Characterization of primordial germ cell-like cells derived from porcine skin stem cells in vitro
Mechansims underlying mammalian germ cell specification and early gametogenesis remain to be clearly defined, and the culture of primordial germ cells (PGCs) continues to present a challenge despite recent advances in the field. This thesis describes the characterization of PGC-like cells generated from porcine fetal skin-derived stem cells 'in vitro', substantiating that somatic stem cells have an intrinsic ability differentiate into germ cell precursors. When skin stem cells were induced to differentiate in an appropriate microenvironment, a subpopulation of morphologically distinct cells resembling PGCs arose. Some of these cells were alkaline phosphatase (AP)-positive, expressed several other key markers indicative of germ cell formation, and underwent imprint erasure. In order to establish a link between 'in vitro'-derived putative PGCs and cells resembling oocytes, the porcine Deleted in Azoospermia-Like ('Dazl') gene promoter was cloned, characterized, and a lentiviral green fluorescent protein (GFP) reporter construct, 'Dazl'-GFP, was generated. The germ cell-restricted expression of 'Dazl' was found to be controlled, at least in part, by the differential methylation of a key 'Sp1' transcription factor binding site within the core promoter. Further evidence supporting the germ cell identity of ' in vitro'-generated PGC-like cells was provided by the finding that these cells, when labelled with the 'Dazl'-GFP reporter, further differentiated into GFP-positive oocyte-like cells (OLCs). In order to improve upon existing 'in vitro' culture conditions, the effect of glial cell line-derived neurotrophic factor (GDNF) was evaluated. First, a novel ovarian function was established for GDNF, which was found to be present in follicular fluid and to enhance oocyte nuclear and cytoplasmic maturation, as well as to promote cumulus cell expansion of porcine cumulus-oocyte complexes (COCs) cultured 'in vitro'. GDNF also stimulated the proliferation of 'in vitro'-derived PGC-like cells. This mitogenic effect was mediated through the GDNF family receptor [alpha]-1 (GFR [alpha]-1). The results presented in this thesis suggest that cells closely resembling PGCs are specified during induced differentiation, and that factors such as GDNF may enhance their numbers for further manipulations.