Effect of serum replacement on murine spermatogonial stem cell cryopreservation.

Cryopreservation of spermatogonial stem cells (SSCs) is a necessity to preserve the genetic information of valuable livestock herds and to produce transgenic animals. However, serum, a key component that allows efficient cryopreservation, has many limitations attributed to its undefined composition, inter-batch variations, and contamination potential. Therefore, we aimed to establish a method for serum-free cryopreservation of SSCs. To evaluate the cryopreservation efficiency of serum replacements, we assessed the recovery rate, relative proliferation potential, proliferation capacity, and apoptosis capacity. SSCs were characterized, and their functional activity was determined through immunofluorescence, RT-qPCR, and spermatogonial transplantation. The efficiency of each serum replacement was compared to that of the negative control (10% DMSO in DPBS) and positive control (10% DMSO and 40% FBS in DPBS). Our results indicated that cryopreservation with 5% human serum albumin (rHSA) exhibited a higher relative proliferation potential (274.0 ± 13.4%) than with DMSO control (100 ± 8.6%), with no significant difference from the 40% FBS (190.0 ± 20.1%). Moreover, early apoptosis also significantly decreased to a greater extent with 5% rHSA (5.1 ± 0.7%) than with DMSO control (12.9 ± 0.8%) and was at a level comparable to the 40% FBS (4.9 ± 0.8%). In addition, the SSCs cryopreserved with 5% rHSA exhibited normal self-renewal and differentiation abilities. In conclusion, 5% rHSA is a potential serum replacement for SSC cryopreservation, with properties comparable to that of serum. These results would contribute to the application of SSCs in improving livestock and in future clinical trials for human infertility treatment.

Effect of Equilibration Time and Temperature on Murine Spermatogonial Stem Cell Cryopreservation.

Cryopreservation of spermatogonial stem cells (SSCs) is essential for preservation of valuable livestock and clinical applications. Although optimal equilibration of cryoprotectants has emerged as a promising approach to improve the cryopreservation efficiency, standard equilibration protocols have not yet been considered in cryopreservation of SSCs. This study aimed to establish a standard equilibration protocol to improve the cryopreservation efficiency of murine germ cells enriched for SSCs. After time- and temperature-dependent equilibration, the germ cells were cryopreserved with 10% dimethyl sulfoxide (DMSO) and 200 mM trehalose. To investigate cryopreservation efficiency at different equilibration conditions, the survival and proliferation rates were assessed after thawing, and then, cytotoxicity and intracellular trehalose quantification were analyzed. Protein (PLZF, GFRα1, VASA, and c-Kit) and gene (Bcl6b, Erm, Dazl, and Sycp1) expression was determined using immunofluorescence and real-time quantitative polymerase chain reaction (RT-qPCR), respectively. The proliferation rate increased significantly following equilibration for 20 minutes at room temperature (RT; 163.7% ± 24.6%) or 4°C (269.0% ± 18.2%). Cytotoxicity was reduced in 10% DMSO with 200 mM trehalose compared with that of 10% DMSO alone. Also, intracellular trehalose was observed after equilibration. The immunofluorescence and RT-qPCR data revealed that the murine germ cells enriched for SSCs retained their self-renewal ability after cryopreservation following equilibration. The most effective protocol was equilibration with 10% DMSO and 200 mM trehalose for 20 minutes at RT or 4°C, which is due to synergistic effects of intracellular and extracellular trehalose. This improved methodology will contribute toward the development of a standardized freezing protocol for murine germ cells enriched for SSCs and thereby expand their application in various fields.

GDNF family receptor alpha 1 is a reliable marker of undifferentiated germ cells in bulls.

Undifferentiated germ cells, including spermatogonial stem cells (SSCs), make up only a very small proportion of germ cells within the testis; for example, 0.03% of germ cells in the mouse testis are SSCs. In this study, we investigated the characteristics of bovine undifferentiated germ cells and developed an enrichment procedure for these cells on the basis of fluorescence-activated cell sorting (FACS), using the specific cell surface marker glial cell line-derived neurotrophic factor family receptor alpha 1 (GFRα1). FACS analysis showed that only 0.6% of the total testicular cells were GFRα1-positive. These GFRα1-positive cells had a significantly higher expression of UCHL1, ZBTB16, and DDX4 (all markers of undifferentiated spermatogonial and germ cells) than that of fresh testicular cells. Quantitative reverse-transcription PCR analyses also indicated that the gene expression of BCL6B and NANOS2 was significantly higher in GFRα1-positive cells. Furthermore, xenogeneic transplantation of bovine testicular cells into immunodeficient mice resulted in 4.4-fold more colonies of GFRα1-positive cells than those of fresh testicular cells, indicating that FACS with antibodies to GFRα1 had efficiently enriched putative SSCs from total testicular cells. Collectively, these results demonstrate that GFRα1 could be used as a marker of bovine undifferentiated germ cells, including putative SSCs, and that its expression on SSCs has important implications for the further development of techniques for enriching stem cells from other species.