Down-regulation of the autophagy gene, ATG7, protects bone marrow-derived mesenchymal stem cells from stressful conditions

BACKGROUND: Mesenchymal stem cells (MSCs) are valuable for cell-based therapy. However, their application is limited owing to their low survival rate when exposed to stressful conditions. Autophagy, the process by which cells recycle the cytoplasm and dispose of defective organelles, is activated by stress stimuli to adapt, tolerate adverse conditions, or trigger the apoptotic machinery. This study aimed to determine whether regulation of autophagy would affect the survival of MSCs under stress conditions. METHODS: Autophagy was induced in bone marrow-derived MSCs (BM-MSCs) by rapamycin, and was inhibited via shRNA-mediated knockdown of the autophagy specific gene, ATG7. ATG7 expression in BM-MSCs was evaluated by reverse transcription polymerase chain reaction (RT-PCR), western blot, and quantitative PCR (qPCR). Cells were then exposed to harsh microenvironments, and a water-soluble tetrazolium salt (WST)-1 assay was performed to determine the cytotoxic effects of the stressful conditions on cells. RESULTS: Of 4 specific ATG7-inhibitor clones analyzed, only shRNA clone 3 decreased ATG7 expression. Under normal conditions, the induction of autophagy slightly increased the viability of MSCs while autophagy inhibition decreased their viability. However, under stressful conditions such as hypoxia, serum deprivation, and oxidative stress, the induction of autophagy resulted in cell death, while its inhibition potentiated MSCs to withstand the stress conditions. The viability of autophagy-suppressed MSCs was significantly higher than that of relevant controls (P<0.05, P<0.01 and P<0.001). CONCLUSION: Autophagy modulation in MSCs can be proposed as a new strategy to improve their survival rate in stressful microenvironments.

Effect of a high fat diet on ovary morphology, in vitro development, in vitro fertilisation rate and oocyte quality in mice

<p><b>INTRODUCTION</b>The aim of this study was to determine the effect of a high-fat diet (HFD) on oocyte maturation and quality in a mouse model.</p><p><b>METHODS</b>Female BALB/c mice were allocated to one of the following groups: (a) control group (n = 40), which received a controlled diet; or (b) HFD group (n = 40), which received an HFD for 12 weeks. Sections of the ovary were examined histologically. The number of follicles and corpora lutea were counted. In vitro maturation and in vitro fertilisation (IVF) were assessed in germinal vesicle (GV) and metaphase II (MII) oocytes, respectively. The expression of bone morphogenetic protein 15 (BMP15) and leptin receptor genes in GV and MII oocytes was evaluated using reverse transcription real-time polymerase chain reactions.</p><p><b>RESULTS</b>In the HFD group, there was a decreased number of primordial and Graafian follicles, as well as corpora lutea (p < 0.05). The rate of oocyte development to the MII stage was also reduced (p < 0.001). Cumulus expansion was observed more frequently in the control group than the HFD group (p < 0.05). The IVF rate in the HFD group was lower than that in the control group (p < 0.05). In the HFD group, BMP15 and leptin receptor genes were upregulated in the GV stage (p > 0.05) and MII stage (p < 0.05), compared to the control group.</p><p><b>CONCLUSION</b>An HFD reduces folliculogenesis in the primordial and Graafian stages, in vitro maturation and in vitro fertilisation rates, as well as oocyte quality in mice.</p>

Protective effects of restricted diet and antioxidants on testis tissue in rats fed with high-fat diet

A high-fat diet [HFD] promotes the oxidative stress formation, which in turn has hazardous effects on reproductive system and fertility. The present study examines the potential positive effects of a restricted high fat diet [RHFD] and antioxidants consumption on sperm parameters and testis tissue in rats. Male rats [n = 48] were divided into four groups [12 in each group]: control group [Cont], HFD group, RHFD, and RHFD with astaxanthin and vitamins E and C group [RHFDA]. After 12 weeks, serum analysis and sperm parameters study were performed. Sections of fixed testes were stained with Hematoxilin and Eosin to study the histological changes. A one-way ANOVA was used to compare the data. HFD fed animals presented significant increase in weight load and serum low density lipoprotein [LDL-C] levels [P < 0.05]. The sperm count in RHFD was lower than three other groups [P < 0.05] and sperm motility of RHFDA group was significantly higher than HFD and RHFD groups [P < 0.05]. The histological study was showed a significant increase in spermatogonium number in RHFDA compared to three other groups [P < 0.05]. The number of spermatocyte I and spermatid in RHFD was significantly [P < 0.05] lower than Cont and HFD groups. HFD and obesity can affect sperm parameters and spermatogenesis and antioxidants consumption may improve their quality. Although the RHFD is a benefit way in weight loss and decrease of LDL-C of serum, but it is suggested that is not effective on sperm quality improvement

Protective effects of antioxidants on sperm parameters and seminiferous tubules epithelium in high fat-fed rats

Prescription of antioxidants might increase the quality of sperm parameters and improve the rate of pregnancy in obese people who suffer from infertility. Therefore, the present study investigated protective effects of vitamin A, E and astaxanthin on sperm parameters and seminiferous tubules epithelium in high-fat diet model. Thirty-six numbers of 3 months old albino Wistar rats were divided to control, high-fat diet and high-fat diet with antioxidants groups. After 12 weeks, levels of LDL-C and HDL-C were detected in the groups. Sperm was obtained from the tail of epididymis and its parameters [count, vitality, motility and morphology] were analyzed. Testes were fixed in 10% formalin and after tissue processing, stained with Hematoxylin and Eosine [H and E] for histological evaluation. Data were analyzed by a one-way ANOVA and p<0.05 was considered significant. Our results indicated that viability, motility and normal morphology of sperm in high-fat diet [HFD] decreased significantly compared to high-fat diet with antioxidant [HFD A] and the control groups [p<0.05]. Also spermatogonium and the number of Sertoli cells increased significantly in HFD+A compared to the control [p<0.05]. As it is shown in our study, application of antioxidants decreased serum triglyceride, cholesterol and HDL-C/LDL-C in high-fat diet model and improved the semen parameters. Therefore, it is suggested that the low quality of sperm can be improved in obese men through antioxidant prescription. Finally, it seems that the antioxidants in obese patients with subfertility or infertility is a new and efficient strategy with few side effects

Melatonin improves development of early mouse embryos impaired by actinomycin-D and TNF-alpha

Melatonin, a reactive oxygen species [ROS] scavenger and an antioxidant, has been shown that can inhibit apoptosis. Administration of melatonin may improve embryo development in assisted reproductive technology [ART]. The aim of this study was to evaluate the role of melatonin in inhibition of spontaneous and induced apoptosis by Tumor Necrosis Factor Alph [TNF-alpha] and actinomycin-D during preimplantation development of mouse embryos. Female BALB/c mice were superovulated with pregnant mare serum gonadotropin [PMSG] followed by human chorionic gonadotropin [HCG], then allowed to mate with male mice. The resultant 2-cell embryos were divided into six groups as follows: control [group I], melatonin [group II], actinomycin-D [group III], actinomycin-D + melatonin [group IV], TNF-alpha [group V], and TNF-alpha + melatonin [group VI]. We recorded the numbers and developmental rates of the 4-cell, 8-cell, morula and blastocyst embryos. Blastocysts were stained with acridine orange in order to assess for the embryo quality. The group IV showed a significantly higher developmental rate of blastocysts compared to group III [p<0.05]. The number of dead blastomers was significantly decreased in group IV in comparison to group III [p<0.05]. Both V and VI groups had a lower developmental rate and lesser quality of blastocysts compared with group I. There was no significant difference in the developmental rate of blastocysts from group II compared to group I [p<0.05]. Supplementation of embryo culture media with melatonin can improve the quality and developmental rate of embryos. Melatonin can prevent cell death that was induced by TNF- alpha and actinomycine-D

MS 14 down-regulates lipocalin2 expression in spinal cord tissue in an animal model of multiple sclerosis in female C57BL/6

Experimental autoimmune encephalomyelitis [EAE] is an animal model of multiple sclerosis, which is a demyelinating and an inflammatory disease of central nervous system. Recent studies have established that some molecules such as Lipocaline2 [LCN2], which expresses during inflammatory conditions, play an important role in EAE pathogenesis and might involve in its treatment process. Recently, it has been proved that MS 14, an herbal-marine drug, has anti-inflammatory properties through reduction of TNF-a and IL-lp. Thus, the present study investigated the effects of MS 14 on the course of EAE and its relation to LCN2 expression in both protein and gene levels. EAE was induced in female C57BL/6 mice using Hooke kits. Animals were scored for clinical signs of the disease according to a 10-point EAE scoring system. On 21[st] and 35[th] days after immunization, mice [n = 4/group] were deeply anesthetized, and the spinal cords were removed. Inflammatory cell infiltration and LCN2 expression in spinal cord were assessed by hematoxylin and eosin staining, immuno-histochemistry, and real-time PCR methods. MS 14 significantly ameliorated EAE symptoms and decreased lymphocyte infiltration into the spinal cord [P<0.05]. Our data also revealed that LCN2 expression was significantly down-regulated in acute and chronic phases of EAE both at protein and gene levels after MS 14 treatment [P<0.05]. The results demonstrated that MS 14 regulatory effect on EAE is accompanied by LCN2 down-regulation after treatment with the herb; however, more studies are required for clarifying the other involved mechanisms

HESA-A exerts its cytoprotective effects through scavenging of free radicals: an in vitro study

Natural medicines have been recently considered more reasonable for human use most notably due to their safety and tolerance. HESA-A is a marine-originated herbal medicine with a variety of healing effects. However, its exact biological mechanism is not clear. The present study aimed at the evaluation of the HESA-A antioxidant effect. Chinese hamster ovary [CHO] and human embryonic kidney [HEK293T] cells were treated with different concentrations of HESA-A and H2O2 followed by cell proliferation assays. The antioxidant effect of the HESA-A preparations was evaluated by an antioxidant assay kit. The viability of CHO and HEK293T cells were about 89% following their incubation with 100 and 200 ng/ml HESA-A, respectively for 1.5 hrs. However, when the cells were incubated with concentrations of 300 ng/ml or more, the cell viability significantly decreased to 48% compare to the control cells. The cytotoxic effects of H2O2 were observed after 2 hrs of incubation of the HEK293T or CHO cells with 10 mM or 16 mM H2O2, respectively, while in the presence of HESA-A the cytotoxicity was significantly decreased. Antioxidant assay revealed that HESA-A scavenges free radicals. The findings indicate that HESA-A had cytoprotective effects in vitro, and that such an effect might be due to antioxidant properties

Down-regulation of metallothionein 1 and 2 after exposure to electromagnetic field in mouse testis

It is proved that testis is sensitive to electromagnetic field [EMF] and its damage results in infertility. Exposure to EMF induces reactive oxygen species production and affects on anti-oxidants defense mechanisms. Metallothionein [MT] is a name for a group of low molecular weight [6-7 kDa], sulfhydryl rich proteins. Expression of MT1 and MT2 genes in testis tissue after EMF exposure was aimed in this study. Male BALB/c mice [8 weeks old] were exposed to 3 MT EMF for 8 weeks, 4 hours/day. After 8 weeks, the mice were sacrificed and the testis tissue was removed. The testis pieces were stained with hematoxylin and eosin and analyzed under an optical microscope. Assessment of MT1 and MT2 genes and also protein expression was performed by real-time PCR and Western-blot, respectively. In light microscopic observation, the number of primary spermatocytes was increased significantly in EMF group [P<0.01]. In addition, in interstitial space, the number of leydig cells was increased significantly in EMF group [P<0.01] and basement membrane thickness was increased as well. MT1 and MT2 genes were down-regulated significantly in testis tissue of mice exposed to EMF both in mRNA and protein level compared to control. It is clear that MT is mediated in testis development and spermatogenesis. Down-regulation of MT1 and MT2 after EMF in mouse testis might be followed by some consequences that result in infertility

High frequency electromagnetic field induces lipocalin 2 expression in mouse liver

Neutrophil gelatinase-associated lipocalin [NGAL/Lcn2], comprise a group of small extracellular proteins with a common beta-sheet-dominated 3-dimensional structure. In the past, it was assumed that the predominant role of lipocalin was acting as transport proteins. Recently it has been found that oxidative stress induces Lcn2 expression. It has been also proved that electromagnetic field [EMF] produces reactive oxygen species [ROS] in different tissues. Expression of Lcn2 following exposure to electromagnetic field has been investigated in this study. Balb/c mice [8 weeks old] were exposed to 3 mT, 50 HZ EMF for 2 months, 4 hr/day. Afterwards, the mice were sacrificed by cervical dislocation and livers were removed. The liver specimens were stained with Haematoxylin-Eosin [H and E] and analyzed under an optical microscope. Total RNA was extracted from liver and reverse transcription was performed by SuperScript III reverse transcriptase with 1 micro g of total RNA. Assessment of Lcn2 expression was performed by semiquantitative and real time-PCR. The light microscopic studies revealed that the number of lymphocyte cells was increased compared to control and dilation of sinosoids was observed in the liver. Lcn2 was up-regulated in the mice exposed to EMF both in mRNA and protein levels. To the extent of our knowledge, this is the first report dealing with up-regulation of Lcn2 in liver after exposure to EMF. The up-regulation might be a compensatory response that involves cell defense pathways and protective effects against ROS. However, further and complementary studies are required in this regards

Effects of leukemia inhibitory factor on gp130 expression and rate of metaphase II development during in vitro maturation of mouse oocyte

Leukemia inhibitory factor [LIF] is a 45-56 kDa glycoprotein that has an important role in proliferation and embryo implantation. Its effect on oocyte maturation and how to exert the function remained to be elucidated. Immature mice superovulated with human menopausal gonadotropin and germinal vesicle [GV] oocytes were obtained from ovary 48 hours after. GV oocytes were cultured in tissue culture medium 199 with 0, 100, 500 and 1,000 U/ml LIF. Cumulus expansion and in vitro maturation [IVM] rate were assessed after culture. For reverse transcriptase PCR, total RNA from GV and metaphase II [Mil] oocytes were extracted by Trizol reagent. The quantity and quality of RNA were determined by spectrophotometry and electrophoresis, respectively. Reverse transcription was performed by Super Script III reverse transcriptase with 1 micro g of total RNA followed by DNase I treatment and heat inactivation. Expression of gp130 was determined by RT-PCR. Our results showed that cumulus expansion was improved with 1,000 U/ml in culture medium compared to others. GV breakdown and Mil rate in groups with LIF were higher than control group and were dose dependant. In 1,000 U/ml, LIF rate of Mil was significantly higher than control group [P<0.05]. Our results also showed that gp130 is expressed neither in GV nor in Mil oocytes during IVM of mouse oocytes. gp130 is expressed in human oocyte but not in mouse. Our results suggest that in mouse, LIF could affect the oocyte via another receptor or via cumulus cells; however, further studies are warranted

influence of meiotic spindle configuration by cysteamine during in vitro maturation of mouse oocytes

The aim of this study was to assess effects of cysteamine as an anti-oxidant on rate of in vitro maturation of oocyte and determination of its effects on spindle size and shape. Pre-mature mice were primed with pregnant mare stimulating gonadotrophin [PMSG] and germinal vesicle [GV] stage oocytes were obtained 48 h after. The oocytes were cultured in tissue culture medium [TCM 199] with 50, 100, 200 and 500 microM cysteamine. Experiments also included a group of ovulated oocytes [in vivo matured] after priming with PMSG and human chorionic gonadotrophin. For spindle immuno-cytochemistry of metaphase II [MII], oocytes alpha and beta tubulin antibody were performed. Chromosome staining was performed with Hoechst. Our results showed that the rate of GV breakdown [GVBD] and MII increased in all cysteamine groups except in group cultured with 500 microM cysteamine. Immuno-cytochemistry analysis showed that spindle area in all in vitro Maturation oocytes increased when compared to in vivo oocytes. Spindle shape and size [spindle area] in 100 microM cysteamine in comparison to in vivo group was insignificant [P>0.05]. Our results revealed that cysteamine improved IVM rate in a dose dependant manner. The size and shape of spindle in the presence of given concentration of cysteamine was similar to in vivo. Therefore, cysteamine improved microtubule organization, rate of GVBD and MIl development