Research from JAMA — Propagation of Human Spermatogonial Stem Cells In Vitro

Testicular scaffolds may be an option for fertility preservation. The aim was to develop various procedures for the decellularization of testicular tissue and to design a bio-ink to construct a bioartificial testis. Ram testicular tissue fragments were decellularized using NaCl buffer, NaCl buffer-Triton, SDS and SDS-Triton. The removal of the cells from the tissues was confirmed by DAPI and H & E staining, as well as the evaluation of the DNA content. Alcian blue, Orcein and Masson's trichrome staining methods were also used to confirm that T-ECM was preserved intact. Then, the optimal decellularization protocol was selected to determine the parameters of the bio-ink and printing of the scaffold. The extracted T-ECM was used to print the hydrogel scaffold in combination with alginate–gelatin. The printability, morphological, mechanical and biological properties of the printed hydrogels were characterized. Decellularization of testicular tissue fragments using the NaCl buffer-Triton protocol was significantly more efficient than other decellularization methods in removing the cellular debris and preserving the T-ECM compounds. The 3D printed scaffold with 5% T-ECM showed a uniform surface morphology with high cell attachment and cyto-biocompatibility properties for spermatogonia stem cells in vitro and in vivo compared to other groups. It is concluded that T-ECM can be used as a biomimetic material to make an artificial testis with possible in vitro sperm production.

Cryopreservation of spermatogonial stem cells (SSCs) is an applicable method for young males seeking fertility preservation before starting a treatment. It increases reactive oxygen species (ROS) formation and oxidative stress, which damages cellular structures. In this study, we added two antioxidants, catalase and α-tocopherol (α-TCP), to the basic freezing medium to evaluate their effects on the efficiency of SSCs. SSCs were isolated from testes of 3- to 6-day-old male mice using enzymatic digestion. The enrichment of isolated cells was evaluated by flow cytometry and Stra8 antibody. Catalase (40 μg/mL), or α-TCP (200 μg/mL) was added to the basic freezing medium. The cell viability was evaluated by the methylthiazoltetrazolium (MTT) assay. After thawing, cells were cultured for 1 month, and the expression pattern of specific genes of SSCs and the ability of the cells to restore spermatogenesis were used to determine the efficiency of the cryopreservation method. The survival rate of the frozen cells in the presence of catalase or α-TCP was significantly higher than the control group (p < 0.05). The number of colonies and their diameter measured after 1 month were significantly higher in the antioxidant groups than in the control group (p < 0.05). Gene expression and resumption of spermatogenesis also followed the same pattern. Thus, adding antioxidants to the basic freezing medium can be helpful in increasing the quality and viability of SSCs after cryopreservation. This new approach to stem cells cryopreservation can also be a promising strategy for fertility preservation in patients who suffer from malignancy.

Background: Cadmium (Cd) is known to cause various disorders in the testis. The general population may be exposed to Cd through ingestion of food and drinking water, inhalation of particulates from ambient air, tobacco smoke and ingestion of contaminated soil and dust. Saffron (Crocus sativus L.) is widely used as a food flavour, and has well known medicinal effects. Objectives: The aim of this study was to determine the effects of saffron on the results of semen parameters (sperm concentration, motility and viability in cauda of epididymis) in rats exposed to cadmium. Materials and Methods: Thirty Wistar male rats were divided into four groups. Cadmium chloride (1 mg/kg body weight) was injected intraperitoneally during 16 days at intervals of 48 hours between subsequent treatments. Crocus sativus L. (100 mg/kg b.w., IP) was pretreated in both control and cadmium-injected rats. Both control and cadmium-injected rats were pretreated with Crocus sativus L. (100 mg/kg b.w., IP). The animals were killed and their sperm count, motility, and vitality were evaluated. Results: Sperm parameters did not differ significantly between control and sham groups. Following contamination with cadmium, sperm count, motility and vitality were significantly decreased in comparison to control group (P < 0.05). In pretreated (saffron) group, the sperm parameters improved significantly in comparison with cadmium group (P ≤ 0.05). A significant decrease in sperm motility was observed in Cd-treated rats compared to the control rats. However, no significant changes were recorded by comparison of the control and saffron treated groups except for the sperm motility parameter. Conclusions: Saffron, as an antioxidant, is positively effective on sperm parameters in rats exposed with cadmium. Keywords: Crocus; Cadmium; Sperm Motility; Sperm Count; Rats