Reductive power generated by Mycobacterium leprae through cholesterol oxidation contributes to lipid and ATP synthesis

Upon infection, Mycobacterium leprae, an obligate intracellular bacillus, induces accumulation of cholesterol-enriched lipid droplets (LDs) in Schwann cells (SCs). LDs are promptly recruited to M. leprae-containing phagosomes, and inhibition of this process decreases bacterial survival, suggesting that LD recruitment constitutes a mechanism by which host-derived lipids are delivered to intracellular M. leprae. We previously demonstrated that M. leprae has preserved only the capacity to oxidize cholesterol to cholestenone, the first step of the normal cholesterol catabolic pathway. In this study we investigated the biochemical relevance of cholesterol oxidation on bacterial pathogenesis in SCs. Firstly, we showed that M. leprae increases the uptake of LDL-cholesterol by infected SCs. Moreover, fluorescence microscopy analysis revealed a close association between M. leprae and the internalized LDL-cholesterol within the host cell. By using Mycobacterium smegmatis mutant strains complemented with M. leprae genes, we demonstrated that ml1942 coding for 3ß-hydroxysteroid dehydrogenase (3ß-HSD), but not ml0389 originally annotated as cholesterol oxidase (ChoD), was responsible for the cholesterol oxidation activity detected in M. leprae. The 3ß-HSD activity generates the electron donors NADH and NADPH that, respectively, fuel the M. leprae respiratory chain and provide reductive power for the biosynthesis of the dominant bacterial cell wall lipids phthiocerol dimycocerosate (PDIM) and phenolic glycolipid (PGL)-I. Inhibition of M. leprae 3ß-HSD activity with the 17ß-[N-(2,5-di-t-butylphenyl)carbamoyl]-6-azaandrost-4-en-3one (compound 1), decreased bacterial intracellular survival in SCs. In conclusion, our findings confirm the accumulation of cholesterol in infected SCs and its potential delivery to the intracellular bacterium. Furthermore, we provide strong evidence that cholesterol oxidation is an essential catabolic pathway for M. leprae pathogenicity and point to 3ß-HSD as a prime drug target that may be used in combination with current multidrug regimens to shorten leprosy treatment and ameliorate nerve damage.

hCG Improves Luteal Function and Promotes Progesterone Output through the Activation of JNK Pathway in the Luteal Granulosa Cells of the Stimulated IVF Cycles†.

Human chorionic gonadotropin (hCG) is a luteotropic hormone that promotes the survival and steroidogenic activity of corpus luteum (CL) by acting through luteinizing hormone receptors (LHRs) expressed on luteinized theca and granulosa cells (GCs). Therefore, it is used to support luteal phase in in vitro fertilization (IVF) cycles to improve clinical pregnancy rates and prevent miscarriage. However, the molecular mechanism underlying this action of hCG is not well characterized. To address this question, we designed an in vitro translational research study on the luteal GCs obtained from 58 IVF patients. hCG treatment at different concentrations and time points activated c-Jun N-terminal kinase (JNK) pathway and significantly increased its endogenous kinase activity along with upregulated expression of steroidogenic enzymes (steroidogenic acute regulatory protein (stAR), 3ß-Hydroxysteroid dehydrogenase (3ß-HSD)) in a dose-dependent manner in the luteal GCs. As a result, in vitro P production of the cells was significantly enhanced after hCG. When JNK pathway was inhibited pharmacologically or knocked-down with small interfering RNA luteal function was compromised, P4 production was declined along with the expression of stAR and 3ß-HSD in the cells. Further, hCG treatment after JNK inhibition failed to correct the luteal defect and promote P4 output. Similar to hCG, luteinizing hormone (LH) treatment improved luteal function as well and this action of LH was associated with JNK activation in the luteal GCs. These findings could be important from the perspective of CL biology and luteal phase in human because we for the first time identify a critical role for JNK signaling pathway downstream LHR activation by hCG/LH in luteal GCs. SUMMARY SENTENCE: JNK signaling pathway plays a central role in the upregulated expression of the steroidogenic enzymes StAR and 3b-HSD and augmented progesterone production by hCG/LH in human luteal granulosa cells.

Correlation between function-morphology of effect of the stress and flunitrazepam administered during gestation in adult mouse ovary / Correlación entre la función y la morfología de los efectos de estrés y flunitrazepam administrado durante la gestación en ovario de ratón adulto

Previously we report long lasting effects on ovary of mice prenatally exposed to flunitrazepam (FNZ), a benzodiazepine with tranquilized action. In this work we find that the FNZ don't prevent the effects on ovary prenatally exposure to stress in mice. We studied adult females born from mothers that had been stressed by immobilization on day 6 ofgestation (GD-6) or group S, and from mothers stressed also by immobilization at GD-6, but which received a single oral dose of FNZ immediately after the stress group FNZS. The control groups were the SS that received the GD-6 saline solution and the group NT non-treated. Their ovaries were extracted for histology studies and to observe the activity of 3b hydroxysteroid dehydrogenase/isomerase (3 b-HSD). The histological analysis revealed high staining affinity ovarian cell of S and FNZS. Double oocytes and apoptotic bodies were found in the secondary atretic follicles, as well as abnormal primordial, primary and secondary follicle populations, as compared to SS and NT groups. The primordial, primary, and secondary follicles were significantly reduced in the experimental groups. But the primary and secondary atretic follicles were higher in both groups, and the number of corpora lutea was lower in both groups. The activity of 3 b-HSD was abnormally increased in both FNZS- and S-groups. These findings suggest that FNZ did not counteract the impairing effects of prenatal stress on adult offspring ovarian follicles, and could rather be responsible for long lasting changes occurring during embryonic programming.

Previamente comprobamos efectos de larga duración sobre el ovario de ratones expuestos prenatalmente a flunitrazepam (FNZ), una benzodiazepina con acción tranquilizante. En este trabajo encontramos que el FNZ, no revierte los efectos producidos por la exposición prenatal a estrés. Estudiamos hembras adultas nacidas de madres que se estresaron por inmovilización el día 6 de la gestación (DG-6) o grupo S, y de madres estresadas también por inmovilización el DG-6, las que recibieron una sola dosis de FNZ inmediatamente después del estrés (grupo FNZS). Los grupos de control fueron el SS al que se le administró solución salina y el NT no tratado. Se extrajeron sus ovarios para su estudio histológico y para observar la actividad de delta 3b-deshidroxiesteroide dehidrogenasa/isomerasa (3 b-HSD). El análisis histológico reveló una gran afinidad tintoreal en los ovarios de los grupos S y FNZS. En los ovarios de los ratones del grupo FNZS se encontraron en los folículos secundarios atrésicos ovocitos dobles y cuerpos apoptóticos así como una población mayor de folículos anormales primordiales, primarios y secundarios en comparación con los grupos SS y NT. Los folículos primarios y secundarios tuvieron una reducción significativa en los grupos experimentales pero los folículos atrésicos primarios y secundarios fueron más en ambos grupos y el número de cuerpos lúteos fue menor en ambos grupos. La actividad de 3 b-HSD aumentó de manera anormal tanto en los grupos FNZ y S. Estos hallazgos sugieren que el FNZ no contrarresta los efectos negativos del estrés prenatal sobre los folículos ováricos de las crías adultas, y podría ser responsable de los cambios largo plazo que ocurren a durante la programación embrionaria.

Effect of ethanol extract of whole plant of Trichosanthes cucumerina var. cucumerina L. on gonadotropins, ovarian follicular kinetics and estrous cycle for screening of antifertility activity in Albino Rats / Efectos del etanol extraído de la planta completa de Trichosanthes cucumerina var. cucumerina L. sobre gonadotropinas, cinética folicular ovárica y ciclo para screening de actividad antifertilidad en Ratas Albinas

Ethanol extract of whole plant of Trichosanthes cucumerina L. var. cucumerina was evaluated for antiovulatory activity in adult rats. The ethanol extract at the doses 200 and 400mg/kg body weight (orally) affected the normal estrous cycle showing a significant increase in estrus and metestrus phases and decrease in diestras and proestrus phases. The extract also significantly reduced the number of healthy follicles (Class I-Class VI) and corpora lutea and increased the number of regressing follicles (Stage IA, Stage IB, Stage IIA, and Stage IIB). The protein and glycogen content in the ovaries were significantly reduced in treated rats. The cholesterol level was significantly increased, whereas, the enzyme activities like 3b-HSD and 17b-HSD were significantly inhibited in the ovary of treated rats. Serum FSH and LH levels were significantly reduced in the treated groups were measured by RIA. In acute toxicity test, neither mortality nor change in the behavior or any other physiological activities in mice were observed in the treated groups. In chronic toxicity studies, no mortality was recorded and there were no significant differences in the body and organ weights were observed between controls and treated rats. Hematological analysis showed no significant differences in any of the parameters examined (RBC, WBC count and Hemoglobin estimation). These observations showed the antiovulatory activity of ethanol extract of whole plant of Trichosanthes cucumerina L. var. cucumerina in female albino rats.

El extracto de etanol de toda la planta de Trichosanthes cucumerina var. cucumerina (L.) se evaluó en cuanto a su actividad antiovulatoria en ratas adultas. El extracto de etanol en dosis de 200 y 400mg/kg de peso corporal (oral) afectó el ciclo normal estral, mostrando un aumentó significativo en las fases de estro y metaestro y la disminución de las fases de diestro y proestro. El extracto también redujo significativamente el número de folículos sanos (Clase I=Clase VI) y cuerpo lúteo y aumentó el número de folículos en regresión (etapa I, etapa IB, etapa II y etapa IIB). La proteína y el contenido de glucógeno en los ovarios se redujeron significativamente en las ratas tratadas. El nivel de colesterol aumentó significativamente, mientras que, actividad de las enzimas 3b-HSD y 17b-HSD se inhibió significativamente en el ovario de ratas tratadas. FSH sérico y los niveles de LH se redujeron significativamente en los grupos tratados y medidos por RÍA. En la prueba de toxicidad aguda, no hubo mortalidad ni cambio en el comportamiento fisiológico o de cualquier otra actividad en los grupos tratados de ratas. En estudios de toxicidad crónica, no se registró mortalidad y no hubo diferencias significativas en el peso corporal o el peso de los órganos entre los controles y las ratas tratadas. Los análisis hematológicos no mostraron diferencias significativas en ninguno de los parámetros examinados (eritrocitos, recuento de glóbulos blancos y estimación de hemoglobina). Estas observaciones mostraron la actividad antiovulatoria del extracto de etanol de toda la planta de Trichosanthes cucumerina var. cucumerina en ratas albinas hembras.