Neurosteroid Synthesis in Adult Female Rat Hippocampus, Including Androgens and Allopregnanolone

Female rat hippocampus synthesizes significant amount of estrogens, including progesterone (PROG), estrone and estradiol (E2). Hippocampal level of PROG and E2 are considerably higher than those in plasma. Female hippocampal estrogens play a significant role in the fluctuation of dendritic spine density across the estrous cycle. Here we extend the study to the investigation of female androgens, including testosterone (T) and Dihydrotestosterone (DHT), in the female rat hippocampus, since female androgens had been largely unknown. By combination of mass-spectrometric analysis with HPLC-purification and picolinoyl-derivatization of sex steroids, we determined the accurate concentration of T and DHT in the hippocampus. The levels of T and DHT in female hippocampus at Proestrus were approximately 1.1 nM and 0.6 nM, respectively, suggesting a significant synthesis of T and DHT. The level of plasma T was approx. 0.1 nM, implying almost no contribution of plasma T to hippocampal T. The concentration of hippocampal DHT had a good correlation with that of hippocampal T, suggesting a significant activity of 5α-reductase (DHT synthase) in the female. Allopregnanolone level was also determined as a useful indicator of 5α-reductase activity. Interestingly mRNA expression level of 5α-reductase and androgen receptor (AR) was not significantly different between the different estrous cycle stages, or between female and male. Nevertheless, sex difference existed with respect to the levels of T, DHT and Allo in hippocampus. Although physiological significance of female hippocampal androgens waits further investigations, the female hippocampus produces T or DHT which may be useful to suppress anxiety, for example. Journal of Steroids & Hormonal Science J o u r n al o f S tero ids & Hoonal Scie n c e


Introduction
In female hippocampus, synthesis and function of androgens have not been well investigated, although estrogens have been extensively studied about synthesis and function. We recently demonstrated that female rat hippocampus synthesizes 17β-estradiol (E2), progesterone (PROG) and estrone [1], which is similar to hippocampus-synthesized steroids in male [2][3][4][5]. The estrous cycle-dependent fluctuation of the spine density in female rat hippocampus had a good correlation with the cyclic fluctuation of hippocampal E2 or PROG level [1]. Hippocampal testosterone (T) level in female rats was almost as high as hippocampal E2 level (~1 nM) [1]. The level of Dihydrotestosterone (DHT), the most potent androgen, in female hippocampus, however, remains to be undetermined, although one study reported the presence of very low level DHT (the numerical concentrations were not written) with radioimmunoassay (RIA) [6]. We should determine the accurate concentration of DHT by improvement of the sensitivity of measurements in female hippocampus, since androgen application to the aged female hippocampus decrease a depression-like behavior of aged mice [7]. Concerning the steroid synthesis systems in female rat hippocampus, mRNA for T synthesis including P450 (17α) and 17β-HSD (type 1 and 3) are expressed [1]. It remains unclear whether 5α-reductase (DHT synthase) and androgen receptor (AR) are expressed in female hippocampus, although one report showed 5α-reductase (type 2) expression with in situ hybridization in female mice brain [8]. In male rat hippocampus, steroidogenic enzymes and steroid receptors are mainly localized in pyramidal neurons in CA1 and CA3, and granule neurons in dentate gyrus [2,4,[9][10][11][12]. Their neuronal localization of female should be clarified. 5α-reductase produces not only DHT from T, but also allopregnanolone [13] from PROG. Concentration of Allo has been demonstrated using RIA [6,14], GC-MS [15,16] and liquid chromatography with tandem-mass-spectrometry (LC-MS/MS) [17]. Although Allo had been extensively investigated with regard to its anxiolytic and anti-depressive effects [18,19] via modulation of gamma-aminobutyric acid A (GABAA) receptors [20], the physiological significance of DHT in female hippocampus is still unknown and should be investigated. We here examine whether DHT synthase or AR expresses in female rat hippocampus.
Localization of P450 (17α), steroidogenic acute regulatory protein (StAR) and AR in female hippocampus is also examined with Immunohistochemical *Corresponding author: Suguru Kawato, Department of Biophysics and Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro, Tokyo 153-8902, Japan, Tel/Fax: 81-3-5454-6517; E-mail: kawato@bio.c.u-tokyo.ac.jp staining. By combination of mass-spectrometric analysis with HPLCpurification and picolinoyl-derivatization of steroids [1,10,21], the accurate concentration of hippocampal DHT and T is determined. Allo in female hippocampus is determined to compare with male Allo [6,[14][15][16][17]. By comparison of hippocampal androgens and Allo between in male and female, we clarified the profile of steroid synthesis in female rat hippocampus, which is different from male.

Animals
Wistar rats (10 weeks old) were purchased from Saitama Experimental Animals Supply (Japan). The estrous cycle of female rats was monitored with morning vaginal smears. Only those rats showing three consecutive 4-day cycles of Pro, Est, D1 and D2 were used at the age of 12 weeks old. Ovariectomy (OVX) and sham operations were performed two-weeks before (at 10w old) the experiments. Male rats were also used at the age of 12 weeks old. All animals were maintained under a 12 h light/12 h dark exposure and free access to food and water. The experimental procedure of this research was approved by the Committee for Animal Research of the University of Tokyo.

Chemicals
T and DHT were purchased from Sigma [22]. Picolinic acid was from Tokyo Chemical Industry (Japan) and, 13 C 3 -T, 13

RT-PCR
The detailed procedures of mRNA analyses are described in elsewhere [2,4,23]. Total RNAs were isolated from the hippocampus adrenal gland, liver, prostate and testis of adult rats, using a SV total RNA Isolation System (Promega, USA). The purified RNAs were treated with RNase-free DNase to eliminate the possibility of genomic DNA contamination, and quantified on the basis of the absorbance at 260/280 nm. The purified RNAs (100 ng) were reverse-transcribed to obtain cDNAs, using a M-MLV Reverse Transcriptase (Promega, USA). PCR was performed by using these cDNAs. The oligonucleotides for PCR amplification were designed as illustrated in Table S1. The PCR protocols comprised application of a 30 s denaturation period at 95°C, a 20 s annealing period at individual temperature for each enzyme, and a 30 s extension at 72°C, for individual number of cycles for each enzyme (Table S1). For semi-quantitative analysis, the RT-PCR products were separated on 2% agarose gels, stained with ethidium bromide, and analyzed with a fluorescence gel scanner (Atto, Japan) and Image J software. In all the cases we first plotted amplification curves in order to choose the linear phase of PCR cycles. The comparison between different estrous cycle stages can be performed after normalization by glyceraldehyde-3-phosphate dehydrogenase (GAPDH, a house keeping gene) as internal standard. It should be noted that GAPDH expression was not changed across the estrous cycle. The optimal cycle number of GAPDH mRNA was determined as 17 from the amplification curve. The comparison of relative abundance between different estrous stages or between female and male was performed by using the normalized expression. As positive control, we used the reference organ (adrenal gland, liver, prostate and testis)

Mass-spectrometric assay of steroids (T, DHT and Allo)
Detailed procedures are described elsewhere [21].
Step 1: Purification of steroids from hippocampi with normal phase HPLC.
A rat was deeply anesthetized and decapitated at 10-10:30 a.m., since at this time window estradiol (E2) surge at Pro occurs in plasma [24]. The whole hippocampi was removed and homogenized. To calculate the recovery of steroids, radioactive steroids (20,000 cpm) were added as internal standards to hippocampal homogenate. To extract steroid metabolites, ethyl acetate/hexane (3:2 vol/vol) was applied to the homogenates which were then mixed. The mixture was centrifuged at 2,500×g and the organic layer was collected. After evaporation, the extracts were dissolved in 1ml of 40% methanol/H 2 O and applied to a C 18 Amprep solid phase column (Amersham Biosciences, USA) to remove contaminating fats. The extracts were dried, dissolved in an elution solvent of HPLC. The steroid metabolites were separated into T and DHT using a normal phase HPLC system (Jasco, Japan) with an elution solvent of hexane: isopropyl alcohol: acetic acid=98:2:1. A silica gel column (0.46×15 cm, Cosmosil 5SL, Nacalai Tesque, Japan) was used. By monitoring 3H-steroids, the recoveries of T, DHT and Allo were 41 ± 7%, 29 ± 5% and 50 ± 8% respectively, after extraction, C 18 column treatment and normal phase HPLC separation. As internal standards, 100 pg of isotope labeled steroids ( 13 C 3 -T, 13 C 3 -DHT and Allo-d 4 ) were added to steroid extracts.

Step 3: Determination of the concentration for T, DHT and Allo using LC-MS/MS.
For determination of the concentration of T and DHT, the LC-MS/ MS system, which consists of a Shimadzu HPLC system and an API-5000 triple stage quadrupole mass spectrometer (Applied Biosystems, USA) were employed. LC chromatographic separation was performed on a Kinetex C 18 column (2.1×150 mm, 1.7 µm, Phenomenex, USA) for T-picolinoyl ester and DHT-picolinoyl ester. For determination of the concentration of Allo, LC chromatographic separation was performed on a preparation column (Unison UK-Phenyl HT (3 µm, 2.0×50 mm, Imtact, Japan)) and an analytical column (Cap cell core C 18 (2.7 µm, 2.1×100 mm, Shiseido Japan)). After elution of Allo-picolinoyl-ester from the preparation column, Allo-picolinoyl-ester was subsequently loaded to the analytical column. The preparation column enables us to separate Allo (3α-hydroxy-5α-pregnan-20-one) from isomers with equivalent molecular weight (3α-hydroxy-5β-pregnan-20-one, 3b-hydroxy-5α-pregnan-20-one and 3b-hydroxy-5β-pregnan-20one). MS analysis was operated with electro spray ionization [25] in the positive-ion mode. The isotope-labeled steroid derivatives were  Figure S1 and Table S2). Here, m and z represent the mass and charge of a steroid derivative, respectively. The limits of quantification for steroids were measured with blank samples, prepared alongside hippocampal samples through the whole extraction, fractionation and purification procedures. The limits of quantification were 1 pg for T and DHT, and 0.5 pg for Allo per 0.1 g of hippocampal tissue, respectively (Table S2). From the calibration curve using standard T or DHT dissolved in blank samples, the linearity was observed between 1.0 pg and 2000 pg for T and DHT, between 5 pg and 1000 pg for Allo, respectively ( Figure S2).

Statistical analysis
Data are expressed as mean ± SEM. For comparison of the mRNA level of steroidogenic enzymes and receptors, we used a 1-way ANOVA followed by Tukey-Kramer posthoc multiple comparisons. For comparison of the concentration of T or DHT in the hippocampus between male and female, we used Student's t-test. A difference was considered significant at a value of *p<0.05, **p<0.01 or ***p<0.001.

Molecular biological analysis of steroidogenic enzymes and steroid receptors in the female hippocampus
The hippocampal expression of steroidogenic enzymes (StAR, 5α-reductase (Types 1 and 2)) and AR were examined across the estrous cycle. Expression levels of mRNA transcripts normalized by GAPDH are shown as bar graphs in Figure 1. Surprisingly, no significant cyclic fluctuations across the estrous cycle were observed for their expression levels. OVX did not change the expression level of any steroidogenic enzyme and AR. Relative number of transcripts, expressed in the hippocampus of adult female rats, was approx. 1/3 of that in the liver for 5α-reductase type 1, approx. 1/300 of that in the male prostate for 5α-reductase type 2, approx. 1/10 of that in the male prostate for AR, and approx. 1/100 of that in the adrenal for StAR (Table 1).

Mass-spectrometric determination of androgen and allopregnanolone levels in the female hippocampus
The concentration of T, DHT and Allo was determined for adult female and male rat hippocampus using a chromatogram analysis of the fragmented ions of steroid-derivatives. We chose the hippocampus at Proestrus stage for steroid determination since RT-PCR analysis revealed no estrous cycle-related fluctuation of the expression for 5α-reductase (types 1 and 2) which is required for synthesis of DHT and Allo. Results are summarized in Figures 2 and 3. T, DHT and Allo were derivatized with picolinoyl before application to LC-MS/MS to increase the accuracy of determination by improving the limit of quantification (LOQ) ( Table S2).
In the chromatographic profiles of the fragmented ion of T-17picolinoyl-ester, DHT-17-picolinoyl-ester and Allo-3-picolinoyl-ester, a single clear peak was observed at 4.12, 4.37 and 8.43 min, respectively (Figurea 2A1, 2B1 and 2C1). For these steroids, the retention time of the observed steroid peak was the same as that of standard steroid. To confirm the assay accuracy, the hippocampal homogenate spiked with known amounts of the steroids was prepared and its concentration of steroid was determined 296 (Table S3). The LOQs were defined in Table S2 as the lowest value with an acceptable accuracy (90-110%) and precision (i.e. RSD<10%). The results of intra-and inter-assay were shown in Table S2. The RSD for intra-and inter-assay was less than 5.1% and 7.2%, respectively.
Hippocampal level of androgens in female were 1.1 nM for T and The time of sample injection to LC system was defined as t=0 min. Note that pre-purification step using normal phase HPLC before injection to LC system is very important to achieve high precision and good reproducibility of LC-MS/MS determination in order to avoid contamination of other steroids and fats. Steroid-derivatives or steroids were further separated with reversed phase LC-column before MS/MS. In the multiple reaction monitoring modes, the instrument monitored the m/z transition (Table S2) (Figure 3a). The level of hippocampal T was much higher than that of plasma T (~0.1 nM) in female. In contrast to androgens, the levels of Allo in the female hippocampus was 16.4 nM which was 16 times higher than that in male ( Figure 3B; t=6.52, df=4, p=0.0029).
In order to compare hippocampal levels of steroids with plasma steroids, we converted ng/g wet weight to nM concentration via the following estimation (Table 2). First, 1 mL of plasma (93% is water) is assumed to have 1 g weight, as 1 mL of water has 1 g weight. Second, we assume that the hippocampal tissue having 1 g of wet weight has an approx. volume of 1 mL, as nearly 78% of the brain tissue consists of water [29]. Consideration of specific volume of protein and lipids (0.7-1.0 mL/g) in the brain further support this assumption [11,30,31]. After dividing by the individual hippocampal volume (0.124 ± 0.002 wet weight for one whole hippocampus of 12 weeks old female rat, n=44), the levels of T, DHT, Allo in the hippocampus were calculated. Based on these considerations, 0.18 ng/g wet weight of DHT in the hippocampus at Pro corresponds to 0.62 nM ( Table 2).

Localization of steroidogenic enzymes and receptors in female hippocampus
The localization and presence of P450 (17α), P450arom and StAR in the hippocampus of adult female rats were demonstrated by Immunohistochemical staining. P450 (17α), P450arom and StAR were mainly localized in pyramidal neurons in CA1-CA3 region as well as granule cells in dentate gyrus [9] (Figure 4A-4C). Immuno staining of glial cells for these enzymes was much weaker than that of principal neurons. In addition to steroidogenic enzymes, we also demonstrated the localization for AR and ERa in hippocampal slices of female. AR and ERa also localized in pyramidal and granule neurons ( Figures  4D and 4E). It should be noted that AR immuno reactivity was the most prominent at CA1pyramidal neurons, whereas region difference in staining pattern of ERa was not observed. The staining pattern of steroidogenic enzymes and receptors in female hippocampus was almost the same as that of male hippocampus [2,4,11,12,32].

No significant sex difference in hippocampal steroidogenic systems (mRNA and protein)
Surprisingly, the expression levels of sex steroidogenic enzymes including 5α-reductase (types 1 and 2) and StAR were not significantly different between female and male hippocampus (Figure 1). Furthermore, no cyclic fluctuation of expression levels of these enzymes was observed across the estrous cycle. Other steroidogenic enzymes which are required for androgen or estrogen synthesis including P450 (17α), P450 arom and 17β-HSD (T synthase) also exhibited neither sex difference nor estrous cycle-dependent fluctuation [1], suggesting the constant catalytic activity for steroids in the hippocampus. It may be possible that the localization of enzymes or receptors is sex different even with the equal level of mRNA expression. Therefore, we investigated the localization of enzymes including P450 (17α) and P450arom or receptors including AR and ERa in female hippocampus with immunohistochemistry in current study, resulting in the same pattern as male ( Figure 4) [2,4,11,12]. These results suggest that hippocampal synthesis activity of androgen or Allo is independent of sex. In fact, 5α-reductase activity was almost equal between in male and female from the data with high correlation between hippocampal T and hippocampal DHT ( Figure 3C). All data of hippocampal T (x-axis) and DHT (y-axis) from both sexes were aligned in a straight line with high correlation (Figure 3C), implying that DHT production was only dependent on substrate T concentration. Here we present analysis at only Proestrus stage, and further analysis at other estrus stages may help better understanding. In addition, we also measured corticosterone (CORT) level in the female hippocampus, and CORT was not considerably elevated upon sacrifice or anesthetics treatment. Only approx. 200 nM CORT was penetrated to the hippocampus, although plasma CORT transiently elevated to approx. 1500 nM, which is similar to the situation in male rat hippocampus [33]. These results suggest that effects by sacrifice -stress on the hippocampus may not be serious.

Sex difference in hippocampal androgen and allo levels
The current study revealed that female hippocampus is able to synthesize androgens (T and DHT) in addition to estrogens. In female, hippocampal T (~1 nM) is much higher (nearly 10 fold) than plasma T (~0.1 nM) [1], implying that the contribution of plasma T to hippocampal T is very weak. This is very different from male in which 70-80% of hippocampal T is derived from plasma T [21]. Since female plasma DHT would be lower than plasma T (~0.1nM), plasma DHT cannot contribute to the production of hippocampal DHT (~0.6 nM) in female. Majority of female hippocampal DHT should be therefore synthesized from hippocampal-synthesized T. Although male hippocampus synthesizes androgens and estrogens [2,11,21,33,34], sex difference exists in the level of hippocampal steroids. The hippocampal level of androgens (T and DHT) is significantly high in male than in the female. The levels of T and DHT in the female hippocampus were 15 times lower than those in male ( Figure 3A). In contrast to androgens, hippocampal Allo level was 16 times higher in female than in male. What generates such a sex difference in hippocampal steroid level? It may be due to the difference in precursor steroids supplied via blood circulation between female and male. Most abundant precursor

Earlier studies on female androgens in the brain
The concentration of T and DHT in adult female hippocampus had not yet been accurately determined in previous studies. Although one study reported the presence of androgens with RIA method [6], the numerical concentrations were not written due to very low levels. On the other hand, a significant concentration of Allo has been demonstrated using RIA [6,14], GC-MS [15,16] and LC-MS/MS [17]. The concentration of Allo was approximately 6 ng/g (~20 nM) in female hippocampus at Proestrus [6,14]. On the other hand Allo was very low approximately 0.4 ng/g (~1 nM) in male adult whole brain [15,16] or undetectable in male cultured hippocampus. These concentrations are qualitatively similar to those observed in the current study. Compared to male, physiological role of androgens in female hippocampus is not clear. One of the possible physiological roles might be suppression of depression, since supplementation of androgens in female decreased a depression-like behavior of aged mice [7]. Extensive studies on physiological roles of female androgen should be needed.