Preg L

Brain Plasma

0.24 ± 0.33 0.06 ± 0.06

1.70 ± 0.32 0.20 ± 0.08

0.41 ± 0.13 9.5 ± 2.7 15.1 ± 1.8 0.18 ± 0.05 0.9 ± 0.5 2.5 ± 0.7

10.5 ± 0.4 2.9 ± 1.5

Rats of the Sprague Dawley Ofa strain (Iffa-Credo, L'Arbresles, France) were killed by decapitation when they were approx 11 wk old (200-220 g body weight), 2-3 h after lights were on. The entire brain was quickly removed, weighed, and immediately processed. Trunk blood was collected in heparinized tubes and centrifuged at 4°C. Results are expressed in terms of ng steroid either unconjugated or released from the sulfate or lipoidal esters. [Data from Young et al. (Z8)]

Rats of the Sprague Dawley Ofa strain (Iffa-Credo, L'Arbresles, France) were killed by decapitation when they were approx 11 wk old (200-220 g body weight), 2-3 h after lights were on. The entire brain was quickly removed, weighed, and immediately processed. Trunk blood was collected in heparinized tubes and centrifuged at 4°C. Results are expressed in terms of ng steroid either unconjugated or released from the sulfate or lipoidal esters. [Data from Young et al. (Z8)]

(9-11). The extracts containing unconjugated steroids were prepared by a differential extraction procedure. The water phase containing steroid sulfates was solvolyzed. The organic phase was taken to dryness and defatted by solvent partition. The 90% methanol phases containing unconjugated steroids were further purified by reverse-phase chromatography on C18 microcolumns. The isooctane phases containing lipoidal derivatives were taken to dryness, saponified, and the steroids released were further purified as were those in the unconjugated fractions.

The steroids recovered from the unconjugated, solvolyzed, and saponified fractions were separated by partition chromatography on a celite microcolumn, thus allowing the separation and radioimmunoassay of PROG, 5a DH-PROG, 3a,5a-TH PROG, dehydroepiandrosterone (DHEA), pregnenolone (PREG), and corticosterone (B) (12). Definitive identification of the steroid moiety was made by gas chromatography/ mass spectrometry.

The quantitation of brain neurosteroids appeared to depend on several environmental and methodological factors. Some factors have been partly documented in rodents, as being lighting schedules, hour of killing, housing conditions (number of animals per cage, rats of the other sex in the same room), and stress. The workup conditions for the removal and initial processing of brain tissue was also critical. Therefore, all physiological and pharmacological experiments were conducted under strictly defined conditions.

The concentration of DHEA (mainly in the form of the sulfate ester) is about 2.5 ng (10/pmol)/g tissue, whereas the concentration of PREG (both unconjugated and esteri-fied) is about 35 ng (100 pmol)/g tissue (see Table 1). Although we found differences in neurosteroid concentrations between selected brains areas, the lack of sensitivity of radioimmunoassays precluded any definitive statement.

Concentrations of both neurosteroids were definitely larger in the rat brain than in plasma, and even, in the case of DHEA S, larger in the posterior brain than in the adrenal glands. This led us to assume that there are independent mechanisms for the formation and/or accumulation of brain 3^-hydroxy-A5-steroids (11). Neither DHEA nor PREG disappeared in the brain 15-45 d after combined adrenalectomy (ADX) and orchiectomy (ORX) (see Fig.1). Brain DHEA S was also unchanged after administration of corticotropin (ACTH) for 3 d.

Further work has described additional neurosteroids. PROG concentrations were measured in several brain areas of immature female rats before and after induction of ovulation with pregnant mare serum gonadotropin (14). The highest preovulatory PROG

CONTROL SHAM ORX/ADX

CONTROL SHAM ORX/ADX

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