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The heavy metal cadmium is proposed to be one of the environmental endocrine disruptors of spermatogenesis. Cadmium-induced inhibition of spermatogenesis is associated with a hormone secretion disorder. Letrozole is an aromatase inhibitor that increases peripheral androgen levels and stimulates spermatogenesis. However, the potential protective effects of letrozole on cadmium-induced reproductive toxicity remain to be elucidated. In this study, male mice were administered CdCl 2 (4 mg/kg BW) orally by gavage alone or in combination with letrozole (0.25 mg/kg BW) for 30 days. Cd exposure caused a significant decreases in body weight, sperm count, motility, vitality, and plasma testosterone levels. Histopathological changes revealed extensive vacuolization and decreased spermatozoa in the lumen. However, in the Cd + letrozole group, letrozole treatment compensated for deficits in sperm parameters (count, motility, and vitality) induced by Cd. Letrozole treatment significantly increased serum testosterone levels, which were reduced by Cd. Histopathological studies revealed a systematic array of all germ cells, a preserved basement membrane and relatively less vacuolization. For a mechanistic examination, RNA-seq was used to profile alterations in gene expression in response to letrozole. Compared with that in the Cd-treated group, RNA-Seq analysis showed that 214 genes were differentially expressed in the presence of letrozole. Gene ontology (GO) enrichment analysis and KEGG signaling pathway analysis showed that steroid biosynthetic processes were the processes most affected by letrozole treatment. Furthermore, we found that the expression of the testosterone synthesis-related genes LHCGR (luteinizing hormone/choriogonadotropin receptor) and Hsd3b6 (3 beta- and steroid delta-isomerase 6) was significantly downregulated in Cd‐treated testes, but these genes maintained similar expression levels in letrozole-treated testes as those in the control group. However, the transcription levels of inflammatory cytokines, such as IL-1β and IL-6, and oxidative stress-related genes (Nrf2, Nqo1, and Ho-1) showed no changes. The present study suggests that the potential protective effect of letrozole on Cd-induced reproductive toxicity might be mediated by the upregulation of LHCGR and Hsd3b6, which would beneficially increase testosterone synthesis to achieve optimum protection of sperm quality and spermatogenesis.

Naegleria fowleri is a free-living amoeba that can also act as an opportunistic pathogen causing severe brain infection, primary amebic meningoencephalitis (PAM), in humans. The high mortality rate of PAM (exceeding 97%) is attributed to (i) delayed diagnosis, (ii) lack of safe and effective anti-N. fowleri drugs, and (iii) difficulty of delivering drugs to the brain. Our work addresses identification of new molecular targets that may link anti-Naegleria drug discovery to the existing pharmacopeia of brain-penetrant drugs. Using inhibitors with known mechanism of action as molecular probes, we mapped the sterol biosynthesis pathway of N. fowleri by GC-MS analysis of metabolites. Based on this analysis, we chemically validated two enzymes downstream to CYP51, sterol C24-methyltransferase (SMT, ERG6) and sterol Δ8-Δ7 -isomerase (ERG2), as potential therapeutic drug targets in N. fowleri. The sterol biosynthetic cascade in N. fowleri displayed a mixture of canonical features peculiar to different domains of life: lower eukaryotes, plants and vertebrates. In addition to the cycloartenol→ergosterol biosynthetic route, a route leading to de novo cholesterol biosynthesis emerged. Isotopic labeling of the de novo-synthesized sterols by feeding N. gruberi trophozoites on the U13C-glucose-containing growth medium identified an exogenous origin of cholesterol, while 7-dehydrocholesterol (7DHC) had enriched 13C-content, suggesting a dual origin of this metabolite both from de novo biosynthesis and metabolism of scavenged cholesterol. Sterol homeostasis in Naegleria may be orchestrated over the course of its life-cycle by a \"switch\" between ergosterol and cholesterol biosynthesis. By demonstrating the growth inhibition and synergistic effects of the sterol biosynthesis inhibitors, we validated new, potentially druggable, molecular targets in N. fowleri. The similarity of the Naegleria sterol Δ8-Δ7 -isomerase to the human non-opioid σ1 receptor, implicated in human CNS conditions such as addiction, amnesia, pain and depression, provides an incentive to assess structurally diverse small-molecule brain-penetrant drugs targeting the human receptor for anti-Naegleria activity.

Regeneration of myelin is mediated by oligodendrocyte progenitor cells—an abundant stem cell population in the central nervous system (CNS) and the principal source of new myelinating oligodendrocytes. Loss of myelin-producing oligodendrocytes in the CNS underlies a number of neurological diseases, including multiple sclerosis and diverse genetic diseases 1 – 3 . High-throughput chemical screening approaches have been used to identify small molecules that stimulate the formation of oligodendrocytes from oligodendrocyte progenitor cells and functionally enhance remyelination in vivo 4 – 10 . Here we show that a wide range of these pro-myelinating small molecules function not through their canonical targets but by directly inhibiting CYP51, TM7SF2, or EBP, a narrow range of enzymes within the cholesterol biosynthesis pathway. Subsequent accumulation of the 8,9-unsaturated sterol substrates of these enzymes is a key mechanistic node that promotes oligodendrocyte formation, as 8,9-unsaturated sterols are effective when supplied to oligodendrocyte progenitor cells in purified form whereas analogous sterols that lack this structural feature have no effect. Collectively, our results define a unifying sterol-based mechanism of action for most known small-molecule enhancers of oligodendrocyte formation and highlight specific targets to propel the development of optimal remyelinating therapeutics. Many small molecules that stimulate oligodendrocyte formation act not through their canonical pathways but by inhibiting enzymes within the cholesterol biosynthesis pathway and thereby inducing the accumulation of 8,9-unsaturated sterols.

Recent studies reported the important role of autophagy in follicular development. However, the underlying molecular mechanisms remain elusive. In this study, we investigated the effect of follicle-stimulating hormone (FSH) on mouse granulosa cells (MGCs). Results indicated that autophagy was induced by FSH, which is known to be the dominant hormone regulating follicular development and granulosa cell (GC) proliferation. The activation of mammalian target of rapamycin (mTOR), a master regulator of autophagy, was inhibited during the process of MGC autophagy. Moreover, MHY1485 (an agonist of mTOR) significantly suppressed autophagy signaling by activating mTOR. The expression of hypoxia-inducible factor 1-alpha (HIF-1 α ) was increased after FSH treatment. Blocking hypoxia-inducible factor 1-alpha attenuated autophagy signaling. In vitro , CoCl 2 -induced hypoxia enhanced cell autophagy and affected the expression of beclin1 and BCL2/adenovirus E1B interacting protein 3 (Bnip3) in the presence of FSH. Knockdown of beclin1 and Bnip3 suppressed autophagy signaling in MGCs. Furthermore, our in vivo study demonstrated that the FSH-induced increase in weight was significantly reduced after effectively inhibiting autophagy with chloroquine, which was correlated with incomplete mitophagy process through the PINK1-Parkin pathway, delayed cell cycle, and reduced cell proliferation rate. In addition, chloroquine treatment decreased inhibin alpha subunit, but enhanced the expression of 3 beta-hydroxysteroid dehydrogenase. Blocking autophagy resulted in a significantly lower percentage of antral and preovulatory follicles after FSH stimulation. In conclusion, our results indicate that FSH induces autophagy signaling in MGCs via HIF-1 α . In addition, our results provide evidence that autophagy induced by FSH is related to follicle development and atresia.

The existence of sphingolipid- and sterol-enriched microdomains, known as lipid rafts, in the plasma membrane (PM) of eukaryotic cells is well documented. To obtain more insight into the lipid molecular species required for the formation of microdomains in plants, we have isolated detergent (Triton X-100)-resistant membranes (DRMs) from the PM of Arabidopsis (Arabidopsis thaliana) and leek (Allium porrum) seedlings as well as from Arabidopsis cell cultures. Here, we show that all DRM preparations are enriched in sterols, sterylglucosides, and glucosylceramides (GluCer) and depleted in glycerophospholipids. The GluCer of DRMs from leek seedlings contain hydroxypalmitic acid. We investigated the role of sterols in DRM formation along the secretory pathway in leek seedlings. We present evidence for the presence of DRMs in both the PM and the Golgi apparatus but not in the endoplasmic reticulum. In leek seedlings treated with fenpropimorph, a sterol biosynthesis inhibitor, the usual D5-sterols are replaced by 9b,19-cyclopropylsterols. In these plants, sterols and hydroxypalmitic acid-containing GluCer do not reach the PM, and most DRMs are recovered from the Golgi apparatus, indicating that D5-sterols and GluCer play a crucial role in lipid microdomain formation and delivery to the PM. In addition, DRM formation in Arabidopsis cells is shown to depend on the unsaturation degree of fatty acyl chains as evidenced by the dramatic decrease in the amount of DRMs prepared from the Arabidopsis mutants, fad2 and Fad31, affected in their fatty acid desaturases.

Aromatase inhibitors (AIs) have been reported to exert their antiproliferative effects in postmenopausal women with hormone receptor-positive breast cancer not only by reducing estrogen production but also by unmasking the inhibitory effects of androgens such as testosterone (TS) and dihydrotestosterone (DHT). However, the role of androgens in AI-resistance mechanisms is not sufficiently understood. 5α-Androstane-3β,17β-diol (3β-diol) generated from DHT by 3β-hydroxysteroid dehydrogenase type 1 (HSD3B1) shows androgenic and substantial estrogenic activities, representing a potential mechanism of AI resistance. Estrogen response element (ERE)-green fluorescent protein (GFP)-transfected MCF-7 breast cancer cells (E10 cells) were cultured for 3 months under steroid-depleted, TS-supplemented conditions. Among the surviving cells, two stable variants showing androgen metabolite-dependent ER activity were selected by monitoring GFP expression. We investigated the process of adaptation to androgen-abundant conditions and the role of androgens in AI-resistance mechanisms in these variant cell lines. The variant cell lines showed increased growth and induction of estrogen-responsive genes rather than androgen-responsive genes after stimulation with androgens or 3β-diol. Further analysis suggested that increased expression of HSD3B1 and reduced expression of androgen receptor (AR) promoted adaptation to androgen-abundant conditions, as indicated by the increased conversion of DHT into 3β-diol by HSD3B1 and AR signal reduction. Furthermore, in parental E10 cells, ectopic expression of HSD3B1 or inhibition of AR resulted in adaptation to androgen-abundant conditions. Coculture with stromal cells to mimic local estrogen production from androgens reduced cell sensitivity to AIs compared with parental E10 cells. These results suggest that increased expression of HSD3B1 and reduced expression of AR might reduce the sensitivity to AIs as demonstrated by enhanced androgen metabolite-induced ER activation and growth mechanisms. Androgen metabolite-dependent growth of breast cancer cells may therefore play a role in AI-resistance.

The hydra mutants of Arabidopsis are characterized by a pleiotropic phenotype that shows defective embryonic and seedling cell patterning, morphogenesis, and root growth. We demonstrate that the HYDRA1 gene encodes a Δ8-Δ7 sterol isomerase, whereas HYDRA2 encodes a sterol C14 reductase, previously identified as the FACKEL gene product. Seedlings mutant for each gene are similarly defective in the concentrations of the three major Arabidopsis sterols. Promoter::reporter gene analysis showed misexpression of the auxin-regulated DR5 and ACS1 promoters and of the epidermal cell file-specific GL2 promoter in the mutants. The mutants exhibit enhanced responses to auxin. The phenotypes can be rescued partially by inhibition of auxin and ethylene signaling but not by exogenous sterols or brassinosteroids. We propose a model in which correct sterol profiles are required for regulated auxin and ethylene signaling through effects on membrane function.

Background:Androgenic anabolic steroids (AASs) are synthetic drugs structurally related to testosterone, with the ability to bind to androgen receptors. Their uncontrolled use by professional and recreational sportspeople is a widespread problem. AAS abuse is correlated with severe damage to the cardiovascular system, including changes in homeostasis and coagulation disorders. AASs alter vascular function by blocking nitric oxide (NO)-mediated dilation, impairing endothelial growth and by potentiating vasoconstrictor signals.Methods:This paper demonstrated that long-term use of AASs (nandrolone and boldenone), negatively affects the basic cell functions of vascular endothelial cells. The susceptibility of endothelial cells to AASs depends on the expression of androgen receptors, although cells without androgen receptors can also be affected by high doses of AASs to a limited extent. Seven-day incubation with AASs diminishes endothelial cell proliferation and migration (determined by transwell and scratch migration assay) and monolayer formation (using transendothelial electrical resistance assay).Results:Disturbances in cell function were accompanied by downregulation of peroxiredoxins (PRDX1 and PRDX2), involved in maintaining the thiol-disulphide balance. In addition, AASs increased oxidation of the non-enzymatic thiol buffer, glutathione (GSH), reduced secretion of thiol oxidoreductase protein disulphide isomerase (PDI) from endothelial cells and affected the thiol pattern of PDI.Conclusions:These changes may be related to a thiol-disulfide imbalance and vascular endothelium dysfunction, that are often correlated with abnormal platelet aggregation, inflammation, increased vascular permeability, and vascular smooth muscle cell proliferation—all of which are observed in athletes who abuse AASs.

Inflammatory bowel disease (IBD) is associated with dysregulation of both innate and adaptive immune response in the intestine. MicroRNA (miR)-155 is frequently expressed and functions in many immune cell types. Besides its function in adaptive immunity, miR-155 is a key regulator of the innate immune response in macrophages, dendritic cells, and even in epithelia cells. Although the roles of miR-155 within T and B lymphocytes in colitis have been reported, its function in innate immune cells has not been thoroughly examined. In this study, the dextran sulfate sodium (DSS)-induced colitis model was established in wild-type (WT) and miR-155 mice. Our results showed that miR-155 deficiency in macrophages recapitulated the alleviated colitis feature of miR-155 mice and appeared to skew toward the alterative M2 phenotype. Notably, the predominance of M2 in colon can result in dampened intestinal immune cell proliferation and inhibit CD4 T cell polarization toward Th1 and Th17. Moreover, C/EBPβ and SOCS1 were demonstrated as two key functional targets in this process. We also provided evidence for use of miR-155 inhibitor to treat colitis. Collectively, the findings highlight the central role of alternative M2 skewing for miR-155 function in colitis and reveal that macrophages might be a main target for therapeutics.

Cell suspension culture has the potential to be a valuable source for the bioactive compound productions. In this study, an optimized procedure was established for callus and cell suspension culture of Physalis alkekengi for the first time, and the impact of static magnetic field (SMF, 6 mT) was studied on the high-value metabolic compounds through investigation of signaling molecules and gene expressions at the late log-to-stationary phase. Results showed that the growth regulators of 6-benzyl amino purine (BAP, 1.5 mg−1 L) and 1-naphthaleneacetic acid (NAA, 0.4 mg−1 L) induced the highest fresh weight, callus rate, callus index, and total withanolides. Cell suspension culture was established in the liquid MS medium supplied with BAP (1.5 mg−1 L) and NAA (0.1 mg−1 L). SMF application decreased slightly the cell growth and viability and enhanced the number of round-shaped cells. The hydrogen peroxide (H2O2) and nitric oxide (NO) levels increased at an all-time series after SMF exposure, and their maximum contents were observed after 12 h. A significant alteration of malondialdehyde content was also identified after 12 h of SMF exposure. The expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), 1-deoxyD-xylulose 5-phosphate synthase (DXS), squalene synthase (SQS), sterol Δ7-reductase (DWF5), and C-7,8 sterol isomerase (HYD1) genes was upregulated significantly after 24 and 48 h. An increase in the total withanolides was related to more activity of HMGR and DXS enzymes in SMF-exposed cells and the maximum physalin A (12.8 mg g−1 DW) and physalin B (1.92 mg g−1 DW) obtained after 24 h compared to controls. Findings suggest that SMF can play a supportive factor in inducing steroidal compounds in P. alkekengi through modulating H2O2 and NO levels and the related-gene expressions.