425also be derived as naïve pluripotent cells in the presence of a specific MAPK inhibi-
tor, a GSK3β inhibitor and LIF (also known as 2i +LIF) conditions, resulting in cells
with similar signaling requirements, imprinting status and DNA methylation pro-
files as naïve mouse ESCs (Leitch et al. 2010 , 2013 ). The link between naïve embry-
onic germ cells and PGCs suggest that these cell types have a common molecular
regulation. In PGCs, this regulatory network helps protect these cells from somatic
inducing signals during epigenetic reprogramming (Leitch and Smith 2013 ).
In the pig, however, both OCT-4 and NANOG are maintained throughout the period
of germ cell specification, migration and gonadal colonization (Wolf et al. 2011 ;
Hyldig et al. 2011a), suggesting that during the specification of the germ line these
genes play different roles as in mice. No data is currently available for other pluripo-
tency genes, such as SOX2, although in humans it was shown that SOX2 is absent
from gonadal PGCs (Perrett et al. 2008 ). This suggests that the absence of an intact
pluripotency network in PGCs of non-rodent mammals may be an underlying reason
to explain the impossibility to establish embryonic germ cells using similar culture
conditions for mouse embryonic cells. In fact the embryonic germ cell lines reported
in other animals fail to form teratomas or to contribute to the germ line in chimeric
animals (Alberio and Perez 2012 ; Turnpenny et al. 2006 ; Petkov et al. 2011 ; Shamblott
et al. 1998 ), suggesting that they are captured in a different state of pluripotency.
8.6.4 Epigenetic Reprogramming of the Mammalian
Germ Line
Shortly after specification mouse PGC embark on their migration through the hind-
gut and the colonization of the genital ridges. An important feature of this period is
the reprogramming of epigenetic marks in these precursors. Genome wide changes
in histone marks and DNA demethylation are thought to be an essential process of
PGC development that enables the elimination of epimutations acquired by the pre-
vious generation. DNA methylation was reported to decline from E8 as measured
by 5-methylcytosine (5-mC) immunostaining and by whole genome sequencing
(Seki et al. 2005 ; Seisenberger et al. 2012 ). The mechanism of DNA demethylation
at this stage seems to be driven primarily by the reduction in DNA methyltransfer-
ase activity in PGCs compared to their somatic neighbors, as demonstrated by the
reduction in Dnmt3a/b expression. At this stage, 5-hydroxymethylcytosine (5-hmC)
staining is not increased, which indicates that conversion into 5-hmC intermediates
is not involved in methylation reprogramming in pre-migratory mouse PGCs
(Hackett et al. 2013 ; Yamaguchi et al. 2013 ). Indeed, it is from E9.5–10.5 that the
peak of tet1/2 (Ten–eleven translocation methylcytosine dioxygenase 1 and 2)
enzymes is detected in mouse PGCs and this is concurrent with an increase in
5-hmC levels (Hackett et al. 2013 ; Yamaguchi et al. 2013 ). In the pig, changes in
DNA methylation were investigated in early migratory PGCs from day 15 (equiva-
lent to day E8.5 in the mouse) until day 28. The study showed that genome-wide
levels of 5-mC are lower in PGCs from day 15 and remain low until day 21,
8 Mechanisms of Vertebrate Germ Cell Determination