Edwin D. Lephart

Effects of Cyclic AMP and Andre-gens on in vitro Brain Aromatase Enzyme Activity During Prenatal Development in the Rat

In the rat, there is a marked but transient increase in hypothalamic aromatase activity during the last week of fetal life. The present study was undertaken to gain insight into the regulation of this developmental pattern. Hypothalamic fragments comprising the medial basal hypothalamus and the suprachiasmatic region (henceforth referred to as preoptic area) were explanted and cultured in serum‐free medium for 2 to 5 days. Aromatase activity was measured by the formation of 3H2O, utilizing either [1ß‐3H]androstene‐dione or [1ß‐3H]testosterone as substrate. Maximal rates of activity were obtained at a saturating concentration of 0.3 μM [1ß‐3H]testosterone. Confirmation of the identity of the [3H]estradiol formed was demonstrated by recrystallization of the derivatized estradiol to constant specific activity following incubation with [1,2,6,7‐3H]testosterone. In agreement with previous reports, in vivo hypothalamic aromatase activity was negligible before gestational day (GD) 16, increased strikingly by GD19 (>5.0 pmol/h/mg protein) and decreased, thereafter, to low levels at GD22 (∼1.0 pmol/h/mg protein). Medial basal hypothalamus‐preoptic area fragments explanted before GD17 failed to develop aromatase activity in vitro. If the tissue was explanted on GD17 or 18 (i.e. when the in vivo rate of activity was increasing), the enzyme activity did not continue to increase, but it was rather maintained for 2 days before decreasing in a manner that closely mimicked the decline observed in vivo. A similar, butimmediate decline was observed when the tissue was explanted on GD19 (i.e. at the time when theactivity peaks in vivo). Exposure of explants to either growth factors (insulin‐like growth factor II, epidermal growth factor, and basic or acidic fibroblast growth factor), or steroids (estradiol‐17ß, progesterone, testosterone, dihydrotestosterone and corticosterone) failed to either increase aromatase activity before the peak at GD19 or ameliorate its perinatal decline. Increase of Ca2+ fluxes with the ionophore A23187 or activation of the cyclic AMP, cyclic GMP, or protein kinase C pathways were similarly ineffective, as was angiotensin II, a recently proposed stimulator of neural aromatase. In contrast, aromatase activity was suppressed 2‐ to 4‐fold by activation of the cyclic AMP pathway (with either forskolin or 8‐bromo‐cyclic AMP) or by the androgens, testosterone and dihydrotestosterone.

Characterization of aromatase cytochrome P-450 mRNA in rat perinatal brain, ovary and a Leydig tumor cell line: evidence for the existence of brain specific aromatase transcripts

The conversion of androgens to estrogens is catalyzed by the aromatase cytochrome P-450 (P-450AROM) in a veriety of tissues and cell types in vertebrates. The manner in which aromatase activity is regulated appears to be quite different, even between different tissues of a single species. In the current study, we have determined the sequence of the 5′ end of the aromatase mRNA in a rat Leydig tumor cell line, R2C, and in three rat tissue [adult ovary, perinatal amygdala (AmY), and medial basal hypothalamus-preoptic area (MBH-POA)], each of which shows different patterns of aromatase expression. S1 nuclease protection and primer-extension analyses establish that the site of transcription initiation of the aromatase mRNA present in rat ovarh and the rat Leydig tumor cell line R2C is located approximately 97 nucleotides upstream from the initiator methionine. By contrast, although aromatase mRNA was detected in S1 nuclease protection experiments using a probe derived from the aromatase open-reading frame, transcripts initiating at this site were absent from RNA samples prepared from perinatal rat AMY and MBH-POA tissue. S1 mapping and sequencing of the 5′ end of AMY and MBH-POA aromatase cDNAs indicate that the aromatase mRNA transcripts present in these rat neural tissues from perinatal animals contain a distinctive 5′ terminus and are derived from a different promoter.

Inverse relationship between ovarian aromatase cytochrome P450 and 5α-reductase enzyme activities and mRNA levels during the estrous cycle in the rat

Abstract In the present study, we examined the changes in enzyme activity and mRNA levels of aromatase cytochrome P 450 ( P 450 AROM ) and 5α-reductase in ovarian tissue from adult cyclic rats. For each stage of the estrous cycle, the enzymatic activities were quantified by means of the 3 H 2 O-release assay in the case of P 450 AROM and thin-layer chromatography in the case of 5α-reductase. Levels of mRNA encoding P 450 AROM and 5α-reductase in the ovary were determined by the Northern blot analysis utilizing 32 P-labeled rat cDNAs as probes. Serum LH levels were determined by RIA. Three P 450 AROM mRNA species were detected (at 1.7, 2.2 and 2.7 kb) in ovarian tissue from cyclic rats. All three P 450 AROM levels were highest during in a co-ordinated fashion throughout the cycle. The P 450 AROM levels were highest during diestrus and proestrus, decreased during estrus while at metestrus the levels were nearly nondetectable. Conversely, one 5α-reductase mRNA species at 2.5 kb was detected in ovarian tissue from cyclic animals. Ovarian 5α-reductase mRNA levels were lowest during diestrus and proestrus, increased at estrus and were most abundant during metestrus; a pattern opposite to that of P 450 AROM . The pattern of change in P 450 AROM and 5α-reductase activities paralleled that of the respective mRNA profiles but lagged behind the mRNA profiles by about 24 h, or one stage of the estrous cycle. Aromatase activity was 1.5 pmol/h/mg protein during diestrus, increased over 3-fold at proestrus (≈5.5 pmol/h/mg protein), decreased at estrus and declined to the lowest values at metestrus (≈1.0 pmol/h/mg protein). In contrast, the 5α-reductase activity pattern was essentially the mirror image of the P 450 AROM activity pattern during the estrous cycle. 5α-Reductase levels were lowest during proestrus (≈5 pmol/h/mg protein) and estrus (≈8 pmol/h/mg protein), increased over 3-fold during metestrus, while the highest activity levels occurred during diestrus (≈36 pmol/h/mg protein). The normalization of the P 450 AROM and 5α-reductase mRNA levels and their respective enzyme activities revealed a correspondence between mRNA abundance and subsequent increases (24 h later) in enzyme activity levels during the estrous cycle. These findings suggest follows the changes in the levels of their respective mRNAs and (b) an inverse pattern exists between P 450 AROM and 5α-reductase in terms of both enzymatic activity and mRNA expression during the estrous cycle in rat.

Pituitary and Brain 5α-Reductase Messenger RNA Levels in Control, Castrated, and Dihydrotestosterone-Treated Rats

In (28- to 40-day-old) male rats, the influence of castration or hormone-replacement treatment with dihydrotestosterone (5alpha-DHT) upon pituitary or hypothalamic 5alpha-reductase was assessed. The efficacy of the treatments was demonstrated by measuring serum LH and ventral prostate weight. Pituitary 5alpha-reductase enzyme activity was estimated by isolating [5alpha-(3)H]DHT by thin-layer chromatography after incubations with [(3)H]testosterone, and the identity of the [5alpha-(3)H]DHT formed was confirmed by recrystallization experiments. Pituitary and hypothalamic 5alpha-reductase mRNA content was determined by RNA blot analysis utilizing a rat 5alpha-reductase (type 1 or type 2) cDNA as probe. Pituitary 5alpha-reductase activity was significantly increased as a function of days after castration, whereas castration plus DHT treatment (at Day 12 post-castration) significantly decreased the activity to less than one-third of control levels. In controls, pituitary 5alpha-reductase mRNA content was barely detectable using the rat 5alpha-reductase (type 1) cDNA as probe. However, castration resulted in a clear increase in mRNA abundance, while in DHT-injected castrated animals pituitary mRNA content was undetectable. Hypothalamic mRNA content was clearly detectable in all treatment groups with the rat 5alpha-reductase type 1 probe. However, there were no apparent changes in hypothalamic mRNA abundance as affected by the treatments. On the other hand, pituitary or hypothalamic 5alpha-reductase mRNA was undetectable using the rat 5alphareductase (type 2) cDNA as probe. These results indicate that in the rat pituitary the content of mRNA encoding 5alpha-reductase is negatively regulated by DHT. The increase in pituitary 5alpha-reductase enzymatic activity in castrates is due, in part, to higher mRNA levels detected by the 5alpha-reductase type 1 probe. Hypothalamic 5alpha-reductase mRNA levels (type 1) encoding this protein are not regulated by DHT, but presumably are, to some extent, under transcriptional control.