This study demonstrates that cholesterol is required for primordial
germ cell survival and motility. Inhibition of HMGCR reduced
cholesterol levels and induced PGC apoptosis in culture. Addition of
cholesterol and farnesol or cholesterol and geranylgeraniol rescued
germ cell survival; however, PGC motility was only rescued by the
latter combination. Additionally, we found that cholesterol is elevated
in the urogenital ridges and present evidence that this asymmetric
distribution can be maintained by differential uptake. In support of
this, embryos lacking the last or penultimate enzyme in cholesterol
biosynthesis do not have germ cell defects but these embryos do not
exhibit cholesterol deficits until late in development [9,10].
We conclude that the cholesterol requirement for early developmental
processes including PGC migration can be met by uptake of maternal
cholesterol.
HMGCR and isoprenoids are required for migration of cardiac progenitors and PGCs in both fly and zebrafish model systems [2,4,31,32].
In these systems, isoprenylation of heterotrimeric G-protein subunits
and/or isoprenylaton of small G-proteins in the Ras superfamily are
thought to be altered by loss of HMGCR activity resulting in the
observed developmental phenotype. Our rescue experiments demonstrate
that isoprenoids may play a similar role during PGC migration in
mammals and demonstrate differential roles for GGOH and FOH. Both
farnesol and geranylgeraniol co-treatments were able to rescue PGC
survival, but only geranylgeraniol co-treatment assisted migration.
This probably reflects differential isoprenylation requirements for
different small GTPase [33].
For instance, Ras proteins are typically farnesylated, but when
farnesylation is inhibited some Ras family members can be
geranylgeranylated. Likewise, the small GTPase RhoB can be modified by
either isoprenyl group, but the selection of group has a profound
effect on its subcellular localization and presumably function. We
propose that either farnesylation or geranylgeranylation can support
signalling via a Ras family member involved in controlling PGC survival
or proliferation. However, geranylgeranyl modification is required to
support the activity of a small GTPase (perhaps in the Rho family)
required for cell motility.
In addition to reflining what has already been shown about the
function of HMGCR and isoprenoids in PGC development, our data also
hints at a function for cholesterol in PGC survival or motility. A role
for cholesterol during gonadal development is not entirely without
precedent. First, genes known to coordinate cholesterol uptake are
elevated within the urogenital ridges (UGRs). Steroidogenic factor 1 (Nr5a1)
is expressed in the UGRs at E9.5 and its expression becomes confined to
the testis by E12.5. NR5A1 is a member of the nuclear receptor family
and controls expression of genes required for cholesterol synthesis (HmgCoA synthase) and uptake (Scarb1) as well as genes required for steroid production [34]. Scarb1 mRNA has been detected in the sexually naive genital ridge as early as E10.5 and like Nr5a1, it later become enriched in the testis [17]. Loss of Nr5a1 results in loss of Scarb1 expression in the UGRs [35] and an absence of gonads and adrenal glands in both male and female mice [36].
Second, in the adult ovary, genes required for cholesterol synthesis
are elevated within the granulosa cells surrounding the oocyte and
cholesterol synthesized by the soma helps support oocyte growth by
metabolic coupling [37].
Curiously, migratory PGCs appear to lack mevalonate kinase and
mevalonate decarboxylase enzymes required for isoprenoid and
cholesterol biosynthesis [38].
This suggests that migratory germ cells are already deficient in
cholesterol synthesis and may rely on interactions with the soma to
supply their metabolic needs.
Cholesterol alters development via its ability to regulate cell-cell
signaling. Cholesterol is covalently attached to members of the
hedgehog (HH) growth factor family and this modification controls
diffusion of the HH proteins [12].
Additionally, cholesterol is required for cells to respond to HH and
evidence suggests that it is this process that is perturbed by
mutations in Dhcr 7 [13]. In flies, HH is a proposed attractant for PGCs [39] but in mice, the expression pattern of hedgehog family members [18,19]
does not suggest a role in PGC guidance. Additionally, a screen for
transcripts expressed in migratory PGCs failed to detect expression of Gli genes [38]
transcription factors required for HH response. We prefer a model in
which changes in cholesterol alter the secretion or reception of growth
factors known to be required for mammalian PGC migration. For instance
cholesterol rich lipid rafts have been shown to be required for
reception of KITL [40] and SDF1 [41] growth factors implicated in PGC guidance [20,21].
Alternatively, the cholesterol rich environment within the genital
ridge might help support PGC survival via metabolic coupling [37] (Figure 8).
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