292 COMPARISON OF DIFFERENTIAL PLATING METHODS TO OBTAIN ENRICHED POPULATIONS OF BOVINE SPERMATOGONIAL STEM CELLS
M. I. Giassetti A , F. R. O. Barros A , R. A. Worst A , G. C. P. Saurin A , A. H. Bruno A , P. V. Moreira A , T. R. Hamilton B A , M. Nichi C , M. E. O. D’Ávila Assumpção A B and J. A. Visintin AA Laboratories of In Vitro Fertilization, Cloning, and Animal Transgenesis Department of Animal Sciences, School of Veterinary Medicine at University of Sao Paulo, Sao Paulo, Sao Paulo, Brazil;
B Laboratory of Biology of Spermatozoa, Department of Animal Sciences, School of Veterinary Medicine at University of Sao Paulo, Sao Paulo, Sao Paulo, Brazil;
C Laboratory of Andrology, Department of Animal Sciences, School of Veterinary Medicine at University of Sao Paulo, Sao Paulo, Sao Paulo, Brazil
Reproduction, Fertility and Development 25(1) 293-294 https://doi.org/10.1071/RDv25n1Ab292
Published: 4 December 2012
Abstract
Spermatogonial stem cells (SSC) represent only 0.1% of all germ cells found in adult animal testicles. The main goal of differential plating treatments is to isolate SSC and enrich the SSC population in a cell subculture. The aim of this study was to determine which differential plating treatment – laminin, lectin from Datura stramonium (DSA), Matrigel (BD Biosciences, San Jose, CA, USA), bovine serum albumin (BSA), or PBS/control – was the most efficient for isolating bovine SSC. Thus, 3 g of testicular parenchyma of adult Nelore bulls was minced and consecutively digested in two steps: collagenase (1 mg mL–1) and trypsin (2.5 mg mL–1). Cells from each testicle were plated (3 × 106 viable cells) in a culture dish coated with 1 mL of each of the following plating treatments: laminin (20 µg mL–1), DSA (20 µg mL–1), Matrigel (5%), BSA (0.5 mg mL–1), or PBS. Cells were cultured for 18 h in a high-humidity atmosphere containing 5% CO2 at 37°C. Supernatants were centrifuged and cell concentration and viability were assessed by Trypan Blue exclusion method. Then, levels of thiobarbituric acid reactive substances (TBARS) were evaluated in a 20 mM ascorbic acid solution and in 25µL of 4 mM ferrous sulfate solution immediately after incubation with 2 × 106 viable cells (n = 30) from each testicle (n = 6) for all treatments. The supernatant was collected and the same volume of 0.1% thiobarbituric acid was added. The TBARS level was quantified by spectrophotometry (532 nm). Approximately 1 × 106 viable cells (n = 55) from each testis (n = 11) for all treatments were fixed with 70% ethanol and incubated with an anti-α six integrin (A6Int) antibody, followed by Alexa Fluor 488 (BioLegends®, San Diego, CA, USA) and propidium iodide labelling. The percentage of marked cells with the anti-A6Int was determined by flow cytometry (Guava Technologies, Hayward, CA, USA). Kruskal-Wallis ANOVA and post hoc Bonferroni tests were performed; P < 0.05 was considered significantly different (Stata version 12.0, Stata Corp., College Station, TX, USA). No treatment side effects were observed on percentage of A6Int-positive cells recovered, TBARS level, or rate of viable cells after and before differential plating. A difference in cell viability was observed between the Matrigel and laminin treatments (P = 0.04). In conclusion, our results showed no quantitative difference between enriched SSC populations purified by differential plating treatments, as was expected. Thus, this experiment needs to be complemented with better characterisation of purified bovine SSC for their molecular markers expression using real time RT-PCR and flow cytometry. Only then will we be able to reach a conclusion on the effectiveness of these treatments.
Financial support: FAPESP (2011/20115-1).
