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Primordial germ cells (PGCs) are the embryonic precursors of the sperm and …

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- Inhibition of HMG CoA reductase reveals an unexpected role for cholesterol during PGC migration in the mouse

Primordial germ cells (PGCs) are the embryonic precursors of gametes. In most model systems, PGCs are migratory and navigate through or around diverse tissues in order to find the site of the developing gonads. PGC migration shares conserved features in many species indicating the process arose in a common ancestor. In vertebrates, however, the majority of factors implicated in PGC guidance are either secreted or membrane bound protein growth factors (e.g. stromal derived factor 1 and Kit ligand); whereas, evidence in Drosophila points to a lipid-based guidance system [1]. A recent study bridged the gap by demonstrating that zebrafish PGCs, like Drosophila PGCs, require 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) for normal migration [2].

HMGCR is the rate limiting enzyme in isoprenoid and cholesterol biosynthesis. In flies, HMGCR was shown to act within the somatic gonadal precursors to control release of a secreted PGC attractant [3]. Drosophila lack enzymes required downstream of HMGCR for cholesterol synthesis indicating that isoprenoids are the relevant downstream effectors [4]. In support of this, Santos and Lehmann demonstrated that mutations in the geranylgeranyl transferase 1 β subunit cause PGC migration defects. It has been proposed that geranylgeranylation of small GTPase in the Ras, Rac, or Rho families regulate secretion of hedgehog [5] or other putative Drosophila PGC attractants [4]. In zebrafish, evidence also points to a role for HMGCR and isoprenoids in PGC migration [2]. Inhibition of HMGCR or geranylgeranyl transferase I cause PGC migration defects. However, in this system it remains unclear whether HMGCR is required in PGCs themselves or within the soma. Additionally, zebrafish unlike flies are capable of de novo cholesterol synthesis and this branch of the pathway was not carefully evaluated.

Cholesterol plays a vital role during vertebrate development. Mutations in genes required for cholesterol biosynthesis cause severe developmental defects. Loss of Hmgcr [6] or squalene synthase [7] result in early embryonic lethality in mouse models. Mutations in 3b-hydroxysterol-Δ7 reductase (Dhcr7) or lathosterol 5-desaturase (Sc5d) cause skeletal, neural, and in some cases urogenital defects in humans [8] and in mice [9,10]. Additionally, mutations in genes required for cholesterol transport are also associated with embryonic lethality or patterning defects [11].

Several models have been evoked in order to explain the role of cholesterol during organogenesis. First, cholesterol is the precursor of steroid hormones, glucocorticoids, and oxysterols, all compounds known to mediate cell-cell signaling via activation of nuclear hormone receptors. Second, cholesterol directly regulates cell-cell signalling by controlling the diffusion [12] or reception [13] of members of the hedgehog growth factor family. Finally, cholesterol is a key structural component of the plasma membrane. Cholesterol controls membrane fluidity and modulates membrane protein interactions. Membrane cholesterol has been shown to influence the activity of growth factor receptors and cell-adhesion molecules by clustering these cell surface proteins into lipid rafts [14]. Of particular note, lipid rafts have been shown to affect epidermal growth factor-induced chemotaxis [15] and migration on fibronectin [16] in cell culture. This suggests that cholesterol levels might also alter cell migration in vivo.

Considering the roles of cholesterol in cell-cell signalling and cell migration, we thought it imperative to test whether this branch of the HMGCR pathway is required for germ cell development in a vertebrate model. In mice, PGCs migrate from the gut to the genital ridges between embryonic day 9.5 (E9.5) and E10.5. Steroid hormones and hedgehog growth factors are unlikely to play a role in this process. Enzymes required to convert cholesterol into steroid hormones are not expressed in the gonads until E11.5 [17]. Likewise, the three vertebrate members of the hedgehog growth factor family are not expressed in the right place or time to play a role in PGC guidance in this system [18,19]. However, changes in cholesterol could very well impact the ability of PGCs to respond to proposed chemoattractants such as SDF1 [20] or KITL [21].

To examine the role of cholesterol in PGC migration, we first measured cholesterol in living tissue dissected from E9.5 embryos and were surprised to find that cholesterol was enriched in the genital ridges relative to the surrounding tissue. This asymmetric distribution appears to be maintained via selective uptake of cholesterol by cells within the genital ridges. We further demonstrate that inhibition of HMGCR reduced total cholesterol and impaired germ cell survival and migration in culture. These defects were rescued by co-addition of geranylgeraniol and cholesterol indicating that both compounds are required. To test if cholesterol biosynthesis is necessary for PGC migration in vivo, we examine the number and distribution of PGCs in embryos lacking Dhcr7 or Sc5d (see Figure 1). PGCs were normally distributed in both lines, but cholesterol levels are only modestly affected in these embryos [9,10]. We conclude that cholesterol is required for PGC migration but that this requirement can be met by uptake of cholesterol from maternal sources. We propose that the asymmetric accumulation of cholesterol within the genital ridges controls signaling interactions required for PGCs to colonize the gonads.

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