Preventive effects of ß-cryptoxanthin against cadmium-induced oxidative stress in the rat testis.

ß-cryptoxanthin (CRY), a major carotenoid of potential interest for health, is obtained naturally from orange vegetables and fruits. A few research studies have reported that CRY could decrease oxidative stress and germ cell apoptosis. The purpose of this study was to examine the effects of CRY on acute cadmium chloride (CdCl 2 )-induced oxidative damage in rat testes. For this study, 24 rats were divided into four groups, one of which serves as a control group that received intraperitoneal (i.p.) injections of corn oil and physiological saline. The other rats were i.p. injected with CRY (10 µg kg-1 ) every 8 h, beginning 8 h before CdCl 2 (2.0 mg kg-1 ) treatment. The pathological and TUNEL findings revealed that CRY ameliorated the Cd-induced testicular histological changes and germ cell apoptosis in the rats. Furthermore, the Cd-induced decrease in the testicular testosterone (T) level was attenuated after CRY administration (P < 0.05). The administration of CRY significantly reversed the Cd-induced increases in the lipid peroxide (LPO) and malondialdehyde (MDA) levels (P < 0.01). The testicular antioxidants superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) were decreased by treatment with Cd alone but were restored by CRY co-treatment. These results demonstrated that the application of CRY can enhance the tolerance of rats to Cd-induced oxidative damage and suggest that it has promised as a pharmacological agent to protect against Cd-induced testicular toxicity.

ß-Cryptoxanthin uptake in THP-1 macrophages upregulates the CYP27A1 signaling pathway.

SCOPE: Mitochondrial sterol 27-hydroxylase (CYP27A1), a mediator of cholesterol homeostasis, is reported to exhibit antiatherogenic properties. Many studies suggested that all-trans retinoic acid can be used to treat atherosclerosis through retinoic acid receptor (RAR)-mediated upregulation of CYP27A1 expression. In this study, we hypothesized that ß-cryptoxanthin (ß-cry), as a natural ligand of RAR, might act as antiatherogenic agent by upregulating CYP27A1. METHODS AND RESULTS: We found that ß-cry treatment significantly upregulated genes involved in the uptake, transport, and metabolism of retinoids and the signaling pathway of CYP27A1 expression in THP-1 macrophages as detected by microarray analysis. Meanwhile, intracellular levels of ß-cry were correlated to the concentration and exposure time of the treatment. The expression of genes, involved in signaling pathway of CYP27A1, was dramatically decreased due to repressed activity of RAR. Higher level of 27-hydroxycholesterol was detected in ß-cry-treated macrophages by HPLC. Docking simulation showed that ß-cry could interact with cellular retinoic acid binding protein 2. These findings were further confirmed through microarray results. CONCLUSION: Our results provide strong evidence that ß-cry can be actively taken up by THP-1 macrophages and exhibits antiatherogenic effect on THP-1 macrophages by inducing CYP27A1 expression via RAR.

The chemopreventive effect of ß-cryptoxanthin from mandarin on human stomach cells (BGC-823).

ß-Cryptoxanthin, a provitaminic carotenoid, present in many fruits and vegetables, has been associated with decreased risk of chronic diseases, including cancer. The influence of ß-cryptoxanthin derived from mandarin on the proliferation of the stomach tumor cell line BGC-823 was tested using MTT and cell count assay at 72 h and dose-response (from 0.01 to 20 µM). ß-Cryptoxanthin suppressed the cell migration by the scratch assay. Furthermore, ß-cryptoxanthin induced an accumulation of cells in the G1/G0 phase of the cell cycle (as detected by flow cytometry), which was in accordance with an increased expression of p21 and down regulations of cyclin D1 and cyclin E, detected by Western blot analysis, and ß-cryptoxanthin increased the mRNA levels of retinoic acid receptor ß (RARß) with the treatment at 10 µM for 24 h. Collectively, the above findings suggest that ß-cryptoxanthin could be therapeutic in the treatment of stomach cancer cell in vitro.

Characterization of the mechanism of inhibin α-subunit gene in mouse anterior pituitary cells by RNA interference.

Inhibin, a member of the transforming growth factor-ß [TGF-ß] superfamily, is a suppressor of follicle-stimulating hormone [FSH] release through pituitary-gonadal negative feedback loop to regulate follicular development. In this study, Inhibin α-subunit [Inha] gene was knocked down successfully in mice primary anterior pituitary cells at both transcriptional and translational levels by RNAi-Ready pSIREN-RetroQ-ZsGreen Vector mediated recombinant pshRNA vectors. The results indicated that inhibin silencing significantly promoted apoptosis by up-regulating Caspase-3, Bax and Bcl-2 genes without affecting p53 both at transcriptional and translational levels. Furthermore, it markedly impaired the progression of G1 phase of cell cycle and decreased the amount of cells in S phase [as detected by flow cytometry]. Inhibin silencing resulted in significant up-regulation of mRNA and protein expressions of Gondotropin releasing hormone receptors [GnRHR] and down-regulated mRNA levels of ß-glycans with parellel change in the amount of its protein expression. Silencing of inhibin-a significantly increased [P<0.05] activin-ß concentration without affecting FSH and LH levels in anterior pituitary cells. These findings revealed that up regulation of GnRH receptors by silencing inhibin a-subunit gene might increase the concentration of activin-ß in the culture medium. Inhibin a silencing resulted in increased mRNA and protein expressions of inhibinß which may demonstrate that both inhibin subunits co-participate in the regulation of reproductive events in anterior pituitary cells. This study concludes that inhibin is a broad regulatory marker in anterior pituitary cells by regulating apoptosis, cellular progression and simultaneously by vital fluctuations in the hormonal signaling.

GDF-9 and bFGF enhance the effect of FSH on the survival, activation, and growth of cattle primordial follicles.

This study aims to investigate the effects of follicle stimulating hormone (FSH) in combination with growth and differentiation factor-9 (GDF-9) or basic fibroblast growth factor (bFGF) on the activation, survival and growth of cattle primordial follicles. Ovarian tissues were cultured for 3, 7, 14, 22 days in α minimum essential medium (α-MEM) supplemented with FSH, FSH+GDF-9 or FSH+bFGF. Non-cultured and cultured ovarian fragments were processed for histological and TUNEL analysis. Compared to the FSH medium, the results showed FSH+GDF-9 medium increased the percentage of primary follicles in all culture periods and secondary follicles after 14 days of culture (P<0.05), meanwhile the diameter of primary and secondary follicles were also observed to increase in this medium after 7 days of cultures (P<0.05). FSH+bFGF medium appeared to increase the percentage of primary follicles after 14 days of culture and secondary follicles at day 14 of culture than FSH medium (P<0.05). Furthermore, the FSH+GDF-9 and FSH+bFGF mediums had a greater percentage of normal follicles, and lesser apoptotic cell rates than FSH medium. The results first indicated that FSH in combination with GDF-9 or bFGF can improve the survival, activation, and growth of cattle primordial follicles after the long-term culture of ovarian cortex.

Action mechanism of inhibin α-subunit on the development of Sertoli cells and first wave of spermatogenesis in mice.

Inhibin is an important marker of Sertoli cell (SC) activity in animals with impaired spermatogenesis. However, the precise relationship between inhibin and SC activity is unknown. To investigate this relationship, we partially silenced both the transcription and translation of the gene for the α-subunit of inhibin, Inha, using recombinant pshRNA vectors developed with RNAi-Ready pSIREN-RetroQ-ZsGreen Vector (Clontech Laboratories, Mountain View, Calif). We found that Inha silencing suppresses the cell-cycle regulators Cyclin D1 and Cyclin E and up-regulates the cell-cycle inhibitor P21 (as detected by Western blot analysis), thereby increasing the number of SCs in the G1 phase of the cell cycle and decreasing the amount in the S-phase of the cell cycle (as detected by flow cytometry). Inha silencing also suppressed Pdgfa, Igf1, and Kitl mRNA levels and up-regulated Tgfbrs, Inhba, Inhbb, Cyp11a1, Dhh, and Tjp1 mRNA levels (as indicated by real-time polymerase chain reaction [PCR] analysis). These findings indicate that Inha has the potential to influence the availability of the ligand inhibin and its antagonist activin in the SC in an autocrine manner and inhibit the progression of SC from G1 to S. It may also participate in the development of the blood-testis barrier, Leydig cells, and spermatogenesis through its effect on Dhh, Tjp1, Kitl, and Pdgfa. Real-time PCR and Western blot analyses of Inha, Inhba, and Inhbb mRNA and Inha levels over time show that Inha plays an important role in the formation of round spermatid during the first wave of spermatogenesis in mice.