Isolation, identification, and culture of goat spermatogonial stem cells using c-kit and PGP9.5 markers

Abstract

Introduction Presently the techniques for making transgenic animals are cumbersome, required costly instruments and trained man-power. The ability of spermatogonial stem cells (SSCs) to integrate foreign genes has provided the opportunity for developing alternate methods for generation of transgenic animals. One of the big challenges in this field is development of the methods to identify and purify donor SSCs by antibody mediated cell sorting. Purpose The present study was aimed to identify goat subpopulations of SSCs using polyclonal antibodies against PGP9.5 and c-kit molecular markers as well as the growth characteristics of SSCs during short term culture. Methods One month old goats' testicular samples were subjected for immunohistochemical and immunocytochemical evaluations. The enzymatically isolated SSCs were cultured in DMEM plus FCS supplemented with (treatment) or without (control) growth factors (GDNF, LIF, FGF, and EGF) for 2 weeks. At the end of culture the morphological characteristics of SSCs colonies and immunocytochemical staining were evaluated. Results The number and size of colonies in treatment groups were significantly (P<0.01) higher than corresponding values in controls. The presence of PGP 9.5 and c-kit antigens was confirmed in immunocytochemical evaluation. In immunocytochemical evaluation, the proportion of c-kit and PGP9.5 positive cells were significantly (P<0.001) higher in control and treatment groups, respectively. Conclusions The presence of PGP9.5 and c-kit antigens was confirmed in goat SSCs. Moreover, culture medium supplementation with growth factors could effectively retain the undifferentiation status of SSCs, reflected as a higher population of PGP9.5 positive cells, after short term culture.

In vitro culture and characterization of spermatogonial stem cells on Sertoli cell feeder layer in goat (Capra hircus)

“…Further, the use of more numbers of enzymes might cause cell injury while less numbers increase the possibility of cell contamination [13,42]. Previously, in farm animals, attempts were made to isolate the SSCs using either four [10,11], three [14] or two [43] enzymes with variable success. In the present study, we performed testicular disaggregation by enzymatic digestion using three enzymes without any reduction in the total cell count.…”

Spermatogonial stem cells: Current biotechnological advances in reproduction and regenerative medicine

“…SSC isolation procedures have been described for several domestic species and humans [7][8][9][10][11][12][13][14] . SSCs are isolated by enzymatic digestion, usually involving two steps of various combinations of proteolytic enzymes (commonly, collagenases I or IV, trypsin, hyaluronidase and DNAse I).…”

“…Culture conditions for SSC propagation in vitro were first developed in mice [16,20] . This was the base for the development of domestic animals SSC culture systems [7][8][9][10][21][22][23][24][25] . Most of these systems have in common the use of GDNF and serum.…”

Transcriptional coactivator undifferentiated embryonic cell transcription factor 1 expressed in spermatogonial stem cells: A putative marker of boar spermatogonia

“…Recent studies have indicated that the genes encoding PGP9.5 (Luo et al, 2006;Lee et al, 2013b), stage-specific embryonic antigen 1 (Kim et al, 2013), and GFR␣-1 (Lee et al, 2013a) are expressed in pig spermatogonia. Among the suggested spermatogonia-specific markers, PGP9.5 has been considered a specific marker for pig spermatogonia because of its consistent gene expression in the spermatogonia of other species, such as mice (Kon et al, 1999), cattle (Wrobel et al, 1996;Herrid et al, 2007), goats (Heidari et al, 2012), monkeys (Tokunaga et al, 1999), and humans (He et al, 2010). In a biomarker screening study of human spermatogonia, PGP9.5 was used as an independent positive control (von Kopylow et al, 2010).…”