BackgroundAlthough routinely used in assisted reproductive technology, human sperm cryopreservation is not an entirely successful procedure. This study determined the effect of nerve growth factor (NGF) supplementation of cryopreservation medium on post-thaw viability, motility, intracellular nitric oxide (NO) concentration, and DNA fragmentation of human spermatozoa in asthenozoospermic men.MethodsSemen samples were collected from 25 asthenozoosprmic men and divided into the following groups (n = 5/group): fresh semen (control); frozen-thawed semen without treatment; frozen-thawed semen with NGF treatment (0.5, 1, and 5 ng/ml). Prior to dividing the asthenozoospermic samples, 200 μl of each sample was collected for NGF content assessment by ELISA and then compared with normozoospermic semen samples (25 normozoospermic men). Sperm motility and viability were assessed according to WHO criteria. Furthermore, intracellular nitric oxide and DNA fragmentation were evaluated by Flow Cytometry.ResultsNGF content was significantly higher in normozoospermic compared with asthenozoospermic men. Cryopreservation of asthenozoospermic semen samples significantly decreased sperm viability and motility, and increased intracellular nitric oxide concentration and DNA damage (p < 0.01). In asthenozoospermic frozen–thawed samples treated with 0.5 ng/ml exogenous NGF, we observed a significantly increased viability, motility, and decreased DNA fragmentation (p < 0.05), but intracellular nitric oxide concentration was not reduced. The other high doses (1 and 5 ng/ml) had no significant effect on the variables.ConclusionSupplementation with exogenous NGF could have partial and limited protective effect during cryopreservation of human spermatozoa but further research is needed to evaluate the possible clinical applications.

Human umbilical Wharton's jelly-derived mesenchymal stem cells (HWJMSCs) are the best candidate to get plentiful stem cells and differentiate them to germ cells under appropriate conditions to treat infertility. We sought to determine under which conditions HWJMSCs could form male germ cells in vitro. So, HWJMSCs were differentiated to male germ cells under a mixture of bone morphogenetic protein-4 (BMP-4) and testicular and placental culture condition (TCC and PCC) medium followed by retinoic acid for 21 d. In the present study, the HWJMSCs were obtained from Wharton's jelly of umbilical cords of male neonates delivered by cesarean section. At the third passage, mesenchymal stem cell markers and differentiation to osteocytes and adipocytes were investigated. Then, HWJMSCs were induced to differentiate into male germ cells in the presence of BMP-4, all-trans retinoic acid, PCC, and TCC for 21 d. The profile of c-Kit, DDX4, Piwil2, and Dazl gene expression was evaluated by qPCR and ICC. Data was analyzed by ANOVA test. After 3 wk of treatment with different reagents, the morphology of these spindle-like cells changed to shiny clusters and germ cell-specific markers in mRNA were upregulated in both TCC + retinoic acid (RA) and BMP-4 + RA. Induction of HWJMSCs with TCC in the presence of RA resulted in significant upregulation (P ≤ 0.05) of all germ cell-specific genes (c-Kit 2.6795 ± 0.75, DDX4 4.3188 ± 1.18, Piwil2 4.9962 ± 1.55, Dazl 6.1199 ± 0.78) compared to control and PCC + RA. Our results indicated that TCC and RA are involved in human germ cell development. Moreover, BMP signaling also induced differentiation. Our findings provide a novel effective approach for generation of germ cells in vitro and studying the interaction of germ cells with their niche. Our work represents an essential step toward gaining knowledge of the molecular properties of HWJMSCs in the field of cell therapy. We demonstrated that under a suitable situation, HWJMSCs have the ability to differentiate into germ cells and this provides an excellent pattern to study infertility cause and cure.

Background: We aimed to assess the effect of sulforaphane (SFN) on breast cancer cell migration and also its effect on the expression of epithelial mesenchymal transition (EMT) markers and β-catenin. Methods: This study was performed in Shahroud University of Medical Sciences, Shahroud, Iran from 2017- 2018. In this experimental study, MDA-MB-231 cells were treated with different concentrations of SFN (5, 10, 20, 30 and 40 μM) at different time points of 24, 48, and 72 h. The control group was untreated cells. The inhibitory effects of different concentrations of SFN on cell migration at different time points were evaluated using scratch assay. Moreover, apoptosis was assessed by using flow cytometric analysis. The expression of βcatenin and EMT markers of ZEB1, fibronectin, and claudin-1 were determined by real-time PCR. Western blotting analysis of β-catenin was applied to determine its changes after SFN treatment. Results: SFN markedly inhibited the migration of cells at concentrations of 10, 20, 30, and 40µM after 24, 48, and 72 h. At relatively, high concentrations (30, 40µM), SFN induced apoptosis. Moreover, SFN reduced the gene expression of ZEB1, fibronectin, and claudin-1 after 72 h. The expression of β-catenin revealed a timedependent decrease at the concentration of 40 µM SFN. Conclusion: Downregulation of EMT markers and β-catenin showed accordance with the inhibition of migration. SFN could be a promising drug candidate to reduce metastasis in breast cancer.

The standard treatment for triple-negative breast cancer (TNBC) is chemotherapy, which is highly toxic to patients; thereby, there is a need to identify safer and more effective therapeutic approaches. Medicinal plants constitute a common alternative for cancer treatment. Pomegranate is a well-known fruit in this context, but its antimetastatic property has not been extensively studied. As breast cancer-related deaths from TNBC are mainly due to metastasis, the present study was designed to investigate the antimigratory effect of pomegranate peel extract (PPE) on TNBC cells. For this purpose, the MDA-MB-231 cells were treated with different concentrations of PPE for 24, 48 and 72 hr. The effects of PPE on cell migration and invasion were determined by wound healing and transwell assays.To address the possible molecular mechanisms underlying the antimetastatic effect of PPE, real-time quantitative PCR analysis of selected epithelial mesenchymal transition (EMT) markers were performed. Moreover, the expression of β-catenin as a critical factor in promoting cancer metastasis was examined. PPE markedly inhibited the migration and invasion of cells at concentrations of 25, 50, 100, 250, 500, and 1000µg/ml. At relatively high concentrations (500, 1000µg/ml), PPE induced apoptosis. Moreover, PPE decreased the gene expression of vimentin, ZEB1, and β-catenin and also increased the expression of E-cadherin in TNBC cells.The protein level of β-catenin, as measured using western analysis, revealed a time-dependent decrease at the concentration of 1000µg/ml PPE. Downregulation of EMT markers and β-catenin showed accordance with the inhibition of migration and invasion. The present data show that PPE could be a promising drug candidate to reduce metastasis in TNBC cells.

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