180 Increasing GfrA1-Positive Spermatogonial Stem Cell Population of Goat

Abstract

Spermatogonial stem cells (SSC) form the basis of spermatogenesis and continuous fertility in male. Their meagre population in the testis is a hindrance in the in vitro study of biological activity of these cells. The objective of the present study was to isolate and characterise goat SSC and increase their number during in vitro culture by different methods. Two goat testes (3 to 4 months of age) were collected from the slaughterhouse and transported to the laboratory. The testes were washed and seminiferous tubules were collected and minced in the laminar flow hood. The seminiferous tubules were washed twice with PBS to remove spermatozoa and subjected to double enzymatic digestion (collagenase, 1 mg mL⁻¹, hyaluronidase, 1 mg mL⁻¹, trypsin, 0.05%, and DNaseI, 10 µg mL⁻¹ for 45 min and second digestion with same set of enzymes except trypsin for 30 min). The isolated cells were filtered sequentially through nylon mesh filters of pore size 70 and 40 µm. The cells were plated on DSA-lectin coated dishes for 4 h and the unattached cells were cultured on a Sertoli cell feeder layer prepared by treating with mitomycin-C for 3 h. The cells were cultured in DMEM/F-12 supplemented with human recombinant growth factors (glial cell-derived neurotrpic factor, 10 ng mL⁻¹, fibroblast growth factor FGF, 10 ng mL⁻¹, epidermal growth factor, 20 ng mL⁻¹), 10% fetal bovine serum, and antibiotics. The expression of pluripotency markers (Oct4, Nanog, Sox2) and SSC-specific markers (Thy1, GfrA1, and Uchl1) in the SSC colonies was determined by RT-PCR and immunostaining, after in vitro culture of 3 weeks. The SSC population was enhanced by differential plating, Percoll density gradient (on Day 1) and SSC passaging (by passaging SSC colonies on Day 20). The cells were tagged with GfrA1 antibody and their population was tested by flow cytometry. The SSC colonies started appearing after 7 days and continued to grow in size and number until 3 weeks. The SSC colonies were positive for the pluripotency markers Oct4, Nanog, and Sox2 by RT-PCR and immunostaining. The SSC were also positive for the SSC-specific markers Thy1, GfrA1, and Uchl1 by RT-PCR and immunostaining. Flow cytometry showed that the GfrA1-positive population in the SSC enriched by the differential plating was 11.23%, Percoll density gradient was 23.57%, and by passaging of SSC colonies, after picking and trypsinising with 0.05% trypsin, was 91.23%. In vitro culture of the SSC enriched by these methods also revealed that the number of SSC colonies appearing in the cells enriched by passaging was higher than the other methods. From the results of present study, we conclude that SSC are positive for markers of pluripotency and SSC-specific markers. The SSC population can be enhanced to a very high level following SSC passaging, which is an inexpensive method and does not require expensive instruments like fluorescence- or magnetic-activated cell sorting.