Semi-quantitative determination of designer steroids by high-performance liquid chromatography with ultraviolet detection in the absence of reference material.

New designer steroids are continually being encountered in dietary supplements that claim to increase muscle mass, but quantitative analysis of such ingredients is challenging due to the availability, quality, or cost of commercial reference materials. Although standard reference material typically becomes available for these emerging compounds, laboratories often face the challenge of finding properly certified materials from accredited suppliers, due to traceability requirements. Several of these designer steroids have been isolated and identified using multiple structural elucidation tools. Structural characteristics of these compounds of interest were evaluated and molar absorptivity data was collected and compared to several readily available steroid standards using ultraviolet/visible spectroscopy. This approach was used to find suitable compounds for use as surrogate reference materials in the semi-quantitative determination of two designer steroids, 1-dehydroepiandrosterone (1-androsterone) and 6ß-chloro-4-androsten-17ß-ol-3-one (6ß-chlorotestosterone). Laboratory-fortified matrix samples and dietary supplement samples were analyzed using this method for the estimation of 1-androsterone and 6ß-chlorotestosterone by HPLC-UV. Assay values obtained for the estimation of 1-androsterone in a dietary supplement sample using a prasterone or dehydroepiandrosterone (DHEA) standard curve were 100% of those obtained using a 1-androsterone reference standard, once it became commercially available. Estimations for 6ß-chlorotestosterone in laboratory-fortified matrix samples using a testosterone standard curve were 92%-93% of those obtained using isolated 6ß-chlorotestosterone as "reference material."

Methyl Jasmonate Ameliorates Testosterone Propionate-induced Prostatic Hyperplasia in Castrated Wistar Rats.

Benign prostate hyperplasia (BPH) is a progressive disease that is related to age. Known therapeutic agents used in the treatment of BPH are associated with toxicity. Therefore, chemoprevention could be an effective approach. We investigated the ameliorative effects of methyl jasmonate (MeJA) in testosterone propionate (TP)-induced BPH in castrated rats. Castration was performed by removing both testes through the scrotum sack under ketamine anesthesia. Rats were assigned into seven groups of seven animals each: non-castrated control, castrated control, castrated rats that received TP, castrated rats that received TP and MeJA, castrated rats that received TP and finasteride, castrated rats that received MeJA, and castrated rats that received finasteride. Results indicate that BPH rats had significantly (p < 0.05) elevated prostate weight and relative weight of prostate relative to control. Also, BPH rats had significantly (p < 0.05) increased activities of prostatic acid and alkaline phosphatases, levels of zinc, and malondialdehyde. Further, levels of enzymic and non-enzymic antioxidative indices were significantly (p < 0.05) reduced in BPH. Histology of prostate revealed hyperplasia of transition lobe, increased expression of PSA, and Ki67 in BPH. Treatment with MeJA and finasteride attenuated the activities of the phosphatases and levels of antioxidants in BPH. Overall, MeJA ameliorates BPH via antioxidative mechanism. Copyright © 2017 John Wiley & Sons, Ltd.

Blood and seminal plasma concentrations of selenium, zinc and testosterone and their relationship to sperm quality and testicular biometry in domestic cats.

The aim of this study was to assess seminal plasma (SP) and serum concentrations of zinc (Zn), selenium (Se) and testosterone (T) in domestic cats and determine whether these are related to sperm quality and testicular biometry. Six tomcats were collected using an artificial vagina and sperm analysis included motility by CASA, morphology, plasma membrane integrity, and sperm count. Serum and SP were submitted to total T concentration determination using a solid-phase radioimmunoassay technique while Zn and Se were measured by atomic absorption spectroscopy. Serum T concentrations were greater compared to SP concentrations, but both values were significantly correlated. Se concentrations were higher in serum, whereas SP had greater Zn values. Concentrations of Se, Zn and T were not correlated with each other either in serum or SP. Negative correlations were detected between Se concentrations in SP and total sperm head defects, and between Se concentrations in serum and VAP, VSL, STR, and LIN. Serum concentrations of Zn were negatively correlated with total abnormal sperm and midpiece defects and positively related to progressive motility. Both serum and SP concentrations of T had no relationship with sperm quality. Concentrations of Se exhibited a negative correlation with total testicular weight, whereas T concentrations in SP and serum were correlated with total testicular volume and weight. In conclusion, both Se and Zn concentrations in serum were correlated to sperm quality variables in the domestic cat, thus, making these potential candidates for fertility markers.

Effects of E/Z isomers of lycopene on experimental prostatic hyperplasia in mice.

AIM: Lycopene is a member of the carotenoid family and has strong anti-oxidant properties. Lycopene occurs in tomato-based food products primarily as an all-E isomer (80-97%),but its Z-isomers accounts for 79 to 88% of total lycopene in benign or malignant prostate tissues, while the specific biological functions of Z-isomers are still not clarified at present. This study was to examine the bioactive potency of Z-isomers on benign prostatic hyperplasia (BPH) in mice and to make a comparison of effective inhibition between Z-isomers and all-E isomer. METHOD: Mice were divided into the Saline group, Vehicle control group and testosterone propionate induced BPH mice group (BPH model group, vehicle BPH model group, lycopene treated (5 mg/kg and 2.5 mg/kg), Z-isomers (57%) treated, Z-isomers (86%) treated, finasteride treated). The drugs were orally administered once a day consecutively for 30 days. The inhibitory effects on BPH of all-E lycopene and Z-isomers were evaluated by prostatic index, prostatic acid phosphatase (PAP), estradiol, testosterone and dihydrotestosterone (DHT) levels in serum and histopathology examination. RESULTS: Compared with the BPH model group, E/Z isomers exhibited significant differences in prostatic index, PAP, estradiol, testosterone and DHT levels in serum and similar histological aspects observed in the mice of the control group. The present research also shows that Z-isomers may be more potent inhibitors than all-E isomers in BPH treatment.

Application of bar adsorptive microextraction (BAµE) for anti-doping control screening of anabolic steroids in urine matrices.

This study proposes a new analytical methodology for the determination of trace levels of testosterone (T) and epitestosterone (E) in urine matrices using bar adsorptive microextraction combined with liquid desorption followed by high-performance liquid chromatography with diode array detection (BAµE-LD/HPLC-DAD). The comparison of different sorbent coatings (five activated carbons, one styrene-divinylbenzene, two modified pyrrolidone, one ciano and one n-vinylpyrrolidone polymers) through BAµE showed that the latter phase presented much higher selectivity and capacity offering multiple mechanisms of interaction. Assays using this phase were performed on 25mL of water samples spiked at the 8.0µg/L level, yielded average recoveries of 92.1 and 93.4% for T and E, respectively, under optimized experimental conditions; BAµE (n-vinylpyrrolidone): 16h (1000rpm), pH 5.5; LD: acetonitrile, 30min under sonication treatment. From the developed analytical methodology, suitable detection limits were achieved (0.4µg/L) and good linear dynamic ranges (1.4-16.0µg/L) with remarkable determination coefficients (r(2)>0.9978). By using the standard addition methodology, the application of the present analytical approach on urine samples revealed good sensitivity. The proposed method, which operated under the floating sampling technology, proved to be a suitable sorption-based static microextraction alternative for screening T, E and the T/E ratio in urine samples for doping control purposes. The methodology showed to be easy to implement, demonstrating good reproducibility, sensitivity and robustness, allowing the possibility to choose the most selective sorbent coating according to the compounds of interest.

Hypothalamus-pituitary axis: an obligatory target for endocannabinoids to inhibit steroidogenesis in frog testis.

Endocannabinoids - primarily anandamide (AEA) and 2-arachidonoylglycerol (2-AG) - are lipophilic molecules that bind to cannabinoid receptors (CB1 and CB2). They affect neuroendocrine activity inhibiting gonadotropin releasing hormone (GnRH) secretion and testosterone production in rodents, through a molecular mechanism supposed to be hypothalamus dependent. In order to investigate such a role, we choose the seasonal breeder, the anuran amphibian Rana esculenta, an experimental model in which components of the endocannabinoid system have been characterized. In February, at the onset of a new spermatogenetic wave, we carried out in vitro incubations of frog testis with AEA, at 10(-9)M dose. Such a treatment had no effect on the expression of cytochrome P450 17alpha hydroxylase/17,20 lyase (cyp17) nor 3-ß-hydroxysteroid dehydrogenase/Δ-5-4 isomerase (3ß-HSD), key enzymes of steroidogenesis. To understand whether or not the functionality of the hypothalamus-pituitary axis could be essential to support the role of endocannabinoids in steroidogenesis, frogs were injected with AEA, at 10(-8)M dose. Differently from in vitro experiment, the in vivo administration of AEA reduced the expression of cyp17 and 3ß-HSD. Whereas the effect on 3ß-HSD was counteracted by SR141716A (Rimonabant) - a selective antagonist of CB1, thus indicating a CB1 dependent modulation - the effect on cyp17 was not, suggesting a possible involvement of receptors other than CB1, probably the type-1 vanilloid receptor (TRPV1), since AEA works as an endocannabinoid and an endovanilloid as well. In conclusion our results indicate that endocannabinoids, via CB1, inhibit the expression of 3ß-HSD in frog testis travelling along the hypothalamus-pituitary axis.

Comparison of chemical compositions and osteoprotective effects of different sections of velvet antler.

ETHNOPHARMACOLOGICAL RELEVANCE: Velvet antlers (VA) have been claimed for centuries to have numerous medical benefits including strengthen bones. To investigate and compare the anti-osteoporotic activities from different sections of VA. MATERIALS AND METHODS: Fresh VA prepared from farmed sika deers (Cervus nippon) was divided into upper (VAU), middle (VAM), and basal (VAB) sections. The chemical constituents and anti-osteoporotic effect of different sections from VA were evaluated using ovariectomized rats. RESULTS: Levels of water-soluble extracts, diluted alcoholic extract, amino acids, testosterone, insulin-like growth factor (IGF)-1 and testosterone plus estradiol significantly differed among the different sections. Levels of these constituents were significantly higher in the upper section than in the basal section. Moreover, levels of testosterone and IGF-1 of the VAM were also significantly higher than those of the VAB. Calcium level increased downward from the tip with statistical significance. The strength of vertebrae increased in all VA-treated groups compared to the control, but only treatment with VAU and VAM increased the strength of the femur and the microarchitecure of the trabecular bone. Alkaline phosphatase levels of VAU- and VAM-treated groups significantly decreased, but osteocalcin did not significantly change. Moreover, VAU and VAM dose-dependently increased proliferation and mineralization of MC3T3-E1 cells. CONCLUSION: Our study provides strong evidence for the regional differences in the effectiveness of velvet antler in treating osteoporosis. However, further studies are needed to elucidate the bioactive chemical constituents associated with the anti-osteoporotic effects of velvet antler.

[Study on preparation of testosterone undecanoate ethosomes and its in vitro transdermal penetration].

Ethosomes, as a new vector for transdermal drug delivery, could obviously improve the transdermal penetration of drugs. In this study, we prepared testosterone undecanoate ethosomes, with TU ethosomes as the basic remedy, to determine its appearance, particle size, entrapment efficiency (EE) and membrane fluidity. Meanwhile, a transdermal test was conducted in mice, in order to determine the permeability characteristics of ethosomes as a vector for transdermal drug delivery, and compare transdermal behaviors of TU ethosomes, liposomes and their ethanol solutions.

Hydrogel blends with adjustable properties as patches for transdermal delivery.

The effect of different preparation parameters were analyzed with respect to the rheological and pharmaceutical characteristics of hydrogel blend patches, as transdermal delivery formulation. Mixtures of pectin and gelatin were employed for the production of patches, with adjustable properties, following a two-step gelation procedure. The first gelation, a thermal one, is trigged by the presence of gelatin, whereas, the second gelation, an ionic one, is due to the formation of the typical egg box structure of pectin. In particular, the patch structural properties were assessed by oscillation stress sweep measurements which provided information concerning their viscolelastic properties. In addition, different modalities for drug loading were analyzed with respect to drug homogeneous distribution; testosterone was employed as model drug for transdermal administration. Finally, the performances of the produced transdermal patches were studied, in term of reproducibility and reliability, by determination of in vitro drug release profiles.

Effect of threonine on immunity and reproductive performance of male mice infected with pseudorabies virus.

The experiment was conducted to evaluate the effects of dietary threonine (Thr) supplement on reproductive performance and immune function of the male mice challenged with pseudorabies virus (PRV). Kun-Ming male mice were assigned randomly to four groups with different Thr levels (0.70%, 0.88%, 1.10% and 1.30%). Half of the mice in each group were injected with PRV or phosphate-buffered saline (PBS) after 5 weeks' adaptation to diets. The second experiment examined the effects of dietary Thr level on copulation rate, pregnancy rate and average number per litter of PRV- or PBS-challenged male mice that copulated with adult female mice on the 9th day post PRV challenge. Sperm quality and testosterone of mice were decreased after PRV infection, but this effect was attenuated by increasing Thr levels. Copulation and conception rates were increased with increasing Thr levels (P = 0.14), but litter size was not affected (P > 0.05). In the PBS and PRV groups, mice fed higher levels of Thr had increased immunoglobulin (Ig)G, IgA and IgM concentrations. The PRV-specific antibody level, interleukin (IL)-1ß and tumor necrosis factor (TNF)-α concentration in PRV groups enhanced with increasing Thr levels; however, there was no difference in PBS groups. Furthermore, higher toll-like receptor (TLR)2 and TLR9 expressions in testis were observed by PRV challenge compared with PBS groups, and higher Thr supplement attenuated PRV-challenged induced the upregulation effect of TLR2 and TLR9 mRNA expression in testis (P < 0.05). These data suggest that higher Thr consumption was recommended in order to counteract the deleterious effects of virus invasion, possibly through the downregulated expression of TLRs, and thus to improve immunity and reproduction performance of male mice challenged with PRV.

Pharmacokinetic determination of ephedrine in Herba Ephedrae and Wu Tou Tang decoctions in rats using ultra performance liquid chromatography tandem mass spectrometry.

A rapid and sensitive ultra performance liquid chromatography tandem mass spectrometry method was developed and validated for the determination and quantification of ephedrine in rat plasma samples. An Acquity UPLC BEH C18 column (1.7 µm, 2.1 mm × 50 mm) was used for chromatographic separation. Electrospray ionization in the positive mode was used, and the precursor-fragment ion pairs of m/z 166/148 and m/z 289/97 were adopted to characterize ephedrine and testosterone (internal standard), respectively. The method was validated using 10, 100 and 500 ng/mL of ephedrine. It demonstrated adequate levels of precision and accuracy, matrix effect, extraction recovery and stability. Linearity over the concentration range of 0.5-2000 ng/mL was acceptable with a correlation coefficient (r²) better than 0.990. To determine the pharmacokinetic behaviour of this sympathomimetic compound in the Sprague-Dawley rats, ephedrine hydrochloride, Herba Ephedrae single-herb and Wu Tou Tang decoctions were administered orally, and ephedrine hydrochloride was also administered by intravenous injection, and blood samples were collected over 24 h. Ephedrine was measured in plasma and pharmacokinetic parameters were determined by using the standard non-compartmental method and calculated by using Practical Pharmacokinetic Program-Version 87/97. The AUC(0-t) and T(max) values were significantly different (p < 0.05). Ephedrine AUC(0-t) values were significantly lower following the Wu Tou Tang decoction compared to the other oral treatments, suggesting that some components in the decoction may reduce the bioavailability of ephedrine.

Qualidade espermática e perfil plasmático de lipídeos em garanhões suplementados com óleo de arroz semirefinado com alto teor de gama-orizanol / Spermatic quality and plasmatic lipids in stallions supplemented with rice bran oil semirefined containing high level of gamma-oryzanol

O gama-orizanol é uma substância natural contida no óleo de arroz, que por suas características hipocolesterolêmicas e antioxidantes, têm sido objeto de estudo em humanos e, mais recentemente, em equinos. Foram utilizados seis garanhões, de várias raças, com idade de 10±5,4 anos e peso inicial de 472,67±90,48 kg. Objetivou-se verificar os efeitos da suplementação da dieta com óleo de arroz semi-refinado, contendo 1,1% de gama-orizanol, sobre os níveis de lipídeos plasmáticos (colesterol total, HDL-C, LDL-C, VLDL-C e triglicérides), de testosterona e da qualidade espermática. Os garanhões foram divididos em dois grupos, recebendo em cada refeição 150 mL de óleo de soja ou de arroz durante 60 dias. Realizaram-se colheitas amostrais de sangue e sêmen a cada 15 dias do período experimental. O delineamento experimental foi inteiramente casualizado, com medidas repetidas no tempo, e as médias comparadas pelo teste F, considerando-se o nível de 5% de significância. Os valores médios de testosterona, colesterol total, HDL-C, LDL-C, VLDL-C e triglicérides foram respectivamente 75,93 ng/dL; 92,73; 61,47; 26,99 e 4,28 mg/dL para o tratamento com óleo de soja; e de 62,13 ng/dL; 110,20; 66,73; 38,44 e 5,02 mg/dL para o tratamento com óleo de arroz. Em relação à qualidade espermática, não foi observada diferença (p<0,05) entre os tratamentos nas variáveis: volume, motilidade, concentração e defeitos. Podemos concluir que a suplementação supracitada não influencia a qualidade espermática nem as concentrações plasmáticas de testosterona, VLDL-C, HDL-C e triglicérides, porém, pode elevar as concentrações plasmáticas de colesterol total e de LDL-C.

Gamma-oryzanol is a natural substance contained in rice bran oil that has been studied for years in humans due to its hypocholeterolemic and antioxidant properties, and in recent years in horses. Using six stallions from different breeds in a randomized experimental design, weighing 472.67±90.48 kg and aging 10±5.4 years, this study was aimed to evaluate the effects of supplementation with rice bran oil containing 1.1% of gamma-oryzanol in plasmatic lipids levels (total cholesterol, HDL-C, LDL-C, VLDL-C and triglycerides), testosterone and sperm quality. Stallions were divided in two groups, one receiving 150 mL of soybean oil, and the other 150 mL of rice bran oil twice a day, during 60 days. Blood and sperm samples were collected every 15 days of the trial period. Data obtained was processed, and means compared using F test, at 5% of confidence level. Mean values of total cholesterol, HDL-C, LDL-C, VLDL-C, triglycerides and testosterone were respectively 92.73; 61.47; 26.99; 4.28 mg/dL and 75.93 ng/dL in the soybean oil supplemented group, and 110.20; 66.73; 38.44; 5.02 mg/dL and 62.13 ng/dL in the rice bran oil one. No difference among treatments (p<0,05) were observed in sperm quality parameters volume, motility, concentration and defects. Supplementation of stallions with rice bran oil containing 1.1% of gamma-oryzanol do not promote any change in sperm quality, testosterone, HDL-C, VLDL-C or triglycerides plasmatic levels, but an increase in total cholesterol and LDL-C.

Seized designer supplement named "1-Androsterone": identification as 3ß-hydroxy-5α-androst-1-en-17-one and its urinary elimination.

New analogues of androgens that had never been available as approved drugs are marketed as "dietary supplement" recently. They are mainly advertised to promote muscle mass and are considered by the governmental authorities in various countries, as well as by the World Anti-doping Agency for sport, as being pharmacologically and/or chemically related to anabolic steroids. In the present study, we report the detection of a steroid in a product seized by the State Bureau of Criminal Investigation Schleswig-Holstein, Germany. The product "1-Androsterone" of the brand name "Advanced Muscle Science" was labeled to contain 100mg of "1-Androstene-3b-ol,17-one" per capsule. The product was analyzed underivatized and as bis-TMS derivative by GC-MS. The steroid was identified by comparison with chemically synthesized 3ß-hydroxy-5α-androst-1-en-17-one, prepared by reduction of 5α-androst-1-ene-3,17-dione with LS-Selectride (Lithium tris-isoamylborohydride), and by nuclear magnetic resonance. Semi-quantitation revealed an amount of 3ß-hydroxy-5α-androst-1-en-17-one in the capsules as labeled. Following oral administration to a male volunteer, the main urinary metabolites were monitored. 1-Testosterone (17ß-hydroxy-5α-androst-1-en-3-one), 1-androstenedione (5α-androst-1-ene-3,17-dione), 3α-hydroxy-5α-androst-1-en-17-one, 5α-androst-1-ene-3α,17ß-diol, and 5α-androst-1-ene-3ß,17ß-diol were detected besides the parent compound and two more metabolites (up to now not finally identified but most likely C-18 and C-19 hydroxylated 5α-androst-1-ene-3,17-diones). Additionally, common steroids of the urinary steroid profile were altered after the administration of "1-Androsterone". Especially the ratios of androsterone/etiocholanolone and 5α-/5ß-androstane-3α,17ß-diol and the concentration of 5α-dihydrotestosterone were influenced. 3α-Hydroxy-5α-androst-1-en-17-one appears to be suitable for the long-term detection of the steroid (ab-)use, as this characteristic metabolite was detectable in screening up to nine days after a single administration of one capsule.

Impact of electro-acupuncture and physical exercise on hyperandrogenism and oligo/amenorrhea in women with polycystic ovary syndrome: a randomized controlled trial.

Polycystic ovary syndrome (PCOS), the most common endocrine disorder in women of reproductive age, is characterized by hyperandrogenism, oligo/amenorrhea, and polycystic ovaries. We aimed to determine whether low-frequency electro-acupuncture (EA) would decrease hyperandrogenism and improve oligo/amenorrhea more effectively than physical exercise or no intervention. We randomized 84 women with PCOS, aged 18-37 yr, to 16 wk of low-frequency EA, physical exercise, or no intervention. The primary outcome measure changes in the concentration of total testosterone (T) at week 16 determined by gas and liquid chromatography-mass spectrometry was analyzed by intention to treat. Secondary outcome measures were changes in menstrual frequency; concentrations of androgens, estrogens, androgen precursors, and glucuronidated androgen metabolites; and acne and hirsutism. Outcomes were assessed at baseline, after 16 wk of intervention, and after a 16-wk follow-up. After 16 wk of intervention, circulating T decreased by -25%, androsterone glucuronide by -30%, and androstane-3α,17ß-diol-3-glucuronide by -28% in the EA group (P = 0.038, 0.030, and 0.047, respectively vs. exercise); menstrual frequency increased to 0.69/month from 0.28 at baseline in the EA group (P = 0.018 vs. exercise). After the 16-wk follow-up, the acne score decreased by -32% in the EA group (P = 0.006 vs. exercise). Both EA and exercise improved menstrual frequency and decreased the levels of several sex steroids at week 16 and at the 16-wk follow-up compared with no intervention. Low-frequency EA and physical exercise improved hyperandrogenism and menstrual frequency more effectively than no intervention in women with PCOS. Low-frequency EA was superior to physical exercise and may be useful for treating hyperandrogenism and oligo/amenorrhea.

Variation in anastrozole metabolism and pharmacodynamics in women with early breast cancer.

Aromatase inhibitors play a prominent role in the management of postmenopausal women with endocrine-sensitive breast cancer, but there is large variability in both efficacy and tolerability. The purpose of our study was to define interindividual variation in anastrozole metabolism and pharmacodynamics among patients treated with the approved daily dose of 1 mg in a standard practice setting as adjuvant therapy for resected early breast cancer. This study was performed in 191 women in whom pretreatment and during anastrozole plasma concentrations of estrone (E1), estradiol (E2), estrone conjugates, androstenedione, and testosterone were determined and correlated with plasma concentrations of anastrozole and anastrozole metabolites. There were large interindividual variations in plasma anastrozole and anastrozole metabolite concentrations, as well as pretreatment and postdrug plasma E1, E2, and E1 conjugate and estrogen precursor (androstenedione and testosterone) concentrations. E1 and E2 concentrations were below the lower limit of quantitation (LLQ) in most patients after anastrozole therapy (83% for both), but those with detectable concentrations had a broad range (1.58-45.2 and 0.635-97.0 pg/mL, respectively). E1 conjugates after anastrozole therapy were above the LLQ in most patients (93%), with wide interpatient variability (3.50-2,990 pg/mL). Two patients seemed to extensively metabolize anastrozole and failed to display substantial decreases in estrogens. Acknowledging the potential factor of variable compliance, our results showed large interindividual variation in anastrozole metabolism and its effect on circulating estrogens in postmenopausal patients. These findings may have implications with regard to efficacy and adverse events and may indicate the need to "individualize" therapy with this drug.

In vitro culture and differentiation of buffalo (Bubalus bubalis) spermatogonia.

The objective of this study was to develop a culture system which could support buffalo spermatogonia differentiation into spermatids in vitro. Testes from 3- to 5-month-old buffaloes were decapsulated and seminiferous tubules were enzymatically dissociated to recover spermatogonia and sertoli cells. The cells were cultured in modified Dulbecco modified Eagle medium supplemented with different concentrations of foetal bovine serum, retinol, testosterone for 2 months at 37 degrees C. Spermatogonia and sertoli cells were identified with an antibody against c-kit or GATA4, respectively. The viability of spermatogonia in the media supplemented with different concentrations of serum was all significantly higher (p < 0.05) compared with that in the medium without serum. A-paired or A-aligned spermatogonia and spermatogonial colonies (AP-positive) were observed after 7-10 days of culture and spermatid-like cells with a flagellum (6-8 microm) appeared after 30 days of culture. For cultured conditions, retinol could not significantly promote the formation of spermatid-like cells (p > 0.05), whereas supplementation of testosterone could significantly promote (p < 0.05) the formation of spermatid-like cells after 41 days of culture. The expression of the spermatid-specific marker gene (PRM2) was identified after 30 days of culture by RT-PCR. Yet, the transition protein 1 (TP1, a haploid makers) was not detected. Meanwhile, spermatids developed in vitro were also confirmed by Raman spectroscopy. These results suggest that buffalo spermatogonia could differentiate into spermatids in vitro based on the analysis of their morphology, PRM2 expression and Raman spectroscopy. Yet, the normality of the spermatid-like cells was not supported by TP1 expression.

Yolk testosterone levels and dietary carotenoids influence growth and immunity of grey partridge chicks.

Early maternal effects in the form of substances accumulated in the egg, such as carotenoids and hormones, can be physiologically relevant for a good development of offspring. It has been found in different species that testosterone (T) can be beneficial to offspring by increasing growth rate, but detrimental by reducing immunocompetence and increasing oxidative stress. Carotenoids on the other hand are suggested to be beneficial because they can counteract the oxidative stress and the immune-depressive effect of T. In this study we analyzed the effect of prenatal T exposure in the grey partridge. We injected eggs with three doses of T (high, intermediate, and physiological). After hatching, chicks exposed to a prenatal high dose of T were fed with two diets (rich or poor) differing in beta-carotene content. We found a significant effect of T on both chick growth and cell-mediated immunity, with high T doses resulting in detrimental effects while low doses were beneficial. Detrimental effects of the high dose of T on immunity were mitigated by beta-carotene consumed in the diet. The differences between groups were observed in the early period of life (age 10 days for mass, and age 10 and 21 days for immunity), and disappeared in the following period, and up to 1 and 2 years later. Overall, our observations show that T in the egg is not detrimental but beneficial, and that negative effects are found only at supraphysiological concentrations. The negative effects of T on immunity could be balanced if chicks could consume a diet rich in beta-carotene.

Moringa oleifera leaf extracts inhibit 6beta-hydroxylation of testosterone by CYP3A4.

BACKGROUND: Moringa oleifera is a tropical tree often used as a herbal medicine, including by people who test positive for HIV. Since herbal constituents may interact with drugs via inhibition of metabolizing enzymes, we investigated the effects of extracts of M. oleifera on the CYP3A4-mediated 6beta-hydroxylation of testosterone. METHODS: Methanolic and aqueous leaf and root of extracts of M. oleifera with concentrations between 0.01 and 10 mg/ml were incubated with testosterone and mixed-sex human liver microsomes in the presence of NADPH. Metabolite concentrations were determined by HPLC. The cytotoxicity of the extracts was tested with HepG2 cells using the MTT formazan assay. RESULTS: Significant CYP3A4 inhibitory effects were found, with IC50 values of 0.5 and 2.5 mg/ml for leaf-methanol and leaf-water extracts, respectively. Root extracts were less active. Cytotoxicity was observed only with the leaf-water extract (IC50 = 6 mg/ml). CONCLUSIONS: Further investigation is warranted to elucidate the potential of M. oleifera for clinically significant interactions with antiretroviral and other drugs.

Formulation and evaluation of a testosterone transdermal spray.

The long-term goal is to develop a spray formulation for transdermal testosterone delivery, and to optimize the drug's skin permeability. Testosterone transport from a series of ethanol/propylene glycol (PG)/water formulations was assessed in vitro across hairless rat skin, and the optimal composition determined. The formulation was then modified for delivery from a mechanical spray, and from an aerosol containing a high percentage of propellant. Drug transport was greatest from a saturated solution in 1:1:1 ethanol/PG/water (1.7 +/- 0.2 microg/cm(2) . h); five spray formulations were then tested, but only 1:1 ethanol/PG achieved a comparable flux. Increasing the % ethanol in the mixture increased evaporation rate but did not alter testosterone delivery. Formulation as an aerosol produced primarily unstable vehicles (phase separation, crystallization). Only 3:1 ethanol/PG remained stable, but no significant improvement in drug transport was observed (testosterone precipitated rapidly at the skin surface). The 1:1:1 ethanol/PG/water saturated solution suggested that some penetration enhancement was possible. Eliminating water to improve sprayability identified 1:1 ethanol/PG as a vehicle, which might allow transient supersaturation (and improved delivery). However, this effect was not improved by using a pressurized aerosol due to instability. Finally, testosterone fluxes were 5 to 10-fold lower than those required for useful transdermal therapy.