Gonadotropin-releasing hormone stimulates the biosynthesis of pregnenolone sulfate and dehydroepiandrosterone sulfate in the hypothalamus.

The sulfated neurosteroids pregnenolone sulfate (Δ(5)PS) and dehydroepiandrosterone sulfate (DHEAS) are known to play a role in the control of reproductive behavior. In the frog Pelophylax ridibundus, the enzyme hydroxysteroid sulfotransferase (HST), responsible for the biosynthesis of Δ(5)PS and DHEAS, is expressed in the magnocellular nucleus and the anterior preoptic area, two hypothalamic regions that are richly innervated by GnRH1-containing fibers. This observation suggests that GnRH1 may regulate the formation of sulfated neurosteroids to control sexual activity. Double labeling of frog brain slices with HST and GnRH1 antibodies revealed that GnRH1-immunoreactive fibers are located in close vicinity of HST-positive neurons. The cDNAs encoding 3 GnRH receptors (designated riGnRHR-1, -2, and -3) were cloned from the frog brain. RT-PCR analyses revealed that riGnRHR-1 is strongly expressed in the hypothalamus and the pituitary whereas riGnRHR-2 and -3 are primarily expressed in the brain. In situ hybridization histochemistry indicated that GnRHR-1 and GnRHR-3 mRNAs are particularly abundant in preoptic area and magnocellular nucleus whereas the concentration of GnRHR-2 mRNA in these 2 nuclei is much lower. Pulse-chase experiments using tritiated Δ(5)P and DHEA as steroid precursors, and 3'-phosphoadenosine 5'-phosphosulfate as a sulfonate moiety donor, showed that GnRH1 stimulates, in a dose-dependent manner, the biosynthesis of Δ(5)PS and DHEAS in frog diencephalic explants. Because Δ(5)PS and DHEAS, like GnRH, stimulate sexual activity, our data strongly suggest that some of the behavioral effects of GnRH could be mediated via the modulation of sulfated neurosteroid production.

Structure-activity relationships of a series of analogs of the endozepine octadecaneuropeptide (ODN(11)(-)(18)) on neurosteroid biosynthesis by hypothalamic explants.

We have previously shown that the endozepine octadecaneuropeptide (ODN) stimulates the biosynthesis of neurosteroids from frog hypothalamic explants. In the present study, we have investigated the structure-activity relationships of a series of analogs of the C-terminal octapeptide of ODN (OP) on neurosteroid formation. We found that OP and its cyclic analog cyclo1-8OP stimulate in a concentration-dependent manner the synthesis of various steroids including 17-hydroxypregnenolone, progesterone, 17-hydroxyprogesterone and dehydroepiandrosterone. Deletion or Ala-substitution of the Arg1 or Pro2 residues of OP did not affect the activity of the peptide. In contrast, deletion or replacement of any of the amino acids of the C-terminal hexapeptide fragment totally abolished the effect of OP on neurosteroid biosynthesis. The present study indicates that the C-terminal hexapeptide of ODN/OP is the minimal sequence retaining full biological activity on steroid-producing neurons.

Expression and localization of estrogenic type 12 17beta-hydroxysteroid dehydrogenase in the cynomolgus monkey.

BACKGROUND: We have recently discovered that human type 12 17beta-HSD (h17beta-HSD12), a homolog of type 3 17beta-HSD, is a new estrogen-specific 17beta-hydroxysteroid dehydrogenase involved in the production of estradiol (E2). To further characterize this estradiol-producing enzyme, we have isolated the corresponding cDNA in the cynomolgus monkey (Macaca fascicularis), characterized its enzymatic activities and performed cellular localization using in situ hybridization. RESULTS: Using HEK-293 cells stably expressing Macaca fascicularis type 12 17beta-HSD (mf17beta-HSD12), we have found that the mf17beta-HSD12 catalyzes efficiently and selectively the transformation of El into E2, in analogy with the h17beta-HSD12. We have also quantified the mf17beta-HSD12 mRNA expression levels in a series of Macaca fascicularis tissues using Quantitative RealTime PCR. The Macaca fascicularis 17beta-HSD12 mRNA is widely expressed with the highest levels tissues found in the cerebellum, spleen and adrenal with moderate level observed in all the other examined, namely the testis, ovary, cerebral cortex, liver, heart, prostate, mammary gland, myometrium, endometrium, skin, muscle and pancreas. To gain knowledge about the cellular localization of the mf17beta-HSD12 mRNA expression, we performed in situ hybridization using a 35S-labeled cRNA probe. Strong labeling was observed in epithelial cells and stromal cells of the mammary gland. In the uterus, the labeling is detected in epithelial cells and stromal cells of the endometrium. CONCLUSION: These results strongly suggest that the Macaca fascicularis 17beta-HSD12 is an essential partner of aromatase in the biosynthesis of estradiol (E2). It strongly suggests that in the estradiol biosynthesis pathway, the step of 17-ketoreduction comes after the step of the aromatization (the aromatization of 4-androstendione to estrone followed by the conversion of estrone into estradiol by estrogen specific l7beta-HSDs) which is in contrast with the hypothesis suggesting that 4-androstenedione is converted to testosterone followed by the aromatization of testosterone.

Isolation and characterization of goat ovarian aromatase cDNA: assessment of the activity using an intact cell system and placental expression.

Goat ovarian follicles produce estrone and estradiol from androgens. The synthesis of C18 estrogens from C19 androgens requires cytochrome P450 aromatase, but little information about this key enzyme is available in the goat. We report here for the first time the cDNA sequence of the goat ovarian aromatase, the activity of the enzyme in a cell system, and its expression in the term goat placenta. A cDNA library from goat ovarian poly(A)+ RNA was constructed. Human aromatase cDNA was selected as probe to screen the library; several clones were isolated, but none was complete. The longest clone was 3.1 kb long, but it lacked the sequence coding for a few amino acids in the NH(2)-terminal. To obtain the missing sequence, we performed reverse amplification of the cDNA end (RACE). Sequence analysis indicated that goat aromatase possessed a very long 3'-untranslated region ( approximately 1790 bp), and a polyadenylation signal (AATAAA) located at position 3320 downstream from the ATG start codon. The coding region of goat cDNA was inserted in an expression vector and transfected into HEK-293 cells that were cultured in presence of [14C]-androstenedione, steroids extracted and further separated by TLC. The transfected cells efficiently transformed [14C]-androstenedione into estrone. This activity was inhibited by 4-hydroxyandrostenedione. We also investigated the presence of mRNA for P450 aromatase in the goat placenta, using reverse transcription-polymerase chain reaction (RT-PCR) and primers derived from the cDNA ovarian sequence and confirmed the expression of the mRNA in term placenta.

3Beta-sulfamate derivatives of C19 and C21 steroids bearing a t-butylbenzyl or a benzyl group: synthesis and evaluation as non-estrogenic and non-androgenic steroid sulfatase inhibitors.

A series of C19 and C21 steroids bearing one or two inhibiting groups (3beta-sulfamate and 17alpha- or 20(S)-t-butylbenzyl or benzyl) were synthesized and tested for inhibition of steroid sulfatase activity. When only a sulfamate group was added to dehydroepiandrosterone, androst-5-ene-3beta,17beta-diol, pregnenolone and 20-hydroxy-pregnenolone, no significant inhibition of steroid sulfatase occurred at concentrations of 0.3 and 3 microM. With only a t-butylbenzyl or a benzyl group, a stronger steroid sulfatase inhibition was obtained in the androst-5-ene than in the pregn-5-ene series. Comparative results from the screening tests and the IC50 values have shown that the effect of a sulfamate moiety as a second inhibiting group can be combined to the t-butylbenzyl or benzyl effect in the C19 and C21 steroid series. The 3beta-sulfamoyloxy-17alpha-t-butylbenzyl-5-androsten-17beta-ol (10) was thus found to be the most active compound with IC50 values of 46 +/- 8 and 14 +/- 1 nM, respectively for the transformations of E1S to E1 and DHEAS to DHEA. The IC50 values of compound 10 are similar to that of 17alpha-t-butylbenzyl-estradiol, which was previously reported by our group as a good steroid sulfatase reversible inhibitor, but remains higher than that of the potent inactivators estrone-3-O-sulfamate (EMATE) and 17alpha-t-butylbenzyl-EMATE. However, contrary to these two latter inhibitors, compound 10 did not induce any proliferative effect on estrogen-sensitive ZR-75-1 cells nor on androgen-sensitive Shionogi cells at concentrations tested, suggesting that this steroid sulfatase inhibitor is non estrogenic and non androgenic.

Human type 1 estrogen sulfotransferase: catecholestrogen metabolism and potential involvement in cancer promotion.

Using purified human type 1 estrogen sulfotransferase (hEST1), we show that the best substrate for this enzyme is 2-hydroxy-catecholestrogen. The enzyme also catalyzes the transformation of 4-hydroxy-estrogens and 16-hydroxy-estrogens, but with a lower affinity. We also present evidence to indicate that estrogen sulfotransferase may play a role in processes other than the detoxification and elimination of steroids. Indeed, hEST1 may also be involved in the production of stable precursors for local steroid biosynthesis or in the activation of promutagenic estrogen metabolites into carcinogens.

Neuropeptide Y inhibits the biosynthesis of sulfated neurosteroids in the hypothalamus through activation of Y(1) receptors.

We have recently shown that hydroxysteroid sulfotransferase (HST), the enzyme responsible for the biosynthesis of pregnenolone sulfate (Delta(5)PS) and dehydroepiandrosterone sulfate (DHEAS), is expressed in neurons located in the anterior preoptic area and the dorsal magnocellular nucleus of the frog diencephalon. As these two nuclei are richly innervated by NPY-immunoreactive fibers, we investigated the possible implication of NPY in the control of Delta(5)PS and DHEAS biosynthesis. Double labeling of frog brain sections revealed that 42% of the HST-immunoreactive perikarya in the diencephalon were contacted by NPY-containing fibers. In situ hybridization studies showed that Y(1) and Y(5) receptor mRNAs are expressed in the anterior preoptic area and the dorsal magnocellular nucleus. Pulse-chase experiments with (35)S-labeled 3'-phosphoadenosine 5'-phosphosulfate as a sulfate donor demonstrated that frog NPY (fNPY) inhibited the conversion of [(3)H]Delta(5)P and [(3)H]dehydroepiandrosterone ([(3)H]DHEA) into [(3)H,(35)S]Delta(5)PS and [(3)H,(35)S]DHEAS by diencephalic explants. The inhibitory effect of fNPY on Delta(5)PS and DHEAS formation was mimicked by (pPYY) and [Leu(31),Pro(34)]pNPY, which is an agonist for non-Y(2) receptors in mammals, and was completely suppressed by the Y(1) receptor antagonist BIBP3226. Conversely, the Y(2) receptor agonist pNPY-(13-36) and the Y(5) receptor agonist [D-Trp(32)]pNPY did not significantly modify the biosynthesis of [(3)H,(35)S]Delta(5)PS and [(3)H,(35)S]DHEAS. The present study provides the first evidence for the innervation of neurosteroid-producing neurons by NPY fibers. Our data also demonstrate that NPY, acting via Y(1) receptors, exerts an inhibitory effect on the biosynthesis of sulfated neurosteroids.

Synthesis and optimization of a new family of type 3 17 beta-hydroxysteroid dehydrogenase inhibitors by parallel liquid-phase chemistry.

Type 3 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) transforms 4-androstene-3,17-dione (Delta(4)-dione) into the androgen testosterone. To produce potent inhibitors of this key steroidogenic enzyme, we performed parallel liquid-phase synthesis of 3beta-substituted androsterone (ADT) libraries (A-D) in good yields and average high-performance liquid chromatography (HPLC) purities of 92-94%. The first library (A) of 3 beta-amidomethyl-ADT derivatives (168 members), including two levels of molecular diversity on the amide (R(1) and R(2)), was synthesized with a parallel liquid-phase method (method I) in less time than with the classic chemistry method. The screening of library A revealed that relatively small hydrophobic chains at R(1) (5-8 carbons) and small hydrophobic substituents at R(2) (1-4 carbons) provided the most potent inhibitors. In accordance with these inhibition results, a second library (B) of 3 beta-amidomethyl-ADT derivatives (56 members) was generated in a very short time using an improved method based on scavenger resins and liquid-phase parallel chemistry. Library B produced more potent inhibitors than library A and provided useful structure-activity relationships that directed the design of a third library (C) of 49 members. Once again, very potent inhibitors were identified from library C and 3 beta-[(N-adamantylmethyl-N-butanoyl)aminomethyl]-3 alpha-hydroxy-5 alpha-androstan-17-one (C-7-3) was identified as the most potent inhibitor of the three libraries with an inhibitory activity (IC(50) = 35 nM) 18-fold higher than that of the natural substrate of the enzyme, Delta(4)-dione, (IC(50) = 650 nM) used itself as inhibitor. Finally, we designed a library (D) of 3-carbamate-ADT derivatives (25 members) using the efficient parallel liquid-phase method III, which allowed the synthesis of more rigid molecules with two levels of molecular diversity (R(1)/R(2) and R(3)) in the local area occupied by the adamantane group of C-7-3. Interestingly, one of the most potent inhibitors of library D, the 3R-spiro-[3'-[3' '-N-morpholino-2' '-(3' "-cyclopentyl-propionyloxy)propyl]-2'-oxo-oxazolidin-5'-yl]-5 alpha-androstan-17-one (D-5-4), showed an inhibitory activity on type 3 17 beta-HSD similar to that of compound C-7-3, while exhibiting a nonandrogenic profile.