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


OBJECTIVE
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.
Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR, P O Box: 19615-1177, Tehran, Iran.
Banafsheh
B
Heidari
Maryam
M
Rahmati-Ahmadabadi
Mohammad Mehdi
MM
Akhondi
Amir Hassan
AH
Zarnani
Mahmood
M
Jeddi-Tehrani
Abolfazl
A
Shirazi
Mohammad Mehdi
MM
Naderi
Bahareh
B
Behzadi
D000818:Animals; D015415:Biomarkers / Q000032:analysis / Q000378:metabolism*; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000187:drug effects; D002469:Cell Separation / Q000379:methods*; D017364:Collagenases / Q000378:metabolism; D003470:Culture Media / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005346:Fibroblast Growth Factors / Q000494:pharmacology; D051100:Glial Cell Line-Derived Neurotrophic Factor / Q000494:pharmacology; D006041:Goats; D007150:Immunohistochemistry; D053605:Leukemia Inhibitory Factor / Q000494:pharmacology; D008297:Male; D019009:Proto-Oncogene Proteins c-kit / Q000378:metabolism*; D015203:Reproducibility of Results; D013093:Spermatogonia / Q000166:cytology*; D013234:Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D013737:Testis / Q000166:cytology; D043222:Ubiquitin Thiolesterase / Q000378:metabolism
D015415:Biomarkers; D003470:Culture Media; D051100:Glial Cell Line-Derived Neurotrophic Factor; D053605:Leukemia Inhibitory Factor; D005346:Fibroblast Growth Factors; D004815:Epidermal Growth Factor; D019009:Proto-Oncogene Proteins c-kit; D043222:Ubiquitin Thiolesterase; D017364:Collagenases; C417249:collagenase 1