Changes in 5α-Pregnane Steroids and Neurosteroidogenic Enzyme Expression in the Perinatal Sheep

Abstract

Pregnane steroids have sedative and neuroprotective effects on the brain as a result of interactions with the steroid-binding site of the GABA A receptor. To determine whether the fetal brain is able to synthesize pregnane steroids de novo from cholesterol, we measured the expression of cytochrome P450 side-chain cleavage (P450scc) and 5␣-reductase type II (5␣RII) enzymes in fetal sheep from 72 to 144 d gestation (term~147 d) and in newborn lambs at 3 and 19-26 d of age. Both P450scc and 5␣RII expression was detectable by 90 d gestation in the major regions of the brain and also in the adrenal glands. Expression increased with advancing gestation and was either maintained at fetal levels or increased further after birth. In contrast, the relatively high content (200-400 pmol/g) of allopregnanolone (5␣-pregnan-3␣-ol-20-one), a major sedative 5␣-pregnane steroid, present throughout the brain from 90 d gestation to term, was reduced significantly (Ͻ50 pmol/g) immediately after birth. These results suggest that although the perinatal brain has the enzymes potentially to synthesize pregnane steroids de novo from cholesterol, either the placenta is a major source of these steroids to the brain or other factors associated with intrauterine life may be responsible for high levels of allopregnanolone production in the fetal brain until birth. Abbreviations AP, allopregnanolone, 5␣-pregnan-3␣-ol-20-one P450scc, P450 side chain cleavage enzyme 5␣RII, 5␣-reductase type II enzyme PMC, primary motor cortex GABA A ,␥-aminobutyric acid/benzodiazepine receptor 5␣-DHP, 5␣-dihydroprogesterone Neuroactive steroids such as allopregnanolone (AP) are potent neuromodulators that modify the excitability of the CNS by interaction with the ␥-aminobutyric acid/benzodiazepine receptor-chloride ionophore (GABA A receptor). In the adult AP is a positive allosteric modulator of the GABA A receptor with potent anxiolytic (1, 2), anticonvulsant (3, 4), sedative/ hypnotic, and anesthetic effects (5) on behavior. Prenatally, neuroactive steroids have been shown to suppress fetal activity in late gestation and seem to have a role in maintaining the low level of arousal-like behavior that typifies fetal life (6, 7). This interaction between AP and the GABA A receptor complex is responsible for the major pharmacologic actions of AP and is distinct from the genomic effects exerted by other steroids such as progesterone (8). In the brain, neuroactive steroids such as AP are synthesized de novo from cholesterol, but a proportion of the pool of steroids may be derived from precursors in blood that enter the brain across the blood-brain barrier (9 -11). Thus, peripheral steroidogenesis may influence neuroactive steroid content in the brain.The cytochrome P450 side-chain cleavage enzyme (P450scc) catalyzes the irreversible conversion of cholesterol to pregnenolone on the inner side of the mitochondrial membrane (12). The primary site of pregnenolone synthesis in the brain seems to be in oligodendrocytes and astrocytes, with significantly less synthesis occ...

Hypothalamic pituitary adrenal axis

“…The antiserum displays low cross-reactivity with related steroids and the charactisation of the antiserum has been reported previously (Bernardi et al 1998, Nguyen et al 2003a. The limit of detection for allopregnanolone was 0·19 0·03 pmol/tube (n=4) and the intra-and interassay coefficients of variance were 5% and 10%, respectively.…”

“…We have previously found that allopregnanolone concentrations are remarkably high in the ovine fetal brain from 0·6 of gestation (90 days, term 148 days), and that this neurosteroid has the effect of suppressing activity of the fetal CNS (Crossley et al 2000, Nguyen et al 2003a. We have also shown that acute fetal asphyxia increased the content of allopregnanolone in brain and adrenal tissues and in the plasma (Nguyen et al 2003b).…”

“…Aliquots of 15 µg protein were separated by SDS-PAGE on 15% separating gels and transferred onto 0·2 µM polyvinylidene difluoride membranes (Osmonics, Westborough, MA, USA) by electroblotting. Membranes were incubated with a 1:3000 dilution of either the P450 scc or 5 RII antibody in Tris-buffered saline Tween, for 1 h at 22 C. The specificity of the P450 scc and 5 RII antibodies has been reported previously (Nguyen et al 2003a). The immune complexes were visualised by chemiluminescence and immunoblots were scanned and analysed using ImageQuaNT software (Amersham, Bucks, England).…”

“…Neurosteroids were extracted from brain or adrenal tissue and plasma as described previously (Barbaccia et al 1992, Nguyen et al 2003a). Briefly, brain or adrenal gland tissue was homogenised three times with 50% methanol containing 1% acetic acid and the supernatants pooled.…”

“…Allopregnanolone was measured by specific radioimmunoassay, as described previously (Billiards et al 2002, Nguyen et al 2003a) using a polyclonal antibody purchased from Dr R H Purdy (San Diego, CA, USA). The antiserum displays low cross-reactivity with related steroids and the charactisation of the antiserum has been reported previously (Bernardi et al 1998, Nguyen et al 2003a.…”