The effects of growth factors on in vitro-cultured porcine testicular cells.
Cell lines from neonate porcine testis were cultured and characterized and the effect of growth factors were investigated, in order to determine the requirements for the establishment of porcine male germ cell lines. In primary cultures, three different colony types with distinctive morphologies could be recognized. From colonies resembling mouse spermatogonial stem cells (SSCs), two cell lines were derived and maintained for nine passages after which proliferation stopped. Growth of these cell lines depended on the growth factors leukemia inhibitory factor (LIF), epidermal growth factor (EGF), glial derived neurotrophic factor (GDNF), and fibroblast growth factor (FGF). In both cell lines NANOG, promyelocytic leukemia zinc-finger (PLZF), and EPCAM, were expressed at higher levels and GFRA1, ITGA6, and THY1 at lower levels than in neonate porcine testis. Primary cultures of neonate pig testis were subjected to a factorial design of the growth factors LIF, GDNF, EGF, and FGF. EGF and FGF had a positive effect on the number and size of the SSC-like colonies. Addition of EGF and FGF to primary cell cultures of neonate pig testis affected the expression of NANOG, PLZF, POU5F1, and GATA4, whereas effects of LIF or GDNF could not be detected. FGF decreased the expression levels of NANOG, a marker for pluripotency also expressed in neonatal porcine male germ cells. FGF decreased expression of PLZF and enhanced the expression of pluripotency-related gene POU5F1 and Sertoli cell marker GATA4. EGF had a positive effect on PLZF expression levels and counteracted the positive effect of FGF on GATA4 expression. These results suggest that FGF can impede successful derivation of porcine SSCs from neonate pig testis.
Kuijk
Ewart W
EW
Department of Farm Animal Health, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 104, 3584 CM Utrecht, The Netherlands.
Colenbrander
Ben
B
Roelen
Bernard A J
BA
D000818:Animals; D000831:Animals, Newborn; D002478:Cells, Cultured; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005346:Fibroblast Growth Factors / Q000494:pharmacology; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000187:drug effects; D051100:Glial Cell Line-Derived Neurotrophic Factor / Q000494:pharmacology; D036341:Intercellular Signaling Peptides and Proteins / Q000494:pharmacology*; D053605:Leukemia Inhibitory Factor / Q000494:pharmacology; D008297:Male; D061251:Primary Cell Culture; D013093:Spermatogonia / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D013552:Swine; D013737:Testis / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology
D051100:Glial Cell Line-Derived Neurotrophic Factor; D036341:Intercellular Signaling Peptides and Proteins; D053605:Leukemia Inhibitory Factor; D005346:Fibroblast Growth Factors; D004815:Epidermal Growth Factor