (line a) or in the presence (line b) of fadrozole, after incubation of slices for 5 h. (D) Reverse-phase HPLC (solvent C) of [3H]testosterone (D1) and [3H]estradiol (D2) taken from peaks T and E2, respectively, in C. (E) Profiles (solvent B) of [3H]testosterone metabolites after incubation of slices for 5 h. (F) Profiles (solvent B) of [3H]estradiol metabolites after a 5-h incubation. The arrows designate the elution peak position of the standard [14C]steroid with abbreviations: E1 (estrone), T (testosterone), and E2 (estradiol). “U” designates unknown metabolites. The vertical axis indicates 3H radioactivity (cpm). The retention time of the (same) standard [14C]steroid differed slightly among C, E, and F due to the different silica gel columns used. More than three independent experiments were performed for each of these analyses.
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