The synergistic effect of trehalose and low concentrations of cryoprotectants can improve post-thaw ram sperm parameters.

The objective of this study was to compare the effects of different concentrations of two different cryoprotectants (glycerol, G and ethylene glycol, EG) and trehalose (T), added to the semen extender, on post-thaw ram sperm parameters. Ejaculates, collected from 6 Merino rams, were pooled and evaluated at 37 °C. The pooled samples were divided into six equal aliquots, and diluted in Tris-based extenders containing 5% G, 3% G + 60 Mm T, 1.5% G + 100 Mm T, 5% EG, 3% EG + 60 mM T, and 1.5% EG + 100 Mm T. Subsequently, the samples were cooled to 5 °C, frozen in 0.25-ml French straws, and stored in liquid nitrogen (LN2). Frozen samples were thawed individually, at 37 °C for 25 s in a water bath, for evaluation. Sperm motility was assessed using a phase-contrast microscope with a warm stage maintained at 37 °C. Acrosome integrity (FITC/PNA-PI), sperm viability (SYBR-14/PI), mitochondrial activity (JC-1/PI), DNA damage (COMET assay) and DNA fragmentation (TUNEL test) were determined. The group of samples diluted in an extender containing 5% of glycerol (Group 5% G) displayed higher percentages of subjective motility, viability and mitochondrial activity of sperm, compared to the other groups (P < 0.05). On the other hand, Group 3% G + 60 mM T yielded the second-best results for subjective motility, viability and mitochondrial activity of sperm, when compared to the other groups. The post-thaw sperm parameters of Group 3% G + 60 Mm T did not show any statistically significant difference from those of Group 5% G. There were no statistically significant differences between the groups for acrosome integrity (P > 0.05). The results of the COMET assay showed that the use of low concentrations of cryoprotectants in combination with trehalose decreased sperm DNA damage. Accordingly, Group 1.5% G + 100 mM T and Group 3% EG + 60 mM T benefited from a significantly stronger cryoprotective effect on DNA integrity, in comparison to Group 5% G (P < 0.05). According to the results of the TUNEL test, the combined use of low concentrations of cryoprotectants with trehalose decreased sperm DNA damage, when compared to the use of 5% glycerol, but this difference was statistically insignificant (P > 0.05). In conclusion, G and EG concentrations can be reduced by adding various amounts of T (60 mM, 100 mM) to the semen extender. The addition of 5% of glycerol and 3% G + 60 mM T to the semen extender did not yield statistically different post-thaw sperm parameters, when compared for protection against cryoinjury. Post-thaw sperm parameters can be improved by the supplementation of the semen extender with 3% G + 60 mM T. Thus, we recommend the use of freezing extenders containing low cryoprotectant concentrations (3% G) combined with trehalose to avoid the high level of toxic and osmotic damage caused by 5% G.

Development of small-molecule-induced fibroblast expansion technologies.

Dermal fibroblasts are responsible from the production of extracellular matrix and take role in the closure of skin wounds. Dermal fibroblasts are major cells of origin in the generation of induced pluripotent stem cells (IPSCs) and are historically being used as feeder layer and biofiller in the restorative surgeries. ex vivo expansion of the dermal fibroblasts provides a suitable model to study skin biology and to engineer bioartifical skins. Thus, development of efficient fibroblast expansion technologies gets outmost importance day by day. We sought to identify small molecules that induce ex vivo fibroblast expansion and understand their mechanisms. We analyzed the effect of 35 small molecules, which are expected to target molecular pathways involving cellular quiescence. We have found that small molecules, especially AS1949490 and SKF96365, increase human dermal fibroblast expansion of at least three different fibroblasts. Cell cycle analysis confirms that these small molecules allow cell cycle progression, as evident by increased percentage of cells in S-G2 -M phase of cell cycle. They led to a lower profile of apoptotic or necrotic fibroblasts. Intriguingly, we have found that identified small molecules could also endogenously induce the expression of IPSC generation, collagen synthesis, and aging-related genes. Identified small molecules may contribute to the induction of collagen synthesis in the biofiller products, the development of fibroblast products with better aging profile, and the improvement of IPSC generation.