Puberty, ovarian cycle, pregnancy, and postpartum ovulation in captive Sichuan golden monkeys (Rhinopithecus roxellana) based on changes in urinary and fecal gonadal steroid metabolites.

The purpose of this study was to evaluate the reproductive status and clarify the reproductive physiology of captive Sichuan golden monkeys. The concentrations of urinary estradiol-3-glucuronide (E2G) and pregnanediol-glucuronide (PdG) or fecal estradiol-17ß (E2) and PdG in two females, and fecal testosterone concentrations in a male, were measured continuously using enzyme immunoassays. On the basis of these hormone profiles, the follicular phase, luteal phase, and ovarian cycle were calculated to be 14.7 ± 4.8, 10.4 ± 2.8, and 25.1 ± 3.3 days, respectively. The first ovulation (puberty) in a female monkey was observed at 5.1 years old, and the first pregnancy was diagnosed at 6.4 years old. For the first 2 months of pregnancy (204 days), fecal E2 and PdG maintained constant high values and then increased until parturition. These profiles were similar to urinary E2G and PdG changes. During the last trimester of a twin pregnancy, fecal PdG was up to approximately three times higher compared with a single pregnancy. Therefore, fecal PdG levels in late pregnancy may be effective for the detection of a twin pregnancy. The first postpartum ovulation occurred 66 (fetal death and artificial rearing), 143 (fetal death), and 189 (natural suckling) days after parturition. The anovulation period of the natural suckling case was longer than the others. Conception and postpartum ovulation were detected between September and January. Fecal testosterone levels of the male were correlated with the fecal E2 level of the nonpregnancy period in exhibited together female. Our results reported that urinary (E2G and PdG) and fecal (E2 and PdG) hormone measurement is effective for monitoring the reproductive status, thereby expanding knowledge of the reproductive endocrinology of this endangered species.

The dynamic character of the BCL2 promoter i-motif provides a mechanism for modulation of gene expression by compounds that bind selectively to the alternative DNA hairpin structure.

It is generally accepted that DNA predominantly exists in duplex form in cells. However, under torsional stress imposed by active transcription, DNA can assume nonduplex structures. The BCL2 promoter region forms two different secondary DNA structures on opposite strands called the G-quadruplex and the i-motif. The i-motif is a highly dynamic structure that exists in equilibrium with a flexible hairpin species. Here we identify a pregnanol derivative and a class of piperidine derivatives that differentially modulate gene expression by stabilizing either the i-motif or the flexible hairpin species. Stabilization of the i-motif structure results in significant upregulation of the BCL2 gene and associated protein expression; in contrast, stabilization of the flexible hairpin species lowers BCL2 levels. The BCL2 levels reduced by the hairpin-binding compound led to chemosensitization to etoposide in both in vitro and in vivo models. Furthermore, we show antagonism between the two classes of compounds in solution and in cells. For the first time, our results demonstrate the principle of small molecule targeting of i-motif structures in vitro and in vivo to modulate gene expression.

Synthesis of Hoodigogenin A, aglycone of natural appetite suppressant glycosteroids extracted from Hoodia gordonii.

14ß-hydroxy pregnane glycosides extracted from Hoodia gordonii, a succulent plant isolated from Apocynaceae are suggested to have appetite suppressant properties in animals and humans. However, limited reports on biological studies concerning the appetite suppressant properties are available in the open literature. One reason for that is the poor availability of these glycosteroids because H. gordonii is a protected plant and the yield of extraction lies between 0.003% and 0.02%. Starting from 3α,12α-diacetoxy-pregnanone 1, we disclose in this report the synthesis of Hoodigogenin A, the aglycone of the natural 14ß-hydroxy pregnane glycosides extracted from H. Gordonii.

Hoodia gordonii: an up-to-date review of a commercially important anti-obesity plant.

Hoodia gordonii is a spiny succulent plant popularly consumed for its purported anti-obesity effect. Traditionally used by the Khoi-San of South Africa and Namibia as a hunger and thirst suppressant while on long hunting trips, the commercialisation of this plant has been highly controversial due to intellectual property rights and benefit sharing issues, as well as the fact that several prominent pharmaceutical companies involved in its development have withdrawn their interest. Quality control has been the main focus of scientific studies as the supply of H. gordonii plant material is limited due to its sparse geographical distribution, slow maturation rate, need for a permit to cultivate or export material as well as high public demand, contributing to adulteration of a large amount of products. Despite the isolation of numerous steroidal glycosides from H. gordonii, the main focus has been on the pregnane glycoside P57, considered to be the active ingredient and marker molecule to determine quality of raw material and products. Publications based on scientific studies of key aspects such as in vivo biopharmaceutics, the biological activity of all chemical constituents, clinical efficacy, and especially safety are insufficient or completely absent causing great concern as H. gordonii is one of the most widely consumed anti-obesity products of natural origin. This review offers an up-to-date overview of all the current available knowledge pertaining to H. gordonii achieved by systematic analysis of the available literature.

Characterization of in vitro pharmacokinetic properties of hoodigogenin A from Hoodia gordonii.

This study was aimed to predict the pharmacokinetic properties of hoodigogenin A, which is the aglycone of the oxypregnane steroidal glycoside P57AS3 (P57) isolated from Hoodia gordonii. A series of in vitro assays was used to predict its gastric, intestinal and metabolic stability, intestinal and blood brain barrier (BBB) transport, protein binding and interaction with major drug metabolising enzymes. In the simulated gastric fluid, hoodigogenin A was stable (2 % degradation in 60 minutes) whereas P57 was unstable (45 % degradation in 30 minutes). In simulated intestinal fluid, P57 was degraded to an extent of 8 % in 180 minutes, while hoodigogenin A was stable. Hoodigogenin A was efficiently transported by passive diffusion across Caco-2 and MDR1-MDCK monolayers with P(app) values in the range of 32 x 10(-6) cm/sec and 22 x 10(-6) cm/sec, respectively. The compound was metabolically unstable in human liver microsomes and S9 fractions with a CL' (int) of 71 and 120 mL/min/kg, respectively and was bound to the plasma proteins to an extent of 92 %. The compound strongly inhibited CYP3A4 activity (IC(50) 3 microM), indicating a possibility of drug-herb/botanical interactions when products containing H. gordonii are used simultaneously with other botanicals/herbs/drugs.

Neurosteroids 3beta, 20 (R/S)-pregnandiols decrease offset rate of the GABA-site activation at the recombinant GABA A receptor.

Neurosteroids directly modulate ligand gated ion channels such as GABA A receptors. Two such molecules, 3beta-OH A-ring reduced pregnane steroids and pregnenolone sulfate (PS), inhibit recombinant GABA A receptor. Using a two-electrode voltage-clamp technique, we compared the effect of 5alpha-pregnan-3beta,20(S)-diol (UC1019), 5beta-pregnan-3beta, 20(R)-diol (UC1020) and PS on the activation onset and offset times of the recombinant GABA A receptor (rat alpha1beta2gamma2L) in Xenopus oocytes. Rapid solution changes allowed the kinetic analysis of GABA-evoked currents. Steroids were co-applied with 30 microM GABA for 10 s, followed by a 80 s washout period. PS (> ir =0.3 microM) moderately increased the slow onset rate (k(on-S)) of GABA-response. PS had no significant effects on the fast onset rate (k(on-F)). UC1019 and UC1020 decreased the k(on-S) of the GABA-response in a concentration-dependent manner with no significant effects on the k(on-F). Like PS, UC1019 and UC1020 decreased the slow offset rates (k(off-S)). In addition, PS increased the fast offset rate (k(off-F)) in a concentration-dependent manner, while UC1019 and UC1020 decreased k(off-F). The EC50 of PS to increase k(off-F) was calculated as 0.47+/-0.1 microM. The corresponding IC50 values of UC1019 and UC1020 to decrease k(off-F) were 5.0+/-0.5 microM and 8.4+/-0.9 microM, respectively. These results suggest differential actions of PS and 3beta, 20(R/S)-pregnandiols on the offset time course of GABA-site activation.

Azasterols as inhibitors of sterol 24-methyltransferase in Leishmania species and Trypanosoma cruzi.

This paper describes the synthesis of some novel azasterols based on (20R,22xi)-5alpha-pregnan-20-(piperidin-2-yl)-3beta,20-diol. These compounds are potential inhibitors of the enzyme sterol 24-methyltransferase (24-SMT), which is a vital enzyme in the biosynthesis of ergosterol and related 24-alkyl sterols. Structure-activity studies were undertaken to understand the important features for activity against the enzyme, with the aim of increasing activity and selectivity. The compounds were evaluated for inhibition of recombinant Leishmania major 24-SMT and the effect of compounds on sterol composition and parasite proliferation. Essentially, compounds which showed good activity against the recombinant enzyme had a significant effect on the sterol composition and growth of parasites. The activity of compounds was found to be related to the basicity and stereochemical location of the nitrogen. Also, presence of an unprotected 3beta-OH seemed to be important for activity. However, some azasterols which were not good inhibitors of 24-SMT also showed antiproliferative activity, suggesting that there may be other modes of actions of these compounds.

Peripartum sex steroid changes and maternal style in rhesus and Japanese macaques.

The subtle and complex relationships between the sequential maturation of the endocrine systems during pregnancy and parturition, and the hormonal role in activating the central nervous system to express maternal behavior in primates, are far from being completely understood. Recent studies on the association between sex steroids and maternal behavior have yielded conflicting results in this group. Here we use a comparative approach to assess the correlation between changes in the peripartum endocrine profiles and maternal styles in two closely related macaque species, housed in analogous environments. We included in this study the first seven Japanese macaque and seven rhesus macaque mother-infant pairs born during the birth season of 2001 at the Primate Research Institute, Kyoto University, Japan. We observed each pair 3h/week (six weekly 30-min observation sessions) during the first 12 weeks of lactation. We collected fecal samples twice a week from each mother, starting 4 weeks before parturition and ending 4 weeks after parturition. We tested the hypothesis that neuroendocrine changes during pregnancy and lactation might specifically contribute to the regulation and timing of infant rejection. Despite their biological similarities, we observed a clear difference in maternal style between the two groups concerning rejection rates: rhesus macaque mothers rejected their infants earlier and more frequently throughout the whole 12 weeks of study. On the other hand, protectiveness showed similar patterns and values in the two groups, and maternal warmth was significantly higher in the rhesus group, but it followed a similar pattern over time. We also confirmed an association between maternal rejection and excreted estrogen, but not excreted progesterone, for Japanese macaques. This association was not apparent for the rhesus macaques. This result, coupled with the observation that rhesus mothers are more rejecting than Japanese macaque mothers, tends to support our hypothesis. As a group, rhesus macaques are less inhibited in the rejection of their infants, and this is paralleled by a less marked change in the primacy of estrogen in the last phase of pregnancy. On the contrary, the Japanese group is characterized by higher levels of E(1)C and the E(1)C/PdG ratio. Therefore, according to our hypothesis, their tendency to increase the rejection rate may be suppressed through a feedback loop that enhances maternal motivation and results in a more tolerant outcome toward the infant.

Purification and characterization of lysozyme-pregnanediol glucuronide conjugates: the effect of the hapten and coupling reagent on the substitution level, sites of acylation and the consequences for the development of future immunoassays.

The preparation of conjugates destined for use in homogeneous enzyme immunoassays requires careful control to maximize the yield of conjugate, limit the level of substitution and obtain highly inhibitable conjugates. Furthermore, previous studies have shown that the acylation position and orientation of haptens in enzyme conjugates is important in determining the recognition and strength of binding of the hapten by anti-hapten antibodies. In this study we have prepared and purified pregnanediol glucuronide conjugates of hen egg white lysozyme by the active ester and mixed anhydride methods and compared the resulting conjugates with those obtained when conjugating with oestrone glucuronide. Conjugation of lysozyme with pregnanediol glucuronide by the mixed anhydride procedure resulted in two major derivatives monoacylated at lysine residues 33 and 97, while acylation by the active ester procedure resulted in six major derivatives acylated at one or more of lysine residues 33, 97 and 116. Comparison of these conjugates with oestrone glucuronide conjugates suggested that when using the more reactive active ester method the protein environment of the amino groups and the acylating agent directed the site(s) of acylation and level of substitution. However, in the case of the less reactive mixed anhydride coupling procedure the chemical nature of the hapten also influenced the final conjugation product makeup. These findings have implications for the design of new, highly inhibitable enzyme conjugate immunoassay systems.

Studies of molecular structure parameters of 20-piperidin-2-yl-5alpha-pregnan-3beta,20-diol and its N-methyl derivative: two inhibitors of Delta24(25) sterol methyl transferase and Delta24(24') sterol methyl reductase of Trypanosoma cruzi.

Molecular structural parameters of two potential drugs against Trypanosoma cruzi epimastigotes, 20-piperidin-2-yl-5alpha-pregnan-3beta,20-diol (1) and 20-N-methylpiperidin-2-yl-5alpha-pregnan-3beta, 20-diol (2) were studied using a combination of a stereoselective synthetic route, spectroscopic characterization and single-crystal X-ray analysis. Both compounds were synthesized with an R configuration at C20. This chirality is a consequence of the stereoselectivity observed during the formation of the intermediate 20-pyridin-2-yl-5alpha-pregnan-3beta,20R-diol (4). NMR data indicated that the six-membered aza ring of (2) is conformationally more restrained, in CDCl3 solution, than (1). X-ray studies showed that maximum deviations among structural molecular parameters of (1) and (2) correspond to torsion angles along the C20-C22 bonds, leading to a different relative orientation of the N atom; a critical structural parameter for the binding properties of aza-sterols to Delta(24(25)) sterol methyl transferase. Cremer-Pople parameters of the five-membered rings of (1) and (2) lie in the observed range for a family of tetracyclic fused ring systems retrieved from the CSD. The phi2 parameter of (1) lies just on the mean of the family, while phi2 of (2) deviates significantly towards the lower limit.

The identification of novel steroid N-acetylglucosaminides in the urine of pregnant women.

A series of pregnanediols and pregnanetriols doubly conjugated with N-acetylglucosamine and glucuronic or sulfuric acid has been identified in urine from pregnant women. Steroid conjugates were separated by ion-exchange chromatography and the glucuronide and monosulfate fractions were analysed by fast atom bombardment mass spectrometry. After removal of the acid moiety, the neutral steroids were isolated, derivatized, and analysed by gas chromatography-mass spectrometry (GC-MS). The analyses revealed the presence of steroids conjugated with N-acetylhexosamine both in the glucuronide and the monosulfate fractions. Following enzyme hydrolysis, the sugar was identified by GC-MS as N-acetylglucosamine (GlcNAc). The major steroid conjugated with GlcNAc both in the glucuronide and monosulfate fractions was identified as 5alpha-pregnane-3alpha,20alpha-diol. 5beta-Pregnane-3alpha,2Oalpha-diol was also present as a GlcNAc conjugate in both fractions whereas a GlcNAc conjugate of 5alpha-pregnane-3beta,20alpha-diol was only found in the sulfate fraction. 5alpha-Pregnane-3alpha,20alpha,21-triol was a double conjugate with GlcNAc in the sulfate fraction whereas a pregnane-2,3,20-triol was a double conjugate in the glucuronide fraction. The positions of conjugation were determined by collision-induced dissociation of the pseudomolecular anions produced by fast atom bombardment ionization. The sulfate and glucuronic acid moieties were located at C-3 and N-acetylglucosamine at C-20. An alternative localization of GlcNAc at C-21 of 5alpha-pregnane-3alpha,20alpha,21-triol cannot be excluded. Judging from the enzymatic hydrolysis of the conjugates, the sugar was attached in beta-glycosidic linkage. The mean excretion of N-acetylglucosaminides of the pregnanediols and pregnanetriols was 32.2 micromol/g creatinine (range 17.9-49.1 micromol) in five healthy women in the 38th-39th week of pregnancy. The mean excretion of 5beta-pregnane-3alpha,20alpha-diol glucuronide in the same women was 71 micromol/g creatinine, (range 27-127 micromol). This indicates that conjugation with N-acetylglucosamine constitutes a quantitatively important pathway of progesterone metabolism in human pregnancy.

Modulation of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity in the equine placenta by pregnenolone and progesterone metabolites.

The purpose of this study was to measure 3beta-HSD activity in the equine placenta and to assess the effect of fetal and maternal blood plasma progestagens on 3beta-HSD activity was measured in 8 late gestation (collected by caesarian section at 250 to 320 days) and 7 term (collected by caesarian section at 250 to 320 days) and 7 term (collected at birth) equine placentae using a tritium release assay with [3alpha-3H] pregnenolone as substrate. Mean +/- s.d. Km(app) and Vmax for term placentae were in general higher than for late gestation placentae (0.129 +/- 0.217 micromol/l and 23.85 +/- 9.1 nmol/mg/h respectively vs. 0.016 +/- 0.048 micromol/l and 17.36 +/- 20.9 nmol/mg/h) but there was no statistical difference between them. Inhibition studies were performed on 3 term placentae and 3 late gestation ones. Steroid concentrations used for inhibition studies were close to blood plasma concentrations (0.5 to 2 micromol/l). 3beta-hydroxy compounds (5alpha-pregnene-3beta, 20alpha-diol and 3beta-hydroxy-5alpha-pregnan-20-one) showed noncompetitive or mixed inhibition. Mean Ki(app) of 0.7 micromol/l. Inhibition was competitive with 20alpha-hydroxy-5alpha-pregnan-3-one with a mean Ki(app) of 0.1 micromol/l.