Cerumenolytic Effects of Carbamide Peroxide in Patients with Ear Wax Obstruction.

Introduction: Accumulated and compacted ear wax or cerumen can cause conductive hearing loss, discomfort and vertigo, and infection. This study investigates the effect of Carbamide peroxide (CP) compared with Phenol glycerin (PG) ear drops on cerumen. Materials and Methods: This experimental study investigated the effect of PG and CP ear drops on cerumen in ex vivo and in vivo phases. In the ex vivo phase cerumen degredation was scored following PG and CP treatments. In the in vivo phase, 29 patients with bilateral cerumen impaction were randomly entered the study. PG and CP were applied 3 times a day (each time 5 drops) for 4 days by patients. After treatments, the time of cerumen removal was measured. Results: Instant changes showing degredation of cerumen (grade 1) was evident when it was exposed to CP, on the other hand degredation changes (grade 1) in cerumen treated with PG was only evident after 20 min incubation at 37 oC, while grade 3 degredation was evident in cerumen treated with CP after the same time incubation. Although the time needed for removal of cerumen was lower in CP treatment (54.10±31.77) compared to PG treatment (67.10±35.54), the difference was not statistically significant. Conclusion: Based on the literature and our results, carbamide peroxide is suggested as a proper treatment for patients with EAC obstruction caused by cerumen compaction, because not only it is significantly effective in cerumen degredation, but also no side effects have been reported.

Enhancing differentiation of menstrual blood-derived stem cells into female germ cells using a bilayer amniotic membrane and nano-fibrous fibroin scaffold.

Three-dimensional nanofiber scaffolds offer a promising method for simulating in vivo conditions within the laboratory. This study aims to investigate the influence of a bilayer amniochorionic membrane/nanofibrous fibroin scaffold on the differentiation of human menstrual blood mesenchymal stromal/stem cells (MenSCs) into female germ cells. MenSCs were isolated and assigned to four culture groups: (i) MenSCs co-cultured with granulosa cells (GCs) using the scaffold (3D-T group), (ii) MenSCs using the scaffold alone (3D-C group), (iii) MenSCs co-cultured only with GCs (2D-T group), and (iv) MenSCs without co-culture or scaffold (2D-C group). Both MenSCs and GCs were independently cultured for two weeks before co-culturing was initiated. Flow cytometry was employed to characterize MenSCs based on positive markers (CD73, CD90, and CD105) and negative markers (CD45 and CD133). Additionally, flow cytometry and immunocytochemistry were used to characterize the GCs. Differentiated MenSCs were analyzed using real-time PCR and immunostaining. The real-time PCR results demonstrated significantly higher levels of VASA expression in the 3D-T group compared to the 3D-C, 2D-T, and 2D-C groups. Similarly, the SCP3 mRNA level in the 3D-T group was notably elevated compared to the 3D-C, 2D-T, and 2D-C groups. Moreover, the expression of GDF9 was significantly higher in the 3D-T group when compared to the 3D-C, 2D-T, and 2D-C groups. Immunostaining results revealed a lack of signal for VASA, SCP3, or GDF9 markers in the 2D-T group, while some cells in the 3D-T group exhibited positive staining for all these proteins. These findings suggest that the combination of a bilayer amniochorionic membrane/nanofibrous fibroin scaffold with co-culturing GCs facilitates the differentiation of MenSCs into female germ cells.

Generation of Haploid Spermatids on Silk Fibroin-Alginate-Laminin-Based Porous 3D Scaffolds.

In vitro production of sperm is a desirable idea for fertility preservation in azoospermic men and prepubertal boys suffering from cancer. In this study, a biocompatible porous scaffold based on a triad mixture of silk fibroin (SF), alginate (Alg), and laminin (LM) is developed to facilitate the differentiation of mouse spermatogonia stem cells (SSCs). Following SF extraction, the content is analyzed by SDS-PAGE and stable porous 3D scaffolds are successfully prepared by merely Alg, SF, and a combination of Alg-SF, or Alg-SF-LM through freeze-drying. Then, the biomimetic scaffolds are characterized regarding the structural and biological properties, water absorption capacity, biocompatibility, biodegradability, and mechanical behavior. Neonatal mice testicular cells are seeded on three-dimensional scaffolds and their differentiation efficiency is evaluated using real-time PCR, flow cytometry, immunohistochemistry. Blend matrices showed uniform porous microstructures with interconnected networks, which maintained long-term stability and mechanical properties better than homogenous structures. Molecular analysis of the cells after 21 days of culture showed that the expression of differentiation-related proteins in cells that are developed in composite scaffolds is significantly higher than in other groups. The application of a composite system can lead to the differentiation of SSCs, paving the way for a novel infertility treatment landscape in the future.

Cannabidiol impairs the rewarding effects of methamphetamine: Involvement of dopaminergic receptors in the nucleus accumbens.

Cannabidiol, as component of cannabis, can potentially hinder the rewarding impact of drug abuse; however, its mechanism is ambiguous. Moreover, the nucleus accumbens (NAc), as a key area in the reward circuit, extensively receives dopaminergic projections from the ventral tegmentum area. To elucidate the role of accumbal D1 and D2 dopamine receptor families in Cannabidiol's inhibitory impact on the acquisition and expression phases of methamphetamine (MET), the conditioned place preference (CPP) procedure as a common method to assay reward characteristics of drugs was carried out. Six groups of rats were treated by various doses of SCH23390 or Sulpiride (0.25, 1, and 4 µg/0.5 µL) in the NAc as D1 or D2 dopamine receptor family antagonists, respectively, prior to infusion of Cannabidiol (10 µg/5 µL) in the lateral ventricle (LV) over conditioning phase in the acquisition experiments. In the second step of the study, animals received SCH23390 or Sulpiride in the NAc before Cannabidiol (50 µg/5 µL) infusion into the LV in the expression phase of MET to illuminate the influence of SCH23390 or Sulpiride on the inhibitory impact of Cannabidiol on the expression of MET-induced CPP. Intra-NAc administration of either SCH23390 or Sulpiride impaired Cannabidiol's suppressive impact on the expression phase, while just Sulpiride could suppress the Cannabidiol's impact on the acquisition phase of the MET-induced CPP. Also, the inhibitory impact of Sulpiride was stranger in both phases of MET reward. It seems that Cannabidiol prevents the expression and acquisition phases of MET-induced CPP partly through the dopaminergic system in the NAc.

The Role of MicroRNA 143 and MicroRNA 206 in The Regulation of Apoptosis in Mouse Lukemia Cancer Cells and Spermatogonial Cells.

OBJECTIVE: In cancer treatments, smart gene delivery via nanoparticles (NPs) can be targeted for cancer cells, while concurrently minimizing damage to healthy cells. This study assessed the efficiency of poly lactic-co-glycolic acid (PLGA)-miR 143/206 transfection on apoptosis in mouse leukemia cancer cells (El4) and spermatogonial stem cells (SSCs). MATERIALS AND METHODS: In this experimental study, neonatal mouse spermatogonia cells and EL4 cancer cell lines were used. MicroRNA-PLGA NPs were prepared, characterized, and targeted with folate. Several doses were evaluated to obtain a suitable miR dose that can induce appropriate apoptosis in EL4 cells, while not harming SSCs. Cells were treated separately at 3 doses of each miR (for miR 143, doses of 25, 50 and 75 nmol and for miR 206, doses of 50, 100 and 150 nmol). The experiments were performed at 24, 48 and 72 hours. Viability and apoptosis were investigated by MTT and Annexin Kits. RESULTS: Based on MTT assay results, the optimal dose of miR 143 was 75 nmol (59.87 ± 2.85 % SSC and 35.3 ± 0.78 % EL4) (P≤0.05), and for miR 206, the optimal dose was 150 nmol (54.82 ± 6.7 % SSC and 33.92 ± 3.01% EL4) (P≤0.05). The optimal time was 48 hours. At these doses, the survival rate of the EL4 cells was below the half maximal inhibitory concentration (IC50) and SSC survival was above 50%. Annexin V staining also confirmed the selected doses (for miR 143 total apoptosis was 6.62% ± 1.8 SSC and 37.4% ± 4.2 EL4 (P≤0.05), and miR 206 was (10.98% ± 1.5 SSC and 36.4% ± 3.7 EL4, P≤0.05). CONCLUSION: Using intelligent transfection by NPs, we were able to induce apoptosis on EL4 cells and maintain acceptable SSC survival rates.

Human Umbilical Cord Mesenchymal Stem Cells Differentiated into Neuron-Like Cells via Laminin and Schwann Cells.

BACKGROUND: Human umbilical cord mesenchymal stem cells (hUMSCs) have been considered to repair damaged tissues and cells. This study aims to investigate the differentiation efficiency affected by Schwann cells (SCs) and laminin and also compare them to other strategies using chemicals or growth factors. METHODS: SCs and hUMSCs were separated from dorsal root ganglion of rats and newborn human umbilical cords (hUCs), respectively, and then cultured. The marker expressions of mesenchymal stem cells (MSCs), hematopoietic and endothelial for hUMSCs were confirmed by flow cytometry. The hUMSCs were cultured in four groups: 1) control, 2) co-culture with SCs (C), 3) laminin (L), and 4) co-culture with SCs treated by laminin (CL). The expression of protein and gene-related differentiation NSE, MAP2 and ß-tubulin were examined by real-time polymerase chain reaction (PCR) and immunocytochemistry after 12 days. RESULTS: The flow cytometry analysis revealed high expression of mesenchymal and low expression of hematopoietic and endothelial markers, where the SCs expressed S100 at a high level (97.4%±2.25). The expression of NSE, MAP2 and ß-tubulin increased significantly in the C, L and CL groups compared to the control group (P<0.001), where the CL group had the highest expression among the groups [7.59±0.126, 7.87±0.191, 6.36±0.420, respectively, (P<0.01)]. Also, the expression of neural proteins was significantly increased in tested groups in comparison to the control group. CONCLUSION: Combined laminin and SCs co-culturing with hUMCSs could be the most effective strategy for neural differentiation.

Optimization of decellularized human placental macroporous scaffolds for spermatogonial stem cells homing.

Decellularized scaffolds have been found to be excellent platforms for tissue engineering applications. The attempts are still being made to optimize a decellularization protocol with successful removal of the cells with minimal damages to extracellular matrix components. We examined twelve decellularization procedures using different concentrations of Sodium dodecyl sulfate and Triton X-100 (alone or in combination), and incubation time points of 15 or 30 min. Then, the potential of the decellularized scaffold as a three-dimensional substrate for colony formation capacity of mouse spermatogonial stem cells was determined. The morphological, degradation, biocompatibility, and swelling properties of the samples were fully characterized. The 0.5%/30 SDS/Triton showed optimal decellularization with minimal negative effects on ECM (P ≤ 0.05). The swelling ratios increased with the increase of SDS and Triton concentration and incubation time. Only 0.5%/15 and 30 SDS showed a significant decrease in the SSCs viability compared with other groups (P < 0.05). The SSCs colony formation was clearly observed under SEM and H&E stained slides. The cells infiltrated into the subcutaneously implanted scaffold at days 7 and 30 post-implantation with no sign of graft rejection. Our data suggest the %0.5/30 SDS/Triton as an excellent platform for tissue engineering and reproductive biology applications.

Human umbilical cord mesenchymal stem cells express cholinergic neuron markers during co-culture with amniotic membrane cells and retinoic acid induction.

Background: A wide variety of cytokines are released from human amniotic membrane cells (hAMCs), which can increase the rate of differentiation of mesenchymal stem cells into the neurons. We studied the effect of Retinoic Acid (RA) on the differentiation rate of human Umbilical Cord Mesenchymal Stem Cells (hUMSCs) which were co-cultured with hAMCs. Methods: In this experimental study, both hUMSCs and hAMCs were isolated from postpartum human umbilical cords and placenta respectively. The expression of mesenchymal (CD73, CD90 and CD105), hematopoietic and endothelial (CD34 and CD45) markers in hUMSCs were confirmed by flow cytometry. The hUMSCs were cultured in four distinct groups: group 1) Control, group 2) Co-culture with hAMCs, group 3) RA treatment and group 4) Co-culture with hAMCs treated by RA. Twelve days after culturing, the expression of NSE, MAP2 and ChAT differentiation genes and their related proteins were examined by real-time PCR and immunocytochemistry respectively. Results: The flow-cytometry analysis indicated increased expression of mesenchymal markers and a low expression of both hematopoietic and endothelial markers (CD73:98.24%, CD90: 97.32%, CD105: 90.75%, CD34: 2.96%, and CD45:1.74%). Moreover, the expression of both NSE and MAP2 markers was increased significantly in all studied groups in comparison to the control group On the other hand, the expression of ChAT had a significant increase in the group 2 and 4 (RA and RA+ co-culture). Conclusion: RA can be used as an effective inducer to differentiate hUMSCs into cholinergic-like cells, and hAMCs could increase the number of differentiated cells as an effective factor.

Simvastatin accelerates the healing process of burn wound in Wistar rats through Akt/mTOR signaling pathway.

Statins, apart from cholesterol-lowering properties, have wound healing effects. Hereby, we aimed to assess the impact of Simvastatin (SMV), one of the most commonly used statins, on Akt/mTOR signaling pathway during burn wound healing process. After creating a second-degree burn on the dorsal area of adult male Wistar rats (n = 60), they were randomly divided into the control, SMV, vehicle of Simvastatin (SMV-Veh), Rapamycin (RM), vehicle of Rapamycin (RM-Veh), and combined SMV and RM (SMV + RM) groups. The animals were sacrificed on the 7th and 14th post-burn days and wound tissue samples were collected for histologic, immunohistochemical, quantitative real-time polymerase chain reaction (qRT-PCR), and western blot investigations. Rapamycin (RM) was also used to treat animals as an mTOR inhibitor. Topical administration of SMV resulted in a faster healing rate, elevated collagen deposition, and increased myofibroblast population compared to other experimental groups. Moreover, qRT-PCR findings showed that the wounds treated with SMV alone had the highest expression levels of CD31, VEGF, Akt, mTOR, and p70S6K after 7 and 14 days of burn model (p < 0.001). According to western blot findings, daily topical treatment with SMV further increased protein levels of P-AktThr308, P-mTORSer2448, and P-p70S6 KThr389 compared with other treatments, at both follow-up time points (p < 0.001). In contrast, inhibition of Akt/mTOR signaling pathway by RM reduced SMV-induced wound healing process. Seemingly, SMV promotes burn wound healing, at least in part, through activating Akt/mTOR signaling pathway, suggesting topically applied SMV as an alternative therapeutic approach for managing burn wound healing.

Correlation Between Protamine-2 and miRNA-122 in Sperm from Heroin-addicted Men: A Case-Control Study.

PURPOSE: Recreational use of illicit drugs is one of the main factors affecting male fertility. However, the mechanisms of heroin smoke-associated damage to mature spermatozoa are still completely unknown. The aim of this study was to concomitantly examine the levels of protamine-2 gene and protein concentrations, the amount of miRNA-122 in seminal plasma and semen analysis findings in heroin-addicted men. MATERIALS AND METHODS: In a case control study, twenty-four fertile men that lacked any recreational drug abuse were considered as the healthy group, and 24 addicted men who used only heroin for at least four months were selected as the addicted group. Semen samples were gathered by masturbation after 2 - 5 days of sexual abstinence. Following the preparation of a semen analysis by computer-assisted sperm analysis according to WHO (2010), the level of protamine-2 gene expression in sperm and miRNA-122 in seminal plasma was measured using real-time sqPCR. Also, protamine-2 protein concentrations were quantified by nuclear protein extraction, SDS-Page and western blotting. RESULTS: Among the studied variables, body mass index (27.75±0.88 vs. 22.30±0.36, p=0.001), seminal pH (7.79±0.06 vs. 7.58±0.06, p=0.003), white blood cell count in semen (1.69±0.41 vs. 8.61±1.73, p=0.001), motility (65.51±2.57 vs. 41.96±3.58, p=0.001) and survival rate (87.41±1.00 vs. 71.50±4.59, p=0.002) of sperm cells was significantly different between the healthy and addicted groups. In addition, the levels of protamine-2 gene and protein expression in the addicted group (0.05±0.02 and 0.10±0.02, respectively) were significantly lower than the healthy group (3.59±0.94 and 0.27±0.06, respectively) (p=0.002 and p=0.017, respectively). Seminal miRNA-122 levels in addicted men (3.51±0.73) were statistically higher than in healthy men (1.52±0.54) (p=0.034). However, there were some significant relationship between the studied parameters and addiction (p<0.05). CONCLUSION: This is one study on human infertility that evaluates the effects of heroin on protamine deficiency and seminal small RNAs expression levels. Heroin abuse may lead to male infertility by causing leukocytospermia, asthenozoospermia, protamine deficiency, and seminal plasma miRNA profile alteration.

Alteration in Expression of Primordial Germ Cell (PGC) Markers During Induction of Human Amniotic Mesenchymal Stem Cells (hAMSCs).

BACKGROUND: Currently, scientists are looking for a solution to the problem of the couples who have a lack of germ cells by through cell therapy. It is found that human amniotic membrane mesenchymal stem cells (hAMSCs) could be a good candidate for solving this problem. In the present study, an attempt was made to show that hUMSCs can express the PGC markers in the presence of retinoic acid (RA). METHODS: Placenta was obtained from healthy mothers and amniotic stem cells were isolated by enzymatic method from amniotic membrane. The cells were treated by retinoic acid for 14 days. Mesenchymal properties of hAMSCs were assessed by flow-cytometry and expression of PGC markers was established by Q-PCR. RESULTS: Mesenchymal stem cell properties were confirmed by antibodies against mesenchymal stem cell markers (CD73, CD90, and CD105). After that, the expression of the C-kit, Oct4, SSEA4, VASA genes were determined as primordial germ cell markers using quantitative PCR. It was found that the use of retinoic acid led to the highest expression of C-kit, SSEA4, VASA genes and lower expression of Oct4. CONCLUSION: Our study indicates that retinoic acid can be used as a suitable factor for induction of hAMSCs into primordial germ cells (PGCs) and hAMSCs have enough potential to do that.

Differentiation of neonate mouse spermatogonial stem cells on three-dimensional agar/polyvinyl alcohol nanofiber scaffold.

Electrospun nanofiber matrices sufficiently mimic the structural morphology of natural extracellular matrix. In this study, we aimed to examine the effects of agar/polyvinyl alcohol nanofiber (PVA) scaffold on the proliferation efficiency and differentiation potential of neonate mouse spermatogonial stem cells (SCCs). Testicular cells were isolated from testes of 40 mouse pups and were seeded in: 1) 2D cell culture plates in the absence (2D/-GF) or presence (2D/+GF) of growth factors and 2) onto agar/PVA scaffold in the absence (3D/-GF) or presence (3D/+GF) of growth factors. The cells were subsequently cultured for 4 weeks. First 2 weeks were dedicated to proliferative phase, whereas the next 2 weeks emphasized the differentiation phase. The identity of the SCCs was investigated at different time-points by flow cytometry and quantitative reverse transcription PCR (qRT-PCR) analyses against the germ cell markers, including PLZF, Id-4, Gfrα-1, Tekt-1, and Sycp-3. After 2 weeks of culture, the 3D/+GF group showed the highest percentage of PLZF-positive cells among culture systems (P < 0.05). The expression levels of pre-meiotic markers (Id-4 and Gfrα-1) decreased significantly in all groups, particularly in 3D/+GF group after 28 days of culture. Additionally, the cells in the 3D/+GF group displayed the highest expression of meiotic (Sycp-3) and post-meiotic markers (Tekt-1) 14 days after differentiation induction. Seemingly, the combination of the agar/PVA scaffold and growth factor-supplemented medium synergistically increased the differentiation rate of mouse SSCs into meiotic and post-meiotic cells. Thus, agar/PVA nanofiber scaffolds may have the potential for applications in the restoration of infertility, especially in azoospermic males. ABBREVIATIONS: 2D: two dimentional; 3D: three dimentional; bFGF: basic fibroblast growth factor; BMP-4: bone morphogenetic protein 4; DMEM: Dulbecco's modified Eagle's medium; ECM: extracellular matrix; FCS: fetal calf serum; FTIR: Fourier-transform infrared spectroscopy; GDNF: glial cell line-derived neurotrophic factor; GF: growth factors; Gfrα-1, GDNF family co-receptor α1; Id-4, Inhibitor of DNA Binding 4; MTT: methylthiazoltetrazolium; PLZF: promyelocytic leukemia zinc finger; PVA: polyvinyl alcohol; qRT-PCR: quantitative reverse transcription PCR; RA: retinoic acid; SACS: soft agar culture system; SD: standard deviation; SEM: scanning electron microscope; SSCs: spermatogonial stem cells; Sycp-3, Synaptonemal complex protein 3; Tekt-1, Tektin 1.

Evaluation of Enkephalin-Degrading Enzymes in Sperm from Heroin-Addicted Men.

BACKGROUND: The aim of this study was to investigate two enkephalin-degrading enzymes, aminopeptidase N (APN/ CD13) and endopeptidase (NEP/CD10), gene and protein expression levels in sperm samples of fertile and heroinaddicted men, and the correlation between their expressions and semen quality. MATERIALS AND METHODS: In this case-controlled study, semen was collected from 24 normozoospermic healthy (as a control group) and 24 heroin-addicted men donors (as case or addiction group). Sperm cells isolated by Cook Medical gradient (40-80%) and followed up by swim-up techniques were used for real-time quantitative polymerase chain reaction (qPCR) and flow cytometry techniques to assess APN/CD13 and NEP/CD10 genes and proteins subsequently. Semen parameters were analyzed by computer-assisted sperm analysis. RESULTS: The findings revealed that there were significant differences in sperm total motility (41.07 ± 3.63 vs. 63.03 ± 3.31 %, P=0.0001), progressive motility (35.21 ± 2.64 vs. 20.93 ± 3.22%, P=0.001) and viability (69.9 ± 4.69 vs. 86.81 ± 1.26 %, P=0.002) in the addicted group vs. control ones. APN and NEP gene expression levels in the addicted group decreased compared with the control ones (1.00 ± 0.67 vs. 0.36 ± 0.13, P= 0.008 and 1.07 ± 0.11 vs. 0.52 ± 0.12 0.002, respectively). Flow cytometry analysis showed that the average percent of APN/CD13 in heroin consumers significantly decreased compared with the healthy ones, while NEP/CD10 rate between two groups was similar. We also observed that duration of drug dependence is correlated with sperm viability (r=-0.627, P=0.016) and motility (r=-0.410, P=0.05), NEP (r=-0.434, P= 0.049), and APN (r=-0.641, P=0.002) gene expression levels. CONCLUSION: We conclude that semen quality and enkephalin-degrading enzymes were altered in heroin-addicted men. other confirming the internal validity of our estimates.

The Effect of Heroin Addiction on Human Sperm Parameters, Histone-to-Protamine Transition, and Serum Sexual Hormones Levels.

PURPOSE: To investigate the effects of heroin on sperm parameters, histone-to-protamine transition ratios in mature sperm, and serum reproductive hormone levels in active heroin users. MATERIALS AND METHODS: Semen and blood samples were collected from 25 men who used only heroin for at least 12 months and the same number healthy men who did not use any drugs and did not suffer from infertility problems. Computer-based analysis, Aniline blue staining, and hormonal assessment were performed to provide valuable new information on the relationship between addiction and semen profile and serum reproductive hor-mone levels. RESULTS: Our finding showed that semen pH (7.8 vs. 7.75), sperm motility (42.93 ± 3.89% vs. 68.9 ± 2.68%), and viability (73.27 ± 3.85% vs. 86.48 ± 1.05%), and sperm histone replacement abnormalities (32.33 ± 10.89% vs. 5.56 ± 0.85%) were significant differences in addicted group vs. non-exposed ones (P ? .05). In addition, serum sex hormone levels were not significantly differed between groups. There was a correlation between the amount of daily heroin consumption and LH level. We also observed that duration of drug dependence is correlated with sperm abnormalities. CONCLUSION: We concluded that heroin consumption affect sperm maturities such as histone-to-protamine ratio and impair semen profile in general and particularly sperm morphology and motility. Heroin may be considered as one of the idiopathic male infertility reason.

In Vitro Cytotoxicity of Folate-Silica-Gold Nanorods on Mouse Acute Lymphoblastic Leukemia and Spermatogonial Cells.

OBJECTIVE: The purpose of this study was to evaluate in vitro cytotoxicity of gold nanorods (GNRs) on the viability of spermatogonial cells (SSCs) and mouse acute lymphoblastic leukemia cells (EL4s). MATERIALS AND METHODS: In this experimental study, SSCs were isolated from the neonate mice, following enzymatic digestion and differential plating. GNRs were synthesized, then modified by silica and finally conjugated with folic acid to form F-Si-GNRs. Different doses of F-Si-GNRs (25, 50, 75, 100, 125 and 140 µM) were used on SSCs and EL4s. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) proliferation assay was performed to examine the GNRs toxicity. Flow cytometry was used to confirm the identity of the EL4s and SSCs. Also, the identity and functionality of SSCs were determined by the expression of specific spermatogonial genes and transplantation into recipient testes. Apoptosis was determined by flow cytometry using an annexin V/propidium iodide (PI) kit. RESULTS: Flow cytometry showed that SSCs and EL4s were positive for Plzf and H-2kb, respectively. The viability percentage of SSCs and EL4s that were treated with 25, 50, 75, 100, 125 and 140 µM of F-Si-GNRs was 65.33 ± 3.51%, 60 ± 3.6%, 51.33 ± 3.51%, 49 ± 3%, 30.66 ± 2.08% and 16.33 ± 2.51% for SSCs and 57.66 ± 0.57%, 54.66 ± 1.5%, 39.66 ± 1.52%, 12.33 ± 2.51%, 10 ± 1% and 5.66 ± 1.15% for EL4s respectively. The results of the MTT assay indicated that 100 µM is the optimal dose to reach the highest and lowest level of cell death in EL4s and in SSCs, respectively. CONCLUSION: Cell death increased with increasing concentrations of F-Si-GNRs. Following utilization of F-Si-GNRs, there was a significant difference in the extent of apoptosis between cancer cells and SSCs.

In vitro development of embryos from experimentally Kerack-addicted Mice.

BACKGROUND: Prenatal drug exposure, as a common public health concern, is associated with an increased risk of adverse effects on early embryo development. OBJECTIVE: To investigate the in vitro development of - embryo from experimentally Kerack-addicted mice. MATERIALS AND METHODS: Twenty-five female mice were studied in five groups: control, vehicle, and three experimental groups of Kerack-dependent mice (I, II, and III) which received different doses of Kerack for 14 days. After the establishment of addiction model (7 days), experimental groups I, II, and III were given Kerack intraperitoneally at the doses of 5, 35, and 70 mg/kg, twice a day for a period of 7 days, respectively. The vehicle group received normal saline and lemon juice whilst the control group just received water and food. Morulae were obtained through oviduct flashing. The survived embryos were cultured in T6+ 5mg/ml bovine serum albumin. The developmental rates up to hatched stage daily and embryo quality (differential staining and Tunnel staining) were also assessed. RESULTS: The developmental potential of embryos obtained from the addicted mother was significantly decreased in comparison with control group. There was a significant reduction in the rate of blastocyst formation in the high dose Kerack dependent group. However, in addicted mice there was reduction in the total cell number (40.92% vs. 65.08% in control) and, inner cell mass percentage (17.17% vs. 26.15% in control) while apoptotic cells numbers were increased (7.17 vs. 1.46 in control) (p<0.05). CONCLUSION: The Kerack addiction during pregnancy retards preimplantation development and induces apoptosis.

Comparison of Human Amniotic, Chorionic, and Umbilical Cord Multipotent Mesenchymal Stem Cells Regarding Their Capacity for Differentiation Toward Female Germ Cells.

Placenta harbors a plentiful source of various cells with stem cells or stem-like cell properties, which can be used in therapeutic procedures and research. Mesenchymal stem cells (MSCs) have attracted much attention due to their specific differentiation potential and tolerogenic properties. MSCs have been isolated from different parts of placenta; however, in this study, we isolated MSCs from amnion and chorion membrane, as well as umbilical cord (Wharton's jelly [WJ]) and compared their capacity regarding differentiation toward female germ cells under influence of 10 ng/mL BMP4. All placenta samples were collected from delivering mothers by normal cesarean section and cells were isolated by different methods. Results showed that all isolated cells were mostly positive for the MSC markers CD73, CD166, and CD105, and minimally reacted with CD34 and CD45 (hematopoietic markers). After differentiation induction using third passage cultured cells, immunocytochemistry staining showed that cells were positive for germline cell-related genes Ssea4, Oct4, and Ddx4, and oocyte-related gene Gdf9. RT-qPCR results indicated that human chorion MSCs (hCMSCs) had a greater potential to be differentiated into female germline cells. Moreover, the results of this study indicate that human umbilical cord MSCs originated from either male or female umbilical cord have the same differentiation potential into female germline cells. We recommend that for presumptive application of MSCs for infertility treatment and research, hUMSCs are best candidates due to their higher differentiation potential, ease of proliferation and expansion, and low immunogenicity.

Effects of antioxidants, catalase and α-tocopherol on cell viability and oxidative stress variables in frozen-thawed mice spermatogonial stem cells.

Cryopreservation of spermatogonial stem cells is considered as a useful procedure for preserving fertility in children with testis cancer. SSCs were isolated from testes mice, and then antioxidant was added to the freezing medium. The Bax expression level in antioxidant groups was significantly (P ≤ 0.05) lower than the control group and a significant rise of Bcl2 expression was detected in the antioxidant groups. ROS production with antioxidant was significantly lower compared with the control group. Cryopreservation with the addition of the antioxidants can help increase the number of SSCs and improve the quality and viability of these cells after cryopreservation.

Alteration of spermatogenesis following spermatogonial stem cells transplantation in testicular torsion-detorsion mice.

PURPOSE: Testicular ischemia is the main consequence of testicular torsion, in both clinical and experimental aspects. Preservation and auto-transplantation of spermatogonial stem cells (SSCs) could be a new treatment for infertility in testicular ischemia following testicular torsion. METHODS: To apply the idea in this study, animals were randomly divided into four groups of control, sham, with torsion, and with torsion followed by transplantation (TT). Isolated SSCs from neonatal mice were cultured and identified by flow cytometry (C-KIT(-), INTEGRIN ß1 (+)) and RT-PCR (Reverse transcription polymerase chain reaction) for specific spermatogonial cell markers (Oct4, Gfrα-1, Plzf, Vasa, Itgα 6 , and Itgß 1 ). SSCs were transplanted upon a 2-h testicular torsion in the TT group. Cultured cells were transplanted into ischemia reperfusion testicle 2 weeks post-testicular torsion. Eight weeks after SSCs transplantation, the SSCs-transplanted testes and epididymis were removed for sperm analysis, weight and histopathological evaluation, and pre- and post-meiotic gene expression assessment by qRT-PCR. RESULTS: Our findings indicated that all evaluated parameters (epididymal sperm profile, Johnsen score, Plzf, Gfrα-1, Scp-1, Tekt-1 expressions, and histopathological profile) were significantly decreased following testicular torsion (group 3) when compared to the control group (p ≤ 0.05). However, all abovementioned parameters showed a significant increase/improvement in torsion-transplantation group compared to torsion group. However, these parameters in the TT group were significantly lower in the sham and control groups (p ≤ 0.05). CONCLUSION: SSCs transplantation could up-regulate the expression of pre- and post-meiotic genes in testicular ischemia, which resulted in improvement of both testicular function and structure after testicular torsion.

Human Wharton's jelly-derived mesenchymal stem cells express oocyte developmental genes during co-culture with placental cells.

OBJECTIVES: The present day challenge is how to obtain germ cells from stem cells to treat patients with cancer and infertility. Much more efforts have been made to develop a procedure for attaining germ cells in vitro. Recently, human umbilical cord-derived mesenchymal stem cells (HUMSCs) have been introduced with higher efficacy for differentiation. In this work, we tried to explore the efficacy of HUMSCs and some effective products of placental cells such as transforming growth factors. This study is aimed to optimize a co-culture condition for HUMSCs with placental cells to obtain primordial germ cells (PGCs) and reach into oocyte-like cells in vitro. MATERIALS AND METHODS: In this experimental study, HUMSCs and placental cells were co-cultured for 14 days without any external inducer in vitro. Then HUMSCs were assessed for expression of PGC markers; Octamer-binding transcription factor 4(OCT4), Tyrosine-protein kinase Kit (CKIT), Stage specific embryonic antigen 4 (SSEA4), DEAD (Asp-Glu-Ala-Asp) box polypeptide 4(DDX4) and oocyte specific markers; Growth differentiation factor-9(GDF9), Zona pellucida glycoprotein 3(ZP3). The pertinent markers were assessed by immunocytochemistry and Q-PCR. RESULTS: Co-cultured HUMSCs with placental cells (including amniotic and chorionic cells) presented Oct4 and DDX4, primordial germ cells specific markers significantly, but increment in expression of oocyte-like cell specific markers, GDF9 and ZP3 did not reach to statistically significant threshold. CONCLUSION: Placental cell supplements Transforming growth factor (TGF α, ß) and basic fibroblast growth factor (bFGF) in a co-culture model can provide proper environment for induction of HUMSCs into PGCs and expression of oocyte-like markers.