RESUMO
OBJECTIVE: Vitamin D receptor (VDR) is expressed in human spermatozoa. However, the role of vitamin D (VD) in human male reproduction has not yet been clarified. In this study, effects of VD on sperm parameters and its apoptosis in asthenozoospermic and healthy men were evaluated. METHODS: The study was carried out on discharged semen samples of 80 asthenozoospermic and healthy men. The samples were divided into control and experimental groups (received 20 µMol of VD). This study assessed sperm motility using the Makler chamber, their morphology by Diff quick, apoptosis and necrosis by Annexin-V and TUNEL assays, and their chromatin integrity was assessed by Aniline blue and Toluidine blue staining, according to WHO guidelines. RESULTS: The results revealed that: 1) the total number of motile sperms was increased by VD in both groups, but it was only significant in the asthenozoospermia group. 2) The progressive motility was increased with significant difference in both groups.3) Morphology of sperm did not show any changes due to VD in any of the groups. 4) Early apoptosis and necrosis of sperms were reduced in both groups, but the results of late apoptosis showed no statistical difference in these groups. 5) The percentage of positive toluidine blue was significantly decreased after using VD in the asthenozoospermia group. CONCLUSION: VD could improve motility, early apoptosis, and sperm necrosis, especially in asthenozoospermic men and it could be used for therapeutic opportunities.
RESUMO
BACKGROUND AIMS: Sonic hedgehog (Shh) is an intercellular signaling molecule that regulates pancreas development in mammals. Manipulation of Shh signaling pathway can be used as reliable approach to improve the generation of functional insulin-producing cells (IPCs) from mesenchymal stromal cells (MSCs). METHODS: In the present study, a novel differentiation protocol was used to produce IPCs from adipose tissue-derived MSCs (ATDMSCs) based on sequential inhibition and reactivation of Shh pathway. ATDMSCs were differentiated into IPCs via a 14-day basic protocol using 1% insulin transferrin selenium (ITS) and 1% nicotinamide in Dulbecco's Modified Eagle's Medium medium. A mixture of 0.25 µmol/L cyclopamine + 64 ng/mL basic fibroblast growth factor at day 3 of differentiation and 150 ng/mL recombinant Shh at day 11 of differentiation were used, respectively, to promote sequential inhibition and reactivation of Shh pathway. Insulin granule formation, glucose-stimulated insulin secretion and gene expression pattern related to the pancreatic endocrine development and function were analyzed in manipulated and unmanipulated IPCs. RESULTS: IPCs obtained after Shh manipulation secreted higher amounts of insulin in vitro. This phenotype was accompanied by increased expression of both genes critical for ß-cell function and transcription factors associated with their mature phenotype including Pdx1, MafA, Nkx2.2, Nkx6.1, Ngn3, Isl1 and insulin at day 14 of differentiation. CONCLUSIONS: Our findings indicated that the early inhibition and late reactivation of Shh signaling pathway during the differentiation of ATDMSCs improved the functional properties of IPCs, a novel method that could be considered as an alternative approach for cell-based therapy for type 1 diabetes.
