Expression of neural 5 alpha-reductase messenger ribonucleic acid: comparison to 5 alpha-reductase activity during prenatal development in the rat.

The present study was designed to characterize the developmental pattern of 5 alpha-reductase messenger RNA (mRNA) levels and enzyme activity in intact medial basal hypothalamic (MBH) and preoptic area (POA) tissue fragments of rats during fetal development. 5 alpha-Reductase activity was determined using [3H]testosterone as the substrate and quantifying, by TLC, the radiolabeled 5 alpha-reduced metabolites (dihydrotestosterone [DHT] and 5 alpha-androstane-3 alpha,17 beta-diol). Confirmation of the identity of the [3H]5 alpha-DHT formed was demonstrated by recrystallization of the derivatized DHT to constant specific activity. Under saturating substrate conditions (1.7 microM testosterone), there were no significant sex differences (male vs. female) detected in neural 5 alpha-reductase during prenatal, perinatal, or neonatal development. 5 alpha-Reductase activity was low but detectable at gestational day (GD) 15 (approximately 10 pmol/h.mg protein), increased over 3-fold to peak levels at GD 18, and then declined to moderate levels at GD 22 (approximately 22 pmol/h.mg protein) and 4 days after birth (approximately 17 pmol/h.mg protein). In addition, the developmental pattern of 5 alpha-reductase mRNA expression in fetal MBH-POA tissue was determined, by RNA blot hybridization, using a complementary DNA encoding the rat 5 alpha-reductase enzyme. A single mRNA species was detected at 2.5 kilobase in MBH-POA tissue, which is similar in size to adult rat liver and prostate 5 alpha-reductase mRNA. At GD 15, 5 alpha-reductase mRNA was clearly detectable; the abundance increased on GD 17, remained at maximal levels at GDs 18, 19, and 20, and then decreased to moderate levels on GD 22. A correlation calculated between the normalized 5 alpha-reductase mRNA levels and enzyme activities revealed a strong correspondence between mRNA abundance and enzyme activity values during prenatal development, r = 0.80, P = 0.056. These findings indicate that neural 5 alpha-reductase activity may be developmentally regulated, and that the changes in 5 alpha-reductase activity in MBH-POA tissue during late fetal development are regulated, in part, by the levels of mRNA encoding the 5 alpha-reductase enzyme.

Immunocytochemical distribution of aromatase cytochrome P450 in the rat brain using peptide-generated polyclonal antibodies.

Estrogen formation is catalyzed by the aromatase cytochrome P450 (P450AROM) enzyme. Aromatase activity has been detected in several regions in the rat brain. In the present study, we used peptide-generated polyclonal antibodies raised against a 20-amino acid synthetic fragment of the rat P450AROM protein (as deduced from the nucleic acid sequence of the rat P450AROM complementary DNA), to determine the location of this enzyme in rat brain sections. Immunoreactive antisera were titered by means of an enzyme-linked immunosorbent assay and purified by diethylaminoethyl-Affigel Blue chromatography. Specific immunoreactivity was confirmed by Western blot analysis using known aromatase-containing tissue (rat ovary homogenates and microsomal fractions). Evaluation of the distribution of P450AROM immunoreactivity in brain sections of male and female rats (30 and 60 days of age) was performed using the avidin biotin peroxidase immunocytochemical technique and light microscopy. P450AROM immunoreactivity appeared to be localized to neurons, and was present in brain regions and nuclei where enzymatic activity has been reported. For example, intense immunoreactivity was observed in the amygdaloid structures and supraoptic nucleus, whereas moderate to light immunoreactivity was evident in the paraventricular and arcuate nuclei and hippocampus. Surprisingly, neurons in the bed nucleus stria terminalis, medial basal hypothalamic, and preoptic areas displayed little aromatase immunoreactivity. However, P450AROM immunoreactivity was detected in specific brain regions not previously recognized to contain the enzyme (i.e. intense staining was seen in the reticular thalamic nucleus, olfactory tract and piriform cortex, as well as other brain structures). The pattern, distribution, and intensity of P450AROM immunoreactivity was similar regardless of sex or age. In this study, microsomal preparations derived from a new brain area (i.e. the reticular thalamic nucleus; Rt) displaying P450AROM immunoreactivity were observed to contain detectable levels of aromatase enzymatic activity, as determined by the 3H2O-release assay. The activity in the Rt was inhibited by a known aromatase inhibitor, 4-hydroxyandrostenedione. These results confirm histologically the localization of P450AROM to brain regions where aromatase enzymatic activity has been detected and extend the knowledge of its location to areas previously unknown as sites of aromatase activity, which may be involved in the modulation of neuroendocrine function and reproductive behavior.

Androgens regulate aromatase cytochrome P450 messenger ribonucleic acid in rat brain.

The conversion of androgens to estrogens by aromatase cytochrome P450 (P450AROM) is an important step in the mechanism of androgen action in the brain. In adult rats, P450AROM activity (AA) is regulated by androgens in the preoptic area and medial basal hypothalamus, but is constitutive in the amygdala. This study was undertaken to determine the distribution of P450AROM messenger RNA (mRNA) and AA in adult rat brain and examine the effects of steroid treatments on their concentrations in various brain regions. AA was determined by a sensitive assay that measures the production of 3H2O during the conversion of [1 beta-3H]androstenedione to estrone. P450AROM mRNA was measured by a ribonuclease protection assay using a RNA probe complementary to the 5'-coding region of rat P450AROM mRNA. The 32P-labeled P450AROM probe protected two mRNA fragments in brain tissues that expressed AA (preoptic area, medial basal hypothalamus, amygdala, and hippocampus). The larger protected RNA fragment was 430 nucleotides (nt) long and corresponded in size to the full-length protected complementary RNA, whereas the shorter protected RNA fragment was 300 nt long. Brain tissues that did not exhibit AA contained either the smaller protected RNA fragment (cingulate and parietal cortex) or no protected RNA (cerebellum). These results suggest that the 430-nt protected RNA fragment represents mRNA that encodes the functional P450AROM enzyme. In agreement with this conclusion, we found that immature rat ovaries that were stimulated with PMSG to synthesize estrogen contained only the 430-nt protected fragment. The levels of the 430-nt protected RNA fragment differed significantly between brain regions (amygdala > > preoptic area > medial basal hypothalamus > or = hippocampus) and were significantly correlated with AA (r = 0.994; P < 0.001). After castration, the concentrations of P450AROM mRNA and AA decreased significantly in the preoptic area and medial basal hypothalamus (P < 0.05), but not in the amygdala. Treatments with testosterone or dihydrotestosterone maintained P450AROM mRNA and AA at levels approximating those found in intact males. Although 17 beta-estradiol treatment increased AA in the preoptic area, it did not affect the P450AROM mRNA content. These results suggest that the increase in AA observed after exposure to androgens results from regulation of the transcription and/or stability of P450AROM mRNA. In contrast, estradiol appears to exert an effect on AA at the posttranscriptional level.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of burn trauma on adrenal and testicular steroid hormone production.

The effects of burn trauma in men on the production of adrenal and testicular steroids was investigated. Whereas there were significant increases in serum cortisol levels and urinary 17-hydroxycorticosteroid excretion soon after thermal injury, there were significant decreases in serum dehydroepiandrosterone sulfate, dehydroepiandrosterone, androstenedione, and testosterone concentrations during the first 4 weeks following burn trauma. Serum androstenediol and androstenediol sulfate levels also were reduced, though insignificantly, 10-23 days postburn. Serum LH levels were unchanged during the postburn interval. Since urinary 17-ketosteroid excretion was normal or below normal rather than increased, the decline in serum C19-steroid levels probably resulted from decreased glandular secretion rather than increased rates of metabolism and excretion. Low dehydroepiandrosterone sulfate and/or testosterone levels were found in some men several months after recovery from their burns. These data suggest that thermal injury leads to acute inhibition of adrenal and testicular C19-steroid secretion, but stimulation of adrenal glucocorticosteroid production, and that endocrine function in many instances is not normalized after complete healing of the burned surfaces. The mechanisms and physiological consequences of such changes in the steroid milieu of men after burn trauma are unknown.

Age-related changes in brain and pituitary 5 alpha-reductase with finasteride (Proscar) treatment.

Finasteride (Proscar), a 5 alpha-reductase inhibitor, was administered with or without 5 alpha-dihydrotestosterone (DHT) in juvenile (28), peripubertal (48), or young adult (68-day-old) male rats. Luteinizing hormone (LH) levels and ventral prostate (VP) weights monitored the efficacy of the treatments. For each postnatal day tested, LH hormone levels were not significantly altered by finasteride, suggesting that 5 alpha-reductase inhibition does not modulate LH in male rats using this protocol. Finasteride plus DHT treatment significantly decreased LH levels. Conversely, VP weights were significantly decreased in finasteride-treated animals while finasteride plus DHT treatment significantly increased VP values. Finasteride plus DHT treatment significantly decreased pituitary, but not brain, 5 alpha-reductase for each postnatal day tested. In finasteride-treated animals, hypothalamic and pituitary 5 alpha-reductase were significantly inhibited in young adult animals, whereas the activities were not altered in juvenile or peripubertal male rats. These data suggest that a different mechanism may regulate 5 alpha-reductase with the aging of the central nervous system and pituitary.

Calretinin and calbindin-D28K in male rats during postnatal development.

Calcium-binding proteins play potentially important roles in neurogenesis and neuroprotective mechanism(s). Some evidence exists that brain calbindin-D28K (CALB) is regulated by androgens. In the present study, calretinin (CALRET) and CALB patterns were determined by Western analysis in the medial basal hypothalamus (MBH) from male rats along with assaying plasma testosterone levels during postnatal development. Testosterone levels were very low in 7-, 10-, and 30-day-old animals (approximately 0.5 ng/mL), increased in a stair-step fashion to peak levels at 90 days (approximately 3.8 ng/mL), then declined with increasing age to very low levels at 300 days of age (approximately 0.3 ng/mL). At 7 and 10 days, MBH CALRET and CALB levels were low; however, at Day 30 a significant twofold increased was observed. Thereafter, in 60-, 120-, 180-, and 300-day-old animals MBH CALRET and CALB levels were, in general, comparable to 30-day-old values. These findings suggest that there is not a clear correspondence between the androgen status in male rats and the calcium-binding proteins (CALRET & CALB) expressed in the MBH. Therefore, it appears that brain CALRET and CALB are regulated in a developmental fashion with significant increases in expression occurring around the 4th postnatal week.

Rat adrenal 5 alpha-reductase mRNA content and enzyme activity are sex hormone dependent.

To investigate the effects of sex hormones on 5 alpha-reductase, we examined 5 alpha-reductase mRNA content and enzyme activity in the adrenal cortex of peripubertal male and female rats. In male rats, the influence of castration or hormone-replacement treatment with dihydrotestosterone (5 alpha-DHT) on 5 alpha-reductase was assessed. To stimulate ovarian sex hormone production in immature female rats, the effect of a single injection of 5 IU pregnant mare serum gonadotrophin (PMSG) on 5 alpha-reductase was examined. The efficacy of the treatments was demonstrated by measuring serum LH and ventral prostate weight in male rats, and serum oestradiol and ovarian weight in female rats. Growth hormone was also measured across all treatments in male and female rats. Adrenal 5 alpha-reductase mRNA levels were determined by RNA blot analysis utilizing a rat 5 alpha-reductase cDNA as probe. 5 alpha-Reductase enzyme activity was estimated by isolating [3H]5 alpha-DHT by thin-layer chromatography after incubation with [3H]testosterone. The identity of the [3H]5 alpha-DHT formed was demonstrated by recrystallization of the derivatized DHT to constant specific activity. In controls, adrenal cortical 5 alpha-reductase mRNA content was nearly four times higher in immature female rats compared with intact peripubertal males. Castration resulted in a sevenfold increase in adrenal 5 alpha-reductase mRNA content compared with that in intact controls, while in DHT-injected castrated animals the mRNA level was nearly undetectable. The content of adrenal 5 alpha-reductase mRNA in anoestrous rats was nearly four times higher than in PMSG-treated animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of mRNA species encoding steroidogenic enzymes in the rat ovary.

We have examined the levels of expression of mRNA species encoding cholesterol side-chain cleavage cytochrome P-450 (P-450scc), 17 alpha-hydroxylase cytochrome P-450 (P-450(17 alpha), aromatase cytochrome P-450 (P-450AROM) and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) in rat ovaries throughout the oestrous cycle, during pregnancy and in immature animals treated with pregnant mare serum gonadotrophin (PMSG). Total or poly(A)(+)-enriched RNA was prepared from adult rat ovaries throughout the oestrous cycle, from immature rat ovaries 24 and 48 h after treatment and from adult rat ovaries on days 10, 14, 17 and 21 of gestation. Expression of the mRNA species was examined by Northern analysis using specific [32P]cDNA probes. During the oestrous cycle P-450scc mRNA of approximately 1.9 kb was detected at low levels, while 3 beta-HSD mRNA of 1.7 kb was in relatively high abundance throughout the oestrous cycle. While P-450(17) alpha mRNA of 1.9 kb and P-450AROM of 2.7, 2.2 and 1.7 kb were highly abundant during dioestrus, pro-oestrus and oestrus, the levels of these mRNA species decreased markedly to be nearly undetectable during metoestrus. During pregnancy there was considerably more variation in the expression of the mRNA species examined. Expression of P-450scc mRNA was at low, but detectable, levels until day 14, thereafter expression increased to high levels (day 14-21 of gestation). Levels of P-450(17) alpha mRNA on day 10 of gestation were lower than at pro-oestrus during the oestrous cycle and decreased further on days 14 and 17. Expression of 3 beta-HSD was decreased on day 10, but on days 14, 17 and 21 of gestation high mRNA levels were detectable. Ovarian expression of the three P-450AROM species was dramatically increased between days 14 and 17 of pregnancy, but declined by day 21. In immature rats, P-450scc mRNA was detected at low levels in unstimulated animals and increased markedly after treatment with PMSG, while subsequent treatment with human chorionic gonadotrophin (hCG) had a minimal effect on expression. Expression of P-450(17) alpha mRNA was high in unstimulated immature and PMSG-treated rats, but diminished after treatment with hCG. All three P-450AROM mRNA species were undetectable in ovaries from unstimulated immature animals; however, induction of all three was observed in PMSG-treated rats, but this expression decreased to undetectable levels upon subsequent administration of hCG.(ABSTRACT TRUNCATED AT 400 WORDS)

Manipulation of prenatal hormones and dietary phytoestrogens during adulthood alter the sexually dimorphic expression of visual spatial memory.

BACKGROUND: In learning and memory tasks, requiring visual spatial memory (VSM), males exhibit higher performance levels compared to females (a difference attributed to sex steroid hormonal influences). Based upon the results from our companion investigation, this study examined the influence of prenatal sex steroid hormone manipulations on VSM in adulthood, as assessed in the radial arm maze. Additionally, the influence of dietary soy phytoestrogens (i.e., the presence of high or low estrogen-like compounds present in the animal's diet) on VSM was examined in combination with the prenatal hormonal manipulations. RESULTS: Radial arm maze performance on a phytoestrogen-rich diet: 1) females treated prenatally with testosterone were masculinized and acquired/performed in a manner similar to control or oil-treated males and 2) males treated prenatally with an androgen receptor blocker (flutamide) were feminized and acquired/performed in a fashion typical of control or flutamide-treated females. When a diet change was initiated in adulthood, control phytoestrogen-rich fed females outperformed control females switched to a phytoestrogen-free diet. Whereas, in control males the opposite diet effect was identified. Furthermore, flutamide-treated males fed a phytoestrogen-rich diet outperformed flutamide-treated males switched to a phytoestrogen-free diet. CONCLUSIONS: These results suggest that prenatal hormonal manipulations significantly sex-reverse the normal sexually dimorphic expression of VSM. Specifically, VSM was enhanced in females treated with testosterone and inhibited in males treated with flutamide. Finally, dietary soy phytoestrogens set a bias on learning and memory in these hormonally manipulated animals in a predictable manner and these data confirm and extend the findings in our companion paper.

Visual spatial memory is enhanced in female rats (but inhibited in males) by dietary soy phytoestrogens.

BACKGROUND: In learning and memory tasks, requiring visual spatial memory (VSM), males exhibit superior performance to females (a difference attributed to the hormonal influence of estrogen). This study examined the influence of phytoestrogens (estrogen-like plant compounds) on VSM, utilizing radial arm-maze methods to examine varying aspects of memory. Additionally, brain phytoestrogen, calbindin (CALB), and cyclooxygenase-2 (COX-2) levels were determined. RESULTS: Female rats receiving lifelong exposure to a high-phytoestrogen containing diet (Phyto-600) acquired the maze faster than females fed a phytoestrogen-free diet (Phyto-free); in males the opposite diet effect was identified. In a separate experiment, at 80 days-of-age, animals fed the Phyto-600 diet lifelong either remained on the Phyto-600 or were changed to the Phyto-free diet until 120 days-of-age. Following the diet change Phyto-600 females outperformed females switched to the Phyto-free diet, while in males the opposite diet effect was identified.Furthermore, males fed the Phyto-600 diet had significantly higher phytoestrogen concentrations in a number of brain regions (frontal cortex, amygdala & cerebellum); in frontal cortex, expression of CALB (a neuroprotective calcium-binding protein) decreased while COX-2 (an inducible inflammatory factor prevalent in Alzheimer's disease) increased. CONCLUSIONS: Results suggest that dietary phytoestrogens significantly sex-reversed the normal sexually dimorphic expression of VSM. Specifically, in tasks requiring the use of reference, but not working, memory, VSM was enhanced in females fed the Phyto-600 diet, whereas, in males VSM was inhibited by the same diet. These findings suggest that dietary soy derived phytoestrogens can influence learning and memory and alter the expression of proteins involved in neural protection and inflammation in rats.

Dietary soy-phytoestrogens decrease testosterone levels and prostate weight without altering LH, prostate 5alpha-reductase or testicular steroidogenic acute regulatory peptide levels in adult male Sprague-Dawley rats.

Nutritional factors, especially phytoestrogens, have been extensively studied for their potential beneficial effects against hormone-dependent and age-related diseases. The present study describes the short-term effects of dietary phytoestrogens on regulatory behaviors (food/water intake, locomotor activity and body weight), prostate weight, prostate 5alpha-reductase enzyme activity, reproductive hormone levels, and testicular steroidogenic acute regulatory peptide (StAR) levels in adult Sprague-Dawley rats. Animals were fed either a phytoestrogen-rich diet containing approximately 600 microg/g isoflavones (as determined by HPLC) or a phytoestrogen-free diet. After 5 weeks of consuming these diets, plasma phytoestrogen levels were 35 times higher in animals fed the phytoestrogen-rich vs phytoestrogen-free diets. Body and prostate weights were significantly decreased in animals fed the phytoestrogen-rich diet vs the phytoestrogen-free fed animals; however, no significant change in prostate 5alpha-reductase enzyme activity was observed between the treatment groups. Locomotor activity levels were higher in the phytoestrogen-rich vs the phytoestrogen-free animals during the course of the treatment interval. Plasma testosterone and androstenedione levels were significantly lower in the animals fed the phytoestrogen-rich diet compared with animals fed the phytoestrogen-free diet. However, there were no significant differences in plasma LH or estradiol levels between the diet groups. Testicular StAR levels were not significantly different between the phytoestrogen-rich vs the phytoestrogen-free fed animals. These results indicated that consumption of dietary phytoestrogens resulting in very high plasma isoflavone levels over a relatively short period can significantly alter body and prostate weight and plasma androgen hormone levels without affecting gonadotropin or testicular StAR levels. The findings of this study identify the biological actions of phytoestrogens on male reproductive endocrinology and provide insights into the protective effects these estrogen mimics exert in male reproductive disorders such as benign prostatic hyperplasia and prostate cancer.

Ovarian aromatase cytochrome P-450 mRNA levels correlate with enzyme activity and serum estradiol levels in anestrous, pregnant and lactating rats.

We examined the changes in P-450AROM mRNA, aromatase enzyme activity and serum estradiol levels (E2) in anestrous, pregnant mare's serum gonadotropin (PMSG)-treated immature, pregnant, and lactating rats to determine if: (a) the various mRNA species encoding P-450AROM in rat ovarian tissue are differentially expressed during different hormonal states, and (b) a positive relationship exists between P-450AROM mRNA and enzymatic activity in rat ovarian tissue and serum estradiol levels from the same animals. Utilizing three different cDNAs encoding rat P-450AROM, levels of P-450AROM mRNA were determined by RNA blot analysis and scanning densitometry. Probe 1, a 5' probe, detects all three P-450AROM mRNA species in rat ovarian tissue (i.e. at 1.7, 2.2 and 2.7 kb). Probe 2 contains an unspliced intronic sequence in place of the heme-binding domain at its 3' terminus and thus the mRNA detected by this probe must encode a nonfunctional aromatase protein. Only the two smaller (i.e. nonfunctional) mRNA species at 1.7 and 2.2 kb are detected by probe 2. Probe 3 contains the heme-binding region and hybridizes to principally the largest mRNA transcript at 2.7 kb (but hybridizes also to a 1.7 kb mRNA transcript). Aromatase enzyme activity was measured by using a saturating concentration of [1 beta-3H]testosterone as substrate in the [3H]water-release assay while serum estradiol levels were determined by radioimmunoassay. In immature rats (IR) or lactating animals (LA) P-450AROM mRNA was not detectable along with low serum estradiol (IR approximately 2.8 pg/ml; LA approximately 0.2 pg/ml) and aromatase activity levels (IR approximately 0.8 pmol/h per mg protein; LA less than 0.5 pmol/h per mg protein). Anestrous animals treated with 5 IU of PMSG resulted in a clear increase (24 h later) in P-450AROM mRNA levels, in concert with a 4-fold increase in serum E2 (approximately 12.5 pg/ml) and aromatase activity (approximately 4.2 pmol/h per mg protein). During pregnancy, all three mRNA species were clearly detectable, but low serum E2 levels (approximately 0.6 pmol/ml) and P-450AROM mRNA abundance were observed at 3 days of gestation (DG). Levels of all three P-450AROM mRNA species increased markedly at 15 and 18 DG; thereafter, the levels declined at 20 DG and further decreased at 22 DG. However, regardless of the probe utilized (probe 1, 2 or 3) in the RNA blot analyses, the mRNA transcripts detected by each probe were expressed in a concerted fashion with respect to abundance and pattern.(ABSTRACT TRUNCATED AT 400 WORDS)

The structure of cDNA clones encoding the aromatase P-450 isolated from a rat Leydig cell tumor line demonstrates differential processing of aromatase mRNA in rat ovary and a neoplastic cell line.

The conversion of androgens to estrogens is catalyzed by a complex of enzymes that includes a specific cytochrome P-450 aromatase (P-450arom). In this paper we describe the high level expression of aromatase activity in the rat Leydig cell tumor line, R2C. We also report the isolation of cDNA clones encoding the rat aromatase P-450arom from a cDNA library prepared from this cell line. Analysis of these cDNA clones predicts a protein sequence with a high degree of sequence conservation when compared to the chicken and human P-450arom enzymes. Notably, four of the cDNA clones were found to lack the last coding exon that contains the heme-binding domain, a structural feature essential for aromatase activity. These clones were found to contain instead a segment of genomic DNA derived from an unspliced intron. Northern analysis using a fragment of the coding region of the rat P-450arom cDNA as probe revealed that three species of P-450arom mRNa are expressed in rat ovary that are similar to those identified in RNA samples prepared from the rat R2C cell line. Analysis of the same samples of RNA using a probe derived from the 3' terminal intron segment of the rat aromatase cDNA clones that lack the heme-binding domain indicates that two of the species of aromatase mRNA transcripts present in both rat ovary and R2C cell lack the heme-binding domain and thus must encode a nonfunctional aromatase protein. These findings have important implications for the measurement of aromatase mRNA and appear to explain why three sizes of rat P-450arom mRNA exist on Northern analysis and why previous studies failed to demonstrate a clear relationship between aromatase mRNA, protein, and enzymatic activity in the rat ovary.

Dimorphic expression of medial basal hypothalamic-preoptic area calbindin-D(28K) mRNA during perinatal development and adult distribution of calbindin-D(28K) mRNA in Sprague-Dawley rats.

The calcium-binding protein, calbindin (CALB) is: (a) distributed throughout the central nervous system (CNS), (b) abundant in neurons and, (c) thought to act as a buffer by binding intracellular calcium, mediating neurogenesis (cell profileration) and neuronal programmed cell death. Using Northern analysis, CALB mRNA distribution was characterized in 12 different adult brain regions. Additionally, CALB mRNA levels were characterized in the medial basal hypothalamus (MBH) and preoptic area (POA) in perinatal male and female rats, in order to compare this mRNA pattern to the dimorphic MBH-POA CALB protein profile our laboratory previously reported. Three CALB mRNA species were detected (at 1.9, 2.8 and 3.2 kilobase pairs) in all CNS regions. The smallest mRNA transcript (at 1.9 kilobase pairs) was the most abundant of the three CALB mRNAs. To quantify these mRNA signals, CALB mRNA levels were normalized to 18s ribosomal RNA bands. Among the various adult brain sites assayed, the cerebellum expressed the highest CALB mRNA signals. High CALB mRNAs were observed in the MBH-POA, olfactory bulb and hippocampal regions. Moderate CALB mRNA levels were seen in the striatum and frontal cortex while moderate to low CALB mRNA levels were observed in the posterior cortex, entorhinal cortex, midbrain, pons, thalamus and medulla. During perinatal development, MBH-POA CALB mRNA levels were lowest at gestation day (GD) 18, increased four-fold in newborns and remained at moderate levels during early postnatal development. Male CALB mRNA levels were notably greater than female values at GD 18 and in newborns. Whereas, at PND 2, the CALB mRNA levels were approximately equivalent in males and females. These findings suggest that in the adult CNS CALB mRNAs vary among different brain regions. The present male vs. female MBH-POA CALB mRNA levels confirm previously reported dimorphic protein patterns of MBH-POA CALB during perinatal development. Thus, the genesis of sexually dimorphic structures may be influenced by the dimorphic CALB expression in the MBH-POA region.

Brain aromatase cytochrome P-450 messenger RNA levels and enzyme activity during prenatal and perinatal development in the rat.

Aromatase cytochrome P-450 (P-450AROM) enzyme activity catalyzes the conversion of androgens to estrogens in specific brain areas. During development local estrogen formation is thought to influence the sexual differentiation of neural structures (i.e. increase neurite growth and establish neural circuitry) and modulate reproductive functions. This study was undertaken to investigate the ontogeny of the (P-450AROM) enzyme and its messenger RNA (mRNA) in medial basal hypothalamic (MBH) and preoptic area (POA) tissue during late fetal and perinatal development of the rat. Aromatase activity in the MBH-POA was negligible before gestational day (GD) 16 (< 0.1 pmol/h/mg protein), increased over 10-fold at GD 17 and continued to increase (over 5-fold) to peak levels at GD 19 (> 5.0 pmol/h/mg protein), and then declined to low levels at GD 22 and 2 days post-birth (approximately 1 pmol/h/mg protein). The profile of P-450AROM mRNA in the MBH-POA tissue was characterized by a predominant 2.7 kilobase (kb) mRNA species, similar in size to the largest functional P-450AROM mRNA observed in adult rat ovarian tissue. At GD 15, the P-450AROM mRNA was undetectable; low but detectable levels were seen at GD 17, the abundance increased at later time points and remained at peak levels on GDs 18 through 20, decreased slightly by GD 22, and then declined further by 2 days post-birth. The developmental increase in P-450AROM mRNA levels correlated with the ascending pattern of enzyme activity before GD 19, but the marked decrease in enzyme activity seen after GD 19 was not accompanied by a corresponding decline in mRNA levels.(ABSTRACT TRUNCATED AT 250 WORDS)

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 (3) H(2) O, utilizing either [1ß-(3) H]androstene-dione or [1ß-(3) H]testosterone as substrate. Maximal rates of activity were obtained at a saturating concentration of 0.3 µM [1ß-(3) H]testosterone. Confirmation of the identity of the [(3) H]estradiol formed was demonstrated by recrystallization of the derivatized estradiol to constant specific activity following incubation with [1,2,6,7-(3) H]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 Ca(2+) 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. These results suggest that: 1) the appearance of aromatase activity in the rat hypothalamus before GD17 requires the unfolding of extrahypothalamic events, 2) the increase in aromatase activity that occurs before GD19 also requires extrahypothalamic inputs and does not involve any of the known intracellular signal transduction pathways, and 3) the decline in activity observed after GD19 is regulated within the hypothalamus, and appears to be determined, at least in part, by the activation of cyclic AMP formation. A potential role for androgens is discussed.

Molecular aspects of brain aromatase cytochrome P450.

Aromatase cytochrome P450 (P450arom) enzyme activity catalyses the conversion of androgens to estrogens in specific brain areas. During central nervous system (CNS) development local estrogen formation influences sexual differentiation of neural structures, regulates neuroendocrine functions and sexual behavior. A proposed mechanism (and re-examination) of the sexual differentiation of the rodent brain is presented. The metabolic pathway of androgen metabolism by P450arom was characterized in the medial basal hypothalamic (MBH) tissue from male rats during various prenatal and postnatal developmental intervals. The P450arom enzyme activity was determined using a saturating concentration of [3H]testosterone as the substrate, and the rates were quantified by scintillation counting. The MBH P450arom activity was highest during prenatal development (i.e. 3-6 pmol/h/mg protein), declined to moderate levels in newborns and infantile animals (approximately 1 pmol/h/mg protein) and then continued to decline to low activity rates in adult animals (approximately 80 fmol/h/mg protein). Regulation of the P450arom gene was characterized by a series of molecular biology studies where the controlling mechanism for brain P450arom was determined in MBH and amygdaloid tissue sites. Evidence for brain P450arom-specific mRNA in perinatal rats is presented as well as comparisons with rat ovary, a rat Leydig tumor cell line (R2C) and human fetal brain P450arom. Specifically, P450arom gene expression is driven in perinatal rat brain tissue by a different promoter compared to rat ovarian tissue or a R2C cell line, whereas human fetal brain tissue utilizes an almost identical promoter segment to that observed in the rodent. These findings provide an insight into the regulation of brain P450arom gene expression and suggest that there is an additional level of control for the expression of this gene during perinatal development. However, further study is necessary to understand the molecular basis of this complex developmental pattern of brain P450arom expression.

Expression of aromatase cytochrome P450 in rat H540 Leydig tumor cells.

The rat H540 Leydig cell tumor has been shown to express cholesterol side-chain cleavage and 17alpha-hydroxylase cytochrome P450s, 3beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase, and steroid 5alpha-reductase, making it a useful model in which to study steroidogenesis. In the current studies, we report that cultured H540 cells express high levels of aromatase cytochrome P450 (P450arom), which converts androgens to estrogens. Levels of aromatase activity varied from 9.4 to 51.7 pmol/h/mg protein and inhibition of 5alpha-reductase with finasteride did not significantly effect aromatase measurements, indicating that 5alpha-reductase is not competing for the substrate used in the aromatase assays. Aromatase activity was decreased 95% by preincubating the cells with 4-hydroxyandrostenedione, an aromatase inhibitor. Characterization of the aromatase mRNA expressed in the H540 cell line demonstrates that, like R2C cells and rat ovarian tissue, three distinct P450arom mRNA species are detected by Northern analysis, and that these transcripts are derived from the same site of transcription initiation. Despite these similarities, the regulation of aromatase activity by 8-bromo-cAMP in H540 cells differs from both R2C cells and rat ovarian tissue. As the H540 and R2C cell lines appear to have distinct origins, H540 is the second rat Leydig tumor cell line characterized that constitutively expresses high levels of aromatase.

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

In the present study, we examined the changes in enzyme activity and mRNA levels of aromatase cytochrome P450 (P450AROM) and 5 alpha-reductase in ovarian tissue from adult cyclic rats. For each stage of the estrous cycle, the enzymatic activities were quantified by means of the 3H2O-release assay in the case of P450AROM and thin-layer chromatography in the case of 5 alpha-reductase. Levels of mRNA encoding P450AROM and 5 alpha-reductase in the ovary were determined by Northern blot analysis utilizing 32P-labeled rat cDNAs as probes. Serum LH levels were determined by RIA. Three P450AROM mRNA species were detected (at 1.7, 2.2 and 2.7 kb) in ovarian tissue from cyclic rats. All three P450AROM transcripts were expressed in a co-ordinated fashion throughout the cycle. The P450AROM levels were highest during diestrus and proestrus, decreased during estrus while at metestrus the levels were nearly nondetectable. Conversely, one 5 alpha-reductase mRNA species at 2.5 kb was detected in ovarian tissue from cyclic animals. Ovarian 5 alpha-reductase mRNA levels were lowest during diestrus and proestrus, increased at estrus and were most abundant during metestrus; a pattern opposite to that of P450AROM. The pattern of change in P450AROM and 5 alpha-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 (approximately 5.5 pmol/h/mg protein), decreased at estrus and declined to the lowest values at metestrus (approximately 1.0 pmol/h/mg protein). In contrast, the 5 alpha-reductase activity pattern was essentially the mirror image of the P450AROM activity pattern during the estrous cycle. 5 alpha-Reductase levels were lowest during proestrus (approximately 5 pmol/h/mg protein) and estrus (approximately 8 pmol/h/mg protein), increased over 3-fold during metestrus, while the highest activity levels occurred during diestrus (approximately 36 pmol/h/mg protein). The normalization of the P450AROM and 5 alpha-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 that: (a) a temporal relationship exists between the profiles of the enzymatic activities that follows the changes in the levels of their respective mRNAs and (b) an inverse pattern exists between P450AROM and 5 alpha-reductase in terms of both enzymatic activity and mRNA expression during the estrous cycle in rat.

Estrogens and phytoestrogens: brain plasticity of sexually dimorphic brain volumes.

Sexually dimorphic brain volumes (sexually dimorphic nucleus of the preoptic area (SDN-POA) and anteroventral periventricular (AVPV) nucleus) are influenced by estrogens. Phytoestrogens, derived from plants (especially soy products), are molecules structurally and functionally similar to estradiol. The purpose of this study was to examine: the consumption of phytoestrogen (using a phytoestrogen-rich (Phyto-600) versus a phytoestrogen-free (Phyto-free)) diets from conception to adulthood (or changing the diets during adulthood) and characterizing (a) circulating plasma phytoestrogen levels, (b) testosterone levels in males, (c) sexually dimorphic brain volumes (i.e. the SDN-POA and AVPV) and (d) the presence of apoptotic cells in these brain structures in Long-Evans rats. Phyto-600 fed animals displayed total serum phytoestrogens levels 37-fold higher compared to Phyto-free values. Circulating testosterone levels were not significantly altered by the diets. Female SDN-POA volumes were not altered by the diets. Whereas, males fed a Phyto-free diet displayed decreased SDN-POA volumes compared to male Phyto-600 values. Females fed the Phyto-600 diet displayed larger AVPV volumes compared to males on the same diet or females on the Phyto-free diet. Males fed the Phyto-free diet had the largest AVPV values compared to Phyto-600 fed males. When the SDN-POA region was examined in lifelong Phyto-free fed males, apoptotic cells were present versus males fed the Phyto-600 diet and in the AVPV region the opposite results were obtained. In summary, consumption of dietary phytoestrogens (estrogen mimics) can alter hormone-sensitive hypothalamic brain volumes in rodents during adulthood.