Signaling through the TGF Beta-Activin Receptors ALK4/5/7 Regulates Testis Formation and Male Germ Cell Development

Denise C Miles,Patrick S Western,Stephanie I Wakeling,Jessica M Stringer,Dagmar Wilhelm,Andrew H Sinclair,Jocelyn A Van Den Bergen,Jean-Marc A Lobaccaro

doi:10.1371/journal.pone.0054606

Abstract

The developing testis provides an environment that nurtures germ cell development, ultimately ensuring spermatogenesis and fertility. Impacts on this environment are considered to underlie aberrant germ cell development and formation of germ cell tumour precursors. The signaling events involved in testis formation and male fetal germ cell development remain largely unknown. Analysis of knockout mice lacking single Tgfβ family members has indicated that Tgfβ's are not required for sex determination. However, due to functional redundancy, it is possible that additional functions for these ligands in gonad development remain to be discovered. Using FACS purified gonadal cells, in this study we show that the genes encoding Activin's, TGFβ's, Nodal and their respective receptors, are expressed in sex and cell type specific patterns suggesting particular roles in testis and germ cell development. Inhibition of signaling through the receptors ALK4, ALK5 and ALK7, and ALK5 alone, demonstrated that TGFβ signaling is required for testis cord formation during the critical testis-determining period. We also show that signaling through the Activin/NODAL receptors, ALK4 and ALK7 is required for promoting differentiation of male germ cells and their entry into mitotic arrest. Finally, our data demonstrate that Nodal is specifically expressed in male germ cells and expression of the key pluripotency gene, Nanog was significantly reduced when signaling through ALK4/5/7 was blocked. Our strategy of inhibiting multiple Activin/NODAL/TGFβ receptors reduces the functional redundancy between these signaling pathways, thereby revealing new and essential roles for TGFβ and Activin signaling during testis formation and male germ cell development.

Highlights

Spermatogenesis and oogenesis are founded on the development of the male and female germ cell lineages in the fetal testis and ovary, respectively
In this study, using ALK5 and ALK4/ALK5/ALK7 inhibitors in ex vivo gonad cultures we demonstrate that loss of TGFb function depleted SMAD phosphorylation, resulting in almost complete disorganization of the developing testis cords in embryonic XY gonads, and errant entry of germ cells into meiosis
We first used qRT-PCR to assess the expression of Inhba, Inhbb, Tgfb1-3 and Nodal and their respective type I (Alk4, Alk5 and Alk7) and type II (Acrv2a and Acrv2b) receptors in FACS purified XX female and XY male E12.5–E15.5 somatic and germ cells (Figure 1)

Summary

Introduction

Spermatogenesis and oogenesis are founded on the development of the male and female germ cell lineages in the fetal testis and ovary, respectively. Primordial germ cells populate the developing gonads at approximately embryonic day (E)10.5 and differentiate down the spermatogenic or oogenic pathways in response to their respective environments [1,2,3,4]. The molecular pathways directing male and female germ line development are poorly understood, even though these processes are crucial for later fertility and for preventing germ cell tumours. SOX9 promotes differentiation of the supporting cells into Sertoli cells, which proliferate and form cords in response to ligands such as FGF9 (fibroblast growth factor 9). The testis cords enclose the germ cells and define the interstitial space, where Leydig cells differentiate and reside. Sry is normally absent in XX females, allowing pathways driven by Wnt (wingless-related MMTV integration site 4) Rspo (Rspondin homolog), Ctnnb (b catenin) and Foxl to promote ovarian development [8,9,10,11,12,13,14,15]

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