The effect of epididymosomes on the development of frozen-thawed mouse spermatogonial stem cells after culture in a decellularized testicular scaffold and transplantation into azoospermic mice.

PURPOSE: This study examined SSC proliferation on an epididymosome-enriched decellularized testicular matrix (DTM) hydrogel and spermatogenesis induction in azoospermic mice. METHODS: Epididymosomes were extracted and characterized using SEM and western blotting. After cryopreservation, thawed SSCs were cultured in a hydrogel-based three-dimensional (3D) culture containing 10 ng/mL GDNF or 20 µg/mL epididymosomes. SSCs were assessed using the MTT assay, flow cytometry, and qRT-PCR after two weeks of culture. The isolated SSCs were microinjected into the efferent ducts of busulfan-treated mice. DiI-labeled SSCs were followed, and cell homing was assessed after two weeks. After 8 weeks, the testes were evaluated using morphometric studies and immunohistochemistry. RESULTS: The expression of PLZF, TGF-ß, and miR-10b did not increase statistically significantly in the 3D + GDNF and 3D + epididymosome groups compared to the 3D group. Among the groups, the GDNF-treated group exhibited the highest expression of miR-21 (*P < 0.05). Caspase-3 expression was lower in the epididymosome-treated group than in the other groups (***P < 0.001). Compared to the 3D and negative control groups, the 3D + epididymosomes and 3D + GDNF groups showed an increase in spermatogenic cells. Immunohistochemical results confirmed the growth and differentiation of spermatogonial cells into spermatids in the treatment groups. CONCLUSION: The DTM hydrogel containing 20 µg/mL epididymosomes or 10 ng/mL GDNF is a novel and safe culture system that can support SSC proliferation in vitro to obtain adequate SSCs for transplantation success. It could be a novel therapeutic agent that could recover deregulated SSCs in azoospermic patients.

Telocytes response to cardiac growth induced by resistance exercise training and endurance exercise training in adult male rats.

Telocytes are interstitial cells found in different tissues, including cardiac stem cell niches. The purpose of this study was to investigate the response of the telocytes to the cardiac growth that occurs in response to resistance and endurance exercise trainings using rats distributed into control, endurance, and resistance training groups. Results revealed that the ratio of heart weight to body weight, cardiomycyte number, cardiomyocyte area, thickness of the left ventricular wall were significantly higher in the training groups compared to the control group. We observed increment in the cardiomyocytes surface area and thickness of the left ventricular wall in the resistance-training group than endurance-training group. We conclude that both resistance and endurance exercise trainings will lead to an increased number of cardiac telocytes, consequently, promote activity of the cardiac stem cells, and results in physiological cardiac growth, and this response does not seem to depend on the type of exercise.

Improving the process of spermatogenesis in azoospermic mice using spermatogonial stem cells co-cultured with epididymosomes in three-dimensional culture system.

AIMS: This study aimed to explore the effect of epididymosomes on the proliferative efficiency of spermatogonial stem cells (SSCs) in vitro and the resumption of spermatogenesis in the azoospermic mice. MAIN METHODS: The epididymosomes were extracted from the epididymis and characterized. SSCs were cultured in 2D (two-dimensional) and hydrogel-based 3D culture in the presence of 20 µg/mL epididymosome or 10 ng/mL GDNF. After two weeks of culture, the proliferation and purity of the separated SSCs were evaluated using the MTT test and flow cytometry, respectively. qRT-PCR was used to analyze PLZF, caspase-3, TGF-ß, miR-10b, and miR-21 expression levels. Then, SSCs grown in the 3D culture system were labeled by DiI and transplanted into azoospermic mice via the efferent duct. After 2 weeks, tracing of DiI and cell homing were evaluated. Subsequently, histomorphometric studies and immunohistochemistry analysis were performed in testes after eight weeks of transplantation. KEY FINDINGS: The expression of PLZF, TGF-ß, miR-10b, and miR-21 increased significantly (*p < 0.05) in the 3D + GDNF and 3D + epididymosomes groups than in the 2D group. Transplanted SSCs migrated into the seminiferous tubules of recipient mice and the number of spermatogenic cells and protein expression of PLZF, SCP3 and ACRBP in the 3D + GDNF and 3D + epididymosomes groups were considerably higher (∗ ∗ ∗ p < 0.001) compared to the azoospermic group. SIGNIFICANCE: This finding indicates that culturing SSCs on decellularized testicular matrix (DTM) hydrogel with 10 ng/mL GDNF or 20 µg/mL epididymosomes could lead to an increase in SSCs proliferation which provides a sufficient number of SSCs for successful transplantation in azoospermic mice.

The Effect of miR-106b-5p Expression in The Production of iPS-Like Cells from Mice SSCs during The Formation of Teratoma and The Three Embryonic Layers.

Objective: According to the mounting data, microRNAs (miRNAs) may play a key role in reprogramming. miR-106b
is considered as an enhancer in reprogramming efficiency. Based on induced pluripotent stem cells (iPSCs), cell treatments have a huge amount of potential. One of the main concerns about using iPSCs in therapeutic settings is the possibility of tumor formation. It is hypothesized that a procedure that can reprogram cells with less genetic manipulation reduces the possibility of tumorigenicity.
Materials and Methods: In this experimental study, miR-106b-5p transduced by pLV-miRNA vector into mice isolated spermatogonial stem cells (SSCs) to achieve iPS-like cells. Then the transduced cells were cultured in specific conditions to study the formation of three germ layers. The tumorigenicity of these iPS-like cells was investigated by transplantation into male BALB/C mice.
Results: We show that SSCs can be successfully reprogrammed into induced iPS-like cells by pLV-miRNA vector to transduce the hsa-mir-106b-5p into SSCs and generating osteogenic, neural and hepatoblast lineage cells in vitro as a result of pluripotency. Although these iPS-like cells are pluripotent, they cannot form palpable tumors in vivo.
Conclusion: These results demonstrate that infection of hsa-mir-106b-5p into SSCs can reprogram them into iPSCs
and advanced germ cell lineages without tumorigenicity. Also, a novel approach for studying the generation of iPSCs
and the application of iPS or iPS-like cells in regenerative medicine is presented.

miR-106b enhances human mesenchymal stem cell differentiation to spermatogonial stem cells under germ cell profile genes involved in TGF-b signaling pathways.

Mesenchymal stem cells can be differentiated into tissue-specific cells. MicroRNAs (miRNAs) regulate the translation of mRNAs involved in the growth and development of a variety of cells, including primordial germ cells (PGCs). This study evaluated male germ cell differentiation from human MSCs by miR-106b. The MSCs were obtained from human adipose tissue. The differentiation of MSCs into PGCs was accomplished by transfection of a lentiviral vector expressing miR-106b. MSCs were treated with bone morphogenic factor 4 as a control and also as a putative inducer of PGC differentiation. PGC was differentiated into spermatogonial-like cells by retinoic acid. Moreover, Dazl, Plzf, Stra8, Gfra, and Thy1 gene expressions were investigated using real-time PCR. Our results showed that Dazl, Plzf, and Stra8 genes that were treated with BMP4 and miR-106b did not show any significant difference, meaning that miR-106b, like BMP4, is able to differentiate PGC cells from MSCs. In spermatogonial-like cells, Thy1 was significantly unregulated in both the miR-106b and BMP4 groups. Our findings showed that miR-106b regulates the differentiation of MSCs into PGCs. miR-106b influences on the expression of Dazl, Plzf, and Stra8 genes in PGC and Gfra, Stra8, and Thy1 genes.

Differentiation of neonate mouse spermatogonia on two-dimensional and three-dimensional culture systems supplemented with d-Serine and Dizocilpine (MK-801).

N-methyl-d-aspartate (NMDA) modulates the spermatogenesis process through stimulating the steroid hormone biosynthesis. The aim of this study was to evaluate the effects of NMDA receptors agonists (d-Serine) and antagonists (MK801) on spermatogonia differentiation on decellularization testicular matrix (DTM) hydrogel scaffold. Four treatment groups were planned: 2D + D-Serine, 3D + D-Serine, 2D + MK801, and 3D + MK801. Results showed that cell viability was significantly decreased after 48 h in the 3D + D-Serine group and after 24 and 48 h in the 3D + MK801 group compared to the controls. The spermatogonia proliferation after two, four, and eight weeks was significantly increased in the 3D + D-Serine culture, while it was significantly reduced in the 2D + MK801 and 3D + MK801 groups after four and eight weeks. Real-time PCR results demonstrated that pre-meiotic gene (Plzf) expression was significantly increased only in the 3D + D-Serine culture compared to the control groups after four weeks of culture. The meiotic gene (Sycp3) expression was significantly increased in the 2D + D-Serine and 3D + D-Serine compared to the 2D controls after four and eight weeks. The post-meiotic gene (Tnp1) level in the 3D + D-Serine was significantly higher than the other groups. Flow-cytometry results indicated that the protein expression of Plzf (after four and eight weeks), Sycp3 (after eight weeks), and Tnp1 (after eight weeks) in the d-Serine-treated groups was significantly increased compared with the 2D control groups. There were not any significant changes in the gene expression of spermatogenic-related markers in MK801 culture media. However, a significant decrease in the protein levels of Plzf after eight weeks and Sycp3 after four and eight weeks was observed. In conclusion, the addition of NMDARs agonists (d-Serine) could be used to regulate the differentiation of spermatogonia in the 3D culture system.

MiR-106b-5p Regulates the Reprogramming of Spermatogonial Stem Cells into iPSC (Induced Pluripotent Stem Cell)-Like Cells

Background: Recent years have brought notable progress in raising the efficiency of the reprogramming technique so that approaches have evolved from known transgenic factors to only a few miRNAs. Nevertheless, there is a poor understanding of both the key factors and biological networks underlying this reprogramming. The present study aimed to investigate the potential of miR-106b-5p in regulating spermatogonial stem cells (SSCs) to induced pluripotent stem cell (iPSC)-like cells. Methods: We used SSCs because pluripotency is inducible in SSCs under defined culture conditions, and they have a few issues compared to other adult stem cells. As both signaling and post-transcriptional gene controls are critical for pluripotency regulation, we traced the expression of Oct-4, Sox-2, Klf-4, c-Myc, and Nanog (OSKMN). Besides, we considered miR-106b-5p targets using bioinformatic methods. Results: Our results showed that transfected SSCs with miR-106b-5p increased the expression of the OSKMN factors, which was significantly more than negative control groups. Moreover, using the functional miRNA enrichment analysis, online tools, and databases, we predicted that miR-106b-5p targeted a signaling pathway gene named MAPK1/ERK2, related to regulating stem cell pluripotency. Conclusion: Together, our data suggest that miR-106b-5p regulates the reprogramming of SSCs into iPSC-like cells. Furthermore, noteworthy progress in the in vitro development of SSCs indicates promise reservoirs and opportunities for future clinical trials.

miR-106b-5p Intensifies the Proliferative Potential of Spermatogonial Stem Cells as a Prerequisite for Male Infertility Treatment.

Although numerous studies have investigated the molecular basis of male infertility, various aspects of this area have remained uncovered. Over the past years, researchers have reported the significant potential of miRNAs in posttranscriptional regulatory roles. By targeting mRNAs, these notable molecules can modulate the processes related to male infertility. On the other side, the outstanding potential of male germline stem cells, SSCs, includes their application in infertility treatment. SSCs retain normal spermatogenesis and fertility by adjusting both SSC self-renewal and differentiation. Therefore, for the characterization and manipulation of SSCs, effective and efficient in vitro culture methods are essential in supporting their maintenance and development. In this regard, the present investigation was undertaken to evaluate the impact of one of the recently conspicuous miRNAs, miR-106b, in SSCs enrichment. As a result, we first found that the SSCs induced with miR-106b-5p highly express TGF-ß1, which is known as a regulator of epigenetic modifiers and downstream genes. We next sought to show that self-renewal markers, including c-Myc, Oct-4, and Sox2, are increased in the induced SSC group. The intended miRNA also induced the inhibitor of differentiation 4 (ID4) and aided to remain unmethylated in SSCs. Additionally, for the tumorigenicity possibility of the manipulation, we indicated that PTEN, a tumor-suppressor gene, expressed remarkably in the induced SSCs. In conclusion, our findings showed that miR-106b-5p enhances the proliferative potential of SSCs, making it a substantial factor for therapeutic strategies of male infertility.

Effect of NMDA receptor agonist and antagonist on spermatogonial stem cells proliferation in 2- and 3- dimensional culture systems.

BACKGROUND: The main purpose of this study was to investigate the effect of D-serine (DS) and Dizocilpine (MK-801) on the proliferation of spermatogonial stem cells (SSCs) in two-dimensional (2D) and three-dimensional (3D) culture systems. METHODS AND RESULTS: The SSCs of male NMRI mice were isolated by enzymatic digestion and cultured for two weeks. Then, the identity of SSCs was validated by anti-Plzf and anti-GFR-α1 antibodies via immunocytochemistry (ICC). The proliferation capacity of SSCs was evaluated by their culture on a layer of the decellularized testicular matrix (DTM) prepared from mouse testis, as well as two-dimensional (2D) with different mediums. After two weeks of the initiation of proliferation culture on 3D and 2D medium, the pre-meiotic at the mRNA and protein levels were evaluated via qRT-PCR and flow cytometry methods, respectively. The results showed that the proliferation rate of SSCs in 3D culture with 50 mM glutamic acid and 20 mM D-serine was significantly different from other groups after 14 days treatment. mRNA expression levels of promyelocytic leukemia zinc finger (Plzf) in 3D cultures supplemented by 20 mM D-serine and 50 mM glutamic acid were considerably higher than the 3D control group (p < 0.001). The flow cytometry analysis revealed that the amount of Plzf in the 2D-culture groups of SSCs with 20 mM MK-801 was considerably lower compared to the 2D-culture control group (p < 0.001). CONCLUSIONS: This study indicated that decellularized testicular matrix supplemented with D-serine and glutamic acid could be considered a promising vehicle to support cells and provide an appropriate niche for the proliferation of SSCs.

Potentilla reptans L. postconditioning protects reperfusion injury via the RISK/SAFE pathways in an isolated rat heart.

BACKGROUND: Our previous study indicated that Potentilla reptans root has a preconditioning effect by its antioxidant and anti-apoptotic effects in an isolated rat heart ischemia/reperfusion (IR) model. In the present study, we investigated the post-conditioning cardio-protective effects of Potentilla reptans and its active substances. METHODS: The ethyl acetate fraction of P. reptans root (Et) was subjected to an IR model under 30 min of ischemia and 100 min of reperfusion. To investigate the postconditioning effect, Et was perfused for 15 min at the early phase of reperfusion. RISK/SAFE pathway inhibitors, 5HD and L-NAME, were applied individually 10 min before the ischemia, either alone or in combination with Et during the early reperfusion phase. The hemodynamic factors and ventricular arrhythmia were calculated during the reperfusion. Oxidative stress, apoptosis markers, GSK-3ß and SGK1 proteins were assessed at the end of experiments. RESULTS: Et postconditioning (Etpost) significantly reduced the infarct size, arrhythmia score, ventricular fibrillation incidence, and enhanced the hemodynamic parameters by decreasing the MDA level and increasing expression of Nrf2, SOD and CAT activities. Meanwhile, Etpost increased the BCl-2/BAX ratio and decreased Caspase-3 expression. The cardioprotective effect of Etpost was abrogated by L-NAME, Wortmannin (a PI3K/Akt inhibitor), and AG490 (a JAK/STAT3 inhibitor). Finally, Etpost reduced the expression of GSK-3ß and SGK1 proteins pertaining to the IR group. CONCLUSION: P. reptans reveals the post-conditioning effects via the Nrf2 pathway, NO release, and the RISK/SAFE pathway. Also, Etpost decreased apoptotic indexes by inhibiting GSK-3ß and SGK1 expressions. Hence, our data suggest that Etpost can be a suitable natural candidate to protect cardiomyocytes during reperfusion injury.

Mir-106b Cluster Regulates Primordial Germ Cells Differentiation from Human Mesenchymal Stem Cells.

OBJECTIVE: Numerous evidence indicates that microRNAs (miRNAs) are critical regulators in the spermatogenesis process. The aim of this study was to investigate miR-106b cluster regulates primordial germ cells (PGCs) differentiation from human mesenchymal stem cells (MSCs). MATERIALS AND METHODS: In this experimental study, samples containing male adipose (n: 9 samples- age: 25-40 years) were obtained from cosmetic surgeries performed for the liposuction in Imam Khomeini Hospital. The differentiation of MSCs into PGCs was accomplished by transfection of a lentivector expressing miR-106b. The transfection of miR-106b was also confirmed by the detection of a clear green fluorescent protein (GFP) signal in MSCs. MSCs were treated with bone morphogenic factor 4 (BMP4) protein, as a putative inducer of PGCs differentiation, to induce the differentiation of MSCs into PGCs (positive control). After 4 days of transfection, the expression of miR-106b, STELLA, and FRAGILIS genes was evaluated by real-time polymerase chain reaction (PCR). Also, the levels of thymocyte differentiation antigen 1 (Thy1) protein was assessed by the western blot analysis. The cell surface expression of CD90 was also determined by immunocytochemistry method. The cytotoxicity of miR-106b was examined in MSCs after 24, 48, and 72 hours using the MTT assay. RESULTS: MSCs treated with BMP4 or transfected by miR-106b were successfully differentiated into PGCs. The results of this study also showed that the expression of miR-106b was significantly increased after 48 hours from transfection. Also, we showed STELLA, FARGILIS, as well as the protein expression of Thy1, was significantly higher in MSCs transfected by lentivector expressing miR-106b in comparison with MSCs treated with BMP4 (P≤0.05). MTT assay showed miR-106b was no toxic during 72 hours in 1 µg/ml dose, that this amount could elevated germ cells marker significantly higher than other experimental groups (P≤0.05). CONCLUSION: According to this findings, it appears that miR-106b plays an essential role in the differentiation of MSCs into PGCs.

Comparison of two methods for prolong storage of decellularized mouse whole testis for tissue engineering application: An experimental study.

BACKGROUND: Biological scaffolds are derived by the decellularization of tissues or organs. Various biological scaffolds, such as scaffolds for the liver, lung, esophagus, dermis, and human testicles, have been produced. Their application in tissue engineering has created the need for cryopreservation processes to store these scaffolds. OBJECTIVE: The aim was to compare the two methods for prolong storage testicular scaffolds. MATERIALS AND METHODS: In this experimental study, 20 male NMRI mice (8 wk) were sacrificed and their testes were removed and treated with 0.5% sodium dodecyl sulfate followed by Triton X-100 0.5%. The efficiency of decellularization was determined by histology and DNA quantification. Testicular scaffolds were stored in phosphate-buffered saline solution at 4°C or cryopreserved by programmed slow freezing followed by storage in liquid nitrogen. Masson's trichrome staining, Alcian blue staining and immunohistochemistry, collagen assay, and glycosaminoglycan assay were done prior to and after six months of storage under each condition. RESULTS: Hematoxylin-eosin staining showed no remnant cells after the completion of decellularization. DNA content analysis indicated that approximately 98% of the DNA was removed from the tissue (p = 0.02). Histological evaluation confirmed the preservation of extracellular matrix components in the fresh and frozen-thawed scaffolds. Extracellular matrix components were decreased by 4°C-stored scaffolds. Cytotoxicity tests with mouse embryonic fibroblast showed that the scaffolds were biocompatible and did not have a harmful effect on the proliferation of mouse embryonic fibroblast cells. CONCLUSION: Our results demonstrated the superiority of the slow freezing method for prolong storage of testicular scaffolds.

Induction of Apoptosis in the U937 Cell Line Co-cultured with Adipose-derived Stem Cells Secreting Bone Morphogenetic Protein-4.

Transforming growth factor-beta (TGF-ß) plays a significant role in tumorigenesis. MiR-181b is a multifunctional miRNA involved in numerous cellular processes, such as cell fate and cell invasion. This study aimed to examine whether the co-culture of adipose-derived stem cells (ADSCs), highly expressing bone morphogenetic protein-4, with the U937 cell line, which is a human myeloid leukemia cell line, is able to induce cell death in this cancer cell line, considering the potential ability of ADSCs to migrate from tumor sites. Cell surface markers, namely CD73 and CD105, were analyzed to verify the identity of mesenchymal stem cells isolated from adipose tissue. Besides, the osteogenic and adipogenic differentiation potentials of ADSCs were evaluated. The induction of cell death and apoptosis in the U937 cell line was assessed using MTT and annexin V/ PI assays, respectively. The expression levels of miR-181 and TGF-ß were determined in the co-culture system using real-time PCR. The results of MTT and annexin V/ PI assays showed that BMP4-expressing ADSCs could inhibit cell viability and induce apoptosis in U937 cells in the co-culture system. The co-culture of ADSCs, highly expressing BMP-4, with the U937 cell line led to the downregulation of miR-181 and TGF-ß genes in the human cancer cell line. ADSCs may further be studied as a candidate for the treatment of hematological cancers.

Preconditioning and anti-apoptotic effects of Metformin and Cyclosporine-A in an isolated bile duct-ligated rat heart.

Despite all previous studies relating to the mechanism of cirrhotic cardiomyopathy (CCM), the role of cirrhosis on Ischemic Preconditioning (IPC) has not yet been explored. The present study strives to assess the cardioprotective role of IPC in bile duct ligated (BDL) rats as well as the cardioprotective role of Cyclosporin-A (CsA) and Metformin (Met) in CCM. Cirrhosis was induced by bile duct ligation (BDL). Rats' hearts were isolated and attached to a Langendorff Apparatus. The pharmacological preconditioning with Met and CsA was done before the main ischemia. Myocardial infarct size, hemodynamic and electrophysiological parameters, biochemical markers, and apoptotic indices were determined at the end of the experiment. Infarct size, apoptotic indices, arrhythmia score, and incidence of VF decreased significantly in the IPC group in comparison with the I/R group. These significant decreases were abolished in the IPC (BDL) group. Met significantly decreased the infarct size and apoptotic indices compared with I/R (BDL) and normal groups, while CsA led to similar decreases except in the level of caspase-3 and -8. Met and CsA decreased and increased the arrhythmia score and incidence of VF in the BDL groups, respectively. Functional recovery indices decreased in the I/R (BDL) and IPC (BDL) groups. Met improved these parameters. Therefore, the current study depicted that the cardioprotective effect of Met and CsA on BDL rats is mediated through the balance between pAMPK and apoptosis in the mitochondria.

The mouse testis tissue culture could resume spermatogenesis as same as in vivo condition after human spermatogonial stem cells transplantation.

OBJECTIVE: The introduction of alternative systems in vivo is very important for cancer patients who are treated with gonadotoxic treatment. In this study, we examine the progression of the spermatogenesis process after human spermatogonial stem cell (SSCs) transplantation in vivo and in tissue culture conditions. MATERIALS AND METHODS: Human SSCs were obtained from a Testicular Sperm Extractions (TESE) sample, and characterization of these cells was confirmed by detecting the promyelocytic leukemia zinc finger (PLZF) protein. These cells, after being labeled with Di-alkyl Indocarbocyanine (DiI), were transplanted to adult azoospermia mouse testes treated with Busulfan 40mg/kg. The host testicular tissue culture was then considered a test group and in vivo transplant a control group. After 8 weeks, immunohistochemical, morphometric and molecular studies were performed. RESULTS: The results of morphometric studies indicated that the mean number of spermatogonia, spermatocytes, and spermatids in the test groups was significantly lower than in the control group (P<0.05) and most of the cells responded positively to DiI tracing. Immunohistochemical study in both groups revealed expression of PLZF, Synaptonemal complex protein 3 (SCP3) and Acrosin Binding Protein (ACRBP) proteins in spermatogonial cells, spermatocyte and spermatozoa, respectively. Also, PLZF, Transition Protein 1 (TP1) and Tektin-1 (Tekt1) human-specific genes had a significant difference in the between test groups and control groups (P<0.05) in molecular studies. CONCLUSION: These results suggest that the conditions of testicular tissue culture after transplantation of SSCs can support spermatogenesis resumption, as well as in an in vivo condition.

Characterization of embryonic stem-like cells derived from mouse spermatogonial stem cells following low-intensity ultrasound treatment.

OBJECTIVE: Spermatogonial stem cells (SSCs) are able to form embryonic stem-like cells (ES-like cells) and embryonic bodies (EBs). Low-intensity ultrasound stimulation (LIUS) has positive effects on the growth and differentiation of the different cells. In this study, we tried to investigate the effects of LIUS on SSC differentiation to ES-like cells. MATERIALS AND METHODS: SSCs were isolated from neonatal mice and their identification was confirmed by tracking of PLZF, Oct-4, and C-Kit proteins. The SSCs and Sertoli cells were co-cultured in DMEM/F12 supplemented with 15% FBS and LIF. SSCs stimulated by LIUS with 200mW/CM2 intensity. Characterization of obtained ES-like cells was confirmed with Sox2, Oct-4, and SSEA-1 immunofluorescence staining. Also, real-time PCR was performed to analyse the expression of c-Myc and Nanog genes in ES-Like Cells and Stra8, Piwil2 and Plzf genes in SSCs after 21 days of the in vitro culture. RESULTS: Our results showed c-Kit, PLZF and Oct-4 proteins were expressed positively in SSCs and Sox2, Oct-4, SSEA-1 in the ES-like cells by immunocytochemistry. The results of flow cytometry showed a significant increase in expression of c-Myc and Nanog in ES-like cells compared to SSCs (p<.05), whereas the Stra8, Piwil2, and Plzf became down-regulated during 21 days of culture. ES-like markers cell SSEA-1, Sox2 and Oct-4 were increased in the LIUS group compared to the control group (p<.05). CONCLUSION: The results indicated that ES-like cells with pluripotency characteristics were derived from SSCs.

Oxytocin Reduces Brain Injury and Maintains Blood-Brain Barrier Integrity After Ischemic Stroke in Mice.

The present study was designed to determine the effect of different doses of oxytocin (OXT) on neuronal injury, spatial memory, blood-brain barrier (BBB) integrity and to explore possible underlying molecular mechanisms in the early stage of stroke in mice. Stroke model was generated by middle cerebral artery occlusion (MCAO) for 60 min and 24 h reperfusion in mice. OXT at doses of 1, 2, 4 and 8 IU/per mouse was administrated intranasally at the beginning of brain ischemia. Brain injury, BBB integrity, and spatial memory were evaluated by standard methods. Changes in the expression of nuclear factor-kappa B (NF-κB), and TUNEL positive cell were detected by immunohistochemistry. The levels of vascular endothelial growth factor (VEGF), aquaporin-4 (AQP4) and brain-derived neurotrophic factor (BDNF) proteins were determined by western blotting and ELISA methods. OXT at doses of 4 and 8 IU/per mouse reduced the infarct size by 42% and 52%, respectively, and improved spatial memory function (p < 0.001). OXT (8 IU/per mouse) significantly reduced brain edema, BBB disruption and upregulated the AQP4 expression (p < 0.001). Finally, OXT significantly diminished the number of apoptotic, NF-κB positive cells and enhanced the expression of BDNF and VEGF proteins in the brain tissue (p < 0.001). These findings provide important evidences that OXT significantly suppresses neuronal damage in the early stage of stroke by inhibiting apoptotic and NF-κB signaling pathway, increasing the expression of VEGF, AQP4 and BDNF proteins and reducing the BBB leakage.

The Effects of Calligonum Extract and Low-Intensity Ultrasound on Motility, Viability, and DNA Fragmentation of Human Frozen-Thawed Semen Samples.

BACKGROUND: The study aimed to evaluate the impact of Calligonum extract and US radiation on sperm parameters of cryopreserved human semen samples. MATERIALS AND METHODS: In this experimental study, twenty-five semen specimens were obtained from healthy semen donors and incubated in human tubal fluid (HTF) medium supplemented with 10% human serum albumin (HSA) for 45 minutes. Samples were treated with Calligonum extract (10 µg/ml) alone (CGM group) and US radiation (LIPUSexposed group) alone or a combination of both treatments (CGM+LIPUS). The US group received US stimulation (in both continuous and pulsed wave modes) at a frequency of 1 MHZ and intensity of 200 mW/cm2 for 200 seconds. Sperm morphology was assessed by Diff-Quik staining. The DNA fragmentation was evaluated the Halo sperm kit. Sperm parameters was analyzed by a computer-assisted semen analysis system. Reactive oxygen species (ROS) was assessed by flow cytometry. RESULTS: The results showed that the treatment with Calligonum extract significantly (P<0.05) increased the progressive motility of spermatozoa in the CGM group as compared with the control group. The application of low-intensity US significantly (P<0.05) decreased the motility and viability of spermatozoa in the US group when compared with the control group. Our findings also indicated that the use of both low-intensity US in continuous mode and Calligonum extract slightly increased progressive motility; however, such an increase was not statistically significant. The rate of DNA fragmentation was considerably higher (P<0.05) in control and LIPUS-exposed groups than the other groups. CONCLUSION: Treatment of spermatozoa with Calligonum extract slightly improved the sperm parameters due to its antioxidant activity, on the other hand, according to our results, US radiation did not improve sperm parameters which may be due to interference with the motility of sperm, as well as its physical effects on spermatozoa.

Three-Dimensional Culture of Mouse Spermatogonial Stem Cells Using A Decellularised Testicular Scaffold.

OBJECTIVE: Applications of biological scaffolds for regenerative medicine are increasing. Such scaffolds improve cell attachment, migration, proliferation and differentiation. In the current study decellularised mouse whole testis was used as a natural 3 dimensional (3D) scaffold for culturing spermatogonial stem cells. MATERIALS AND METHODS: In this experimental study, adult mouse whole testes were decellularised using sodium dodecyl sulfate (SDS) and Triton X-100. The efficiency of decellularisation was determined by histology and DNA quantification. Masson's trichrome staining, alcian blue staining, and immunohistochemistry (IHC) were done for validation of extracellular matrix (ECM) proteins. These scaffolds were recellularised through injection of mouse spermatogonial stem cells in to rete testis. Then, they were cultured for eight weeks. Recellularised scaffolds were assessed by histology, real-time polymerase chain reaction (PCR) and IHC. RESULTS: Haematoxylin-eosin (H and E) staining showed that the cells were successfully removed by SDS and Triton X-100. DNA content analysis indicated that 98% of the DNA was removed from the testis. This confirmed that our decellularisation protocol was efficient. Masson's trichrome and alcian blue staining respectively showed that glycosaminoglycans (GAGs) and collagen are preserved in the scaffolds. IHC analysis confirmed the preservation of fibronectin, collagen IV, and laminin. MTT assay indicated that the scaffolds were cell-compatible. Histological evaluation of recellularised scaffolds showed that injected cells were settled on the basement membrane of the seminiferous tubule. Analyses of gene expression using real-time PCR indicated that expression of the Plzf gene was unchanged over the time while expression of Sycp3 gene was increased significantly (P=0.003) after eight weeks in culture, suggesting that the spermatogonial stem cells started meiosis. IHC confirmed that PLZF-positive cells (spermatogonial stem cells) and SYCP3-positive cells (spermatocytes) were present in seminiferous tubules. CONCLUSION: Spermatogonial stem cells could proliferate and differentiated in to spermatocytes after being injected in the decellularised testicular scaffolds.

The Combination of Metformin and Disulfiram-Cu for Effective Radiosensitization on Glioblastoma Cells.

OBJECTIVE: Glioblastoma (GBM) is one of the devastating types of primary brain tumors with a negligible response to standard therapy. Repurposing drugs, such as disulfiram (DSF) and metformin (Met) have shown antitumor properties in different cell lines, including GBM. In the present study, we focused on the combinatory effect of Met and DSF-Cu on the induction of apoptosis in U87-MG cells exposed to 6-MV X-ray beams. MATERIALS AND METHODS: In this experimental study, the MTT assay was performed to evaluate the cytotoxicity of each drug, along with the combinatory use of both. After irradiation, the apoptotic cells were assessed using the flow cytometry, western blot, and real-time polymerase chain reaction (RT-PCR) to analyze the expression of some cell death markers such as BAX and BCL-2. RESULTS: The synergistic application of both Met and DSF had cytotoxic impacts on the U87-MG cell line and made them sensitized to irradiation. The combinatory usage of both drugs significantly decreased the cells growth, induced apoptosis, and caused the upregulation of BAX, P53, CASPASE-3, and it also markedly downregulated the expression of the anti-apoptotic protein BCL-2 at the gene and protein levels. CONCLUSION: It seems that the synergistic application of both Met and DSF with the support of irradiation can remarkably restrict the growth of the U87-MG cell line. This may trigger apoptosis via the stimulation of the intrinsic pathway. The combinatory use of Met and DSF in the presence of irradiation could be applied for patients afflicted with GBM.