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.
References
[1]
McLaren A (2003) Primordial germ cells in the mouse. Developmental Biology 262: 1–15.
[2]
Guerquin MJ, Duquenne C, Lahaye JB, Tourpin S, Habert R, et al. (2010) New testicular mechanisms involved in the prevention of fetal meiotic initiation in mice. Dev Biol 346: 320–330.
[3]
Adams IR, McLaren A (2002) Sexually dimorphic development of mouse primordial germ cells: switching from oogenesis to spermatogenesis. Development 129: 1155–1164.
[4]
Kocer A, Reichmann J, Best D, Adams IR (2009) Germ cell sex determination in mammals. Mol Hum Reprod 15: 205–213.
[5]
Behringer RR, Finegold MJ, Cate RL (1994) Müllerian-inhibiting substance function during mammalian sexual development. Cell 79: 415–425.
[6]
Chaboissier M-C, Kobayashi A, Vidal VIP, Lützkendorf S, van de Kant HJG, et al. (2004) Functional analysis of Sox8 and Sox9 during sex determination in the mouse. Development 131: 1891–1901.
[7]
Colvin JS, Green RP, Schmahl J, Capel B, Ornitz DM (2001) Male-to-female sex reversal in mice lacking fibroblast growth factor 9. Cell 104: 875–889.
[8]
Kim Y, Kobayashi A, Sekido R, DiNapoli L, Brennan J, et al. (2006) Fgf9 and Wnt4 act as antagonistic signals to regulate mammalian sex determination. Plos Biology 4: e187.
[9]
Ottolenghi C, Pelosi E, Tran J, Colombino M, Douglass E, et al. (2007) Loss of Wnt4 and Foxl2 leads to female-to-male sex reversal extending to germ cells. Hum Mol Genet 16: 2795–2804.
[10]
Garcia-Ortiz JE, Pelosi E, Omari S, Nedorezov T, Piao Y, et al. (2009) Foxl2 functions in sex determination and histogenesis throughout mouse ovary development. BMC Dev Biol 9: 36.
[11]
Chassot AA, Ranc F, Gregoire EP, Roepers-Gajadien HL, Taketo MM, et al. (2008) Activation of beta-catenin signaling by Rspo1 controls differentiation of the mammalian ovary. Hum Mol Genet 17: 1264–1277.
[12]
Maatouk DM, DiNapoli L, Alvers A, Parker KL, Taketo MM, et al. (2008) Stabilization of beta-catenin in XY gonads causes male-to-female sex-reversal. Human Molecular Genetics 17: 2949–2955.
[13]
Tomizuka K, Horikoshi K, Kitada R, Sugawara Y, Iba Y, et al. (2008) R-spondin1 plays an essential role in ovarian development through positively regulating Wnt-4 signaling. Hum Mol Genet 17: 1278–1291.
[14]
Vainio S, Heikkila M, Kispert A, Chin N, McMahon AP (1999) Female development in mammals is regulated by Wnt-4 signalling. Nature 397: 405–409.
[15]
Yao HH, Matzuk MM, Jorgez CJ, Menke DB, Page DC, et al. (2004) Follistatin operates downstream of Wnt4 in mammalian ovary organogenesis. Dev Dyn 230: 210–215.
[16]
Ewen KA, Koopman P (2010) Mouse germ cell development: from specification to sex determination. Mol Cell Endocrinol 323: 76–93.
[17]
Bowles J, Knight D, Smith C, Wilhelm D, Richman J, et al. (2006) Retinoid signaling determines germ cell fate in mice. Science 312: 596–600.
[18]
Koubova J, Menke DB, Zhou Q, Capel B, Griswold MD, et al. (2006) Retinoic acid regulates sex-specific timing of meiotic initiation in mice. Proc Natl Acad Sci U S A 103: 2474–2479.
[19]
Baltus AE, Menke DB, Hu YC, Goodheart ML, Carpenter AE, et al. (2006) In germ cells of mouse embryonic ovaries, the decision to enter meiosis precedes premeiotic DNA replication.[see comment]. Nature Genetics 38: 1430–1434.
[20]
DiNapoli L, Batchvarov J, Capel B (2006) FGF9 promotes survival of germ cells in the fetal testis. Development 133: 1519–1527.
[21]
Kim Y, Bingham N, Sekido R, Parker KL, Lovell-Badge R, et al. (2007) Fibroblast growth factor receptor 2 regulates proliferation and Sertoli differentiation during male sex determination. Proceedings of the National Academy of Sciences of the United States of America 104: 16558–16563.
[22]
Bagheri-Fam S, Sim H, Bernard P, Jayakody I, Taketo MM, et al. (2008) Loss of Fgfr2 leads to partial XY sex reversal. Dev Biol 314: 71–83.
[23]
Bowles J, Feng CW, Spiller C, Davidson TL, Jackson A, et al. (2010) FGF9 suppresses meiosis and promotes male germ cell fate in mice. Dev Cell 19: 440–449.
[24]
Barrios F, Filipponi D, Pellegrini M, Paronetto MP, Di Siena S, et al. (2010) Opposing effects of retinoic acid and FGF9 on Nanos2 expression and meiotic entry of mouse germ cells. J Cell Sci 123: 871–880.
[25]
Best D, Sahlender DA, Walther N, Peden AA, Adams IR (2008) Sdmg1 is a conserved transmembrane protein associated with germ cell sex determination and germline-soma interactions in mic. Development 135: 1415–1425.
[26]
Kumar S, Chatzi C, Brade T, Cunningham TJ, Zhao X, et al. (2011) Sex-specific timing of meiotic initiation is regulated by Cyp26b1 independent of retinoic acid signalling. Nat Commun 2: 151.
[27]
Western PS, Miles DC, van den Bergen JA, Burton M, Sinclair AH (2008) Dynamic regulation of mitotic arrest in fetal male germ cells. Stem Cells 26: 339–347.
[28]
Western PS, Ralli RA, Wakeling SI, Lo C, van den Bergen JA, et al. (2011) Mitotic arrest in teratoma susceptible fetal male germ cells. PLoS One 6: e20736.
[29]
Spiller CM, Wilhelm D, Koopman P (2010) Retinoblastoma 1 Protein Modulates XY Germ Cell Entry into G1/G0 Arrest During Fetal Development in Mice. Biol Reprod 82: 433–443.
[30]
Western PS, van den Bergen JA, Miles DC, Sinclair AH (2010) Male germ cell differentiation involves complex repression of the regulatory network controlling pluripotency. FASEB J 24: 3026–3035.
[31]
La Salle S, Mertineit C, Taketo T, Moens PB, Bestor TH, et al. (2004) Windows for sex-specific methylation marked by DNA methyltransferase expression profiles in mouse germ cells. Developmental Biology 268: 403–415.
[32]
Western P (2009) Foetal germ cells: striking the balance between pluripotency and differentiation. Int J Dev Biol 53: 393–409.
[33]
Yamaguchi S, Kimura H, Tada M, Nakatsuji N, Tada T (2005) Nanog expression in mouse germ cell development. Gene Expression Patterns 5: 639–646.
[34]
Cook MS, Coveney D, Batchvarov I, Nadeau JH, Capel B (2009) BAX-mediated cell death affects early germ cell loss and incidence of testicular teratomas in Dnd1(Ter/Ter) mice. Dev Biol 328: 377–383.
[35]
Krentz AD, Murphy MW, Kim S, Cook MS, Capel B, et al. (2009) The DM domain protein DMRT1 is a dose-sensitive regulator of fetal germ cell proliferation and pluripotency. Proc Natl Acad Sci U S A 106: 22323–22328.
[36]
Itman C, Mendis S, Barakat B, Loveland KL (2006) All in the family: TGF-beta family action in testis development.[see comment]. Reproduction 132: 233–246.
[37]
Shi Y, Massagué J (2003) Mechanisms of TGF-beta signaling from cell membrane to the nucleus. Cell 113: 685–700.
[38]
Nef S, Schaad O, Stallings NR, Cederroth CR, Pitetti JL, et al. (2005) Gene expression during sex determination reveals a robust female genetic program at the onset of ovarian development. Developmental Biology 287: 361–377.
[39]
Archambeault DR, Yao HH (2010) Activin A, a product of fetal Leydig cells, is a unique paracrine regulator of Sertoli cell proliferation and fetal testis cord expansion. Proc Natl Acad Sci U S A 107: 10526–10531.
[40]
Mendis SH, Meachem SJ, Sarraj MA, Loveland KL (2011) Activin A balances Sertoli and germ cell proliferation in the fetal mouse testis. Biol Reprod 84: 379–391.
[41]
Brown CW, Houston-Hawkins DE, Woodruff TK, Matzuk MM (2000) Insertion of Inhbb into the Inhba locus rescues the Inhba-null phenotype and reveals new activin functions. Nat Genet 25: 453–457.
[42]
Mithraprabhu S, Mendis S, Meachem SJ, Tubino L, Matzuk MM, et al. (2010) Activin bioactivity affects germ cell differentiation in the postnatal mouse testis in vivo. Biol Reprod 82: 980–990.
[43]
Yao HH, Aardema J, Holthusen K (2006) Sexually dimorphic regulation of inhibin beta B in establishing gonadal vasculature in mice. Biol Reprod 74: 978–983.
[44]
Memon MA, Anway MD, Covert TR, Uzumcu M, Skinner MK (2008) Transforming growth factor beta (TGFbeta1, TGFbeta2 and TGFbeta3) null-mutant phenotypes in embryonic gonadal development. Mol Cell Endocrinol 294: 70–80.
[45]
Moreno SG, Attali M, Allemand I, Messiaen S, Fouchet P, et al. (2010) TGFbeta signaling in male germ cells regulates gonocyte quiescence and fertility in mice. Dev Biol 342: 74–84.
[46]
Sarraj MA, Escalona RM, Umbers A, Chua HK, Small C, et al. (2010) Fetal testis dysgenesis and compromised Leydig cell function in Tgfbr3 (beta glycan) knockout mice. Biol Reprod 82: 153–162.
[47]
Inman GJ, Nicolas FJ, Callahan JF, Harling JD, Gaster LM, et al. (2002) SB-431542 is a potent and specific inhibitor of transforming growth factor-beta superfamily type I activin receptor-like kinase (ALK) receptors ALK4, ALK5, and ALK7. Mol Pharmacol 62: 65–74.
[48]
Laping NJ, Grygielko E, Mathur A, Butter S, Bomberger J, et al. (2002) Inhibition of transforming growth factor (TGF)-beta1-induced extracellular matrix with a novel inhibitor of the TGF-beta type I receptor kinase activity: SB-431542. Mol Pharmacol 62: 58–64.
[49]
DaCosta Byfield S, Major C, Laping NJ, Roberts AB (2004) SB-505124 is a selective inhibitor of transforming growth factor-beta type I receptors ALK4, ALK5, and ALK7. Mol Pharmacol 65: 744–752.
[50]
Singh J, Chuaqui CE, Boriack-Sjodin PA, Lee WC, Pontz T, et al. (2003) Successful shape-based virtual screening: the discovery of a potent inhibitor of the type I TGFbeta receptor kinase (TbetaRI). Bioorg Med Chem Lett 13: 4355–4359.
[51]
Sawyer JS, Anderson BD, Beight DW, Campbell RM, Jones ML, et al. (2003) Synthesis and activity of new aryl- and heteroaryl-substituted pyrazole inhibitors of the transforming growth factor-beta type I receptor kinase domain. J Med Chem 46: 3953–3956.
[52]
Vallier L, Mendjan S, Brown S, Chng Z, Teo A, et al. (2009) Activin/Nodal signalling maintains pluripotency by controlling Nanog expression. Development 136: 1339–1349.
[53]
Vallier L, Reynolds D, Pedersen RA (2004) Nodal inhibits differentiation of human embryonic stem cells along the neuroectodermal default pathway. Developmental Biology 275: 403–421.
[54]
Morizane A, Doi D, Kikuchi T, Nishimura K, Takahashi J (2011) Small-molecule inhibitors of bone morphogenic protein and activin/nodal signals promote highly efficient neural induction from human pluripotent stem cells. J Neurosci Res 89: 117–126.
[55]
Hueng DY, Lin GJ, Huang SH, Liu LW, Ju DT, et al. (2011) Inhibition of Nodal suppresses angiogenesis and growth of human gliomas. J Neurooncol 104: 21–31.
[56]
Miles DC, van den Bergen JA, Sinclair AH, Western PS (2010) Regulation of the female mouse germ cell cycle during entry into meiosis. Cell Cycle 9: 408–418.
[57]
Lin Y, Gill ME, Koubova J, Page DC (2008) Germ cell-intrinsic and -extrinsic factors govern meiotic initiation in mouse embryos. Science 322: 1685–1687.
[58]
Miles DC, Western PS (2012) Germ cell sex and cell cycle. Histology and Histopathology 27: 445–457.
[59]
Souquet B, Tourpin S, Messiaen S, Moison D, Habert R, et al. (2012) Nodal signaling regulates the entry into meiosis in fetal germ cells. Endocrinology 153: 2466–2473.
[60]
Mesnard D, Guzman-Ayala M, Constam DB (2006) Nodal specifies embryonic visceral endoderm and sustains pluripotent cells in the epiblast before overt axial patterning. Development 133: 2497–2505.
[61]
Combes AN, Wilhelm D, Davidson T, Dejana E, Harley V, et al. (2009) Endothelial cell migration directs testis cord formation. Dev Biol 326: 112–120.
[62]
Brennan J, Capel B (2004) One tissue, two fates: molecular genetic events that underlie testis versus ovary development. Nat Rev Genet 5: 509–521.
[63]
Miles DC, van den Bergen JA, Wakeling SI, Anderson RD, Sinclair AH, et al. (2012 ) The proto-oncogene Ret is required for fetal germ cell survival. Developmental Biology 365: 101–109.
[64]
van den Bergen JA, Miles DC, Sinclair AH, Western PS (2009) Normalizing Gene Expression Levels in Mouse Fetal Germ Cells*. Biol Reprod 81: 362–370.
[65]
Daggag H, Svingen T, Western PS, van den Bergen JA, McClive PJ, et al. (2008) The rhox homeobox gene family shows sexually dimorphic and dynamic expression during mouse embryonic gonad development. Biology of Reproduction 79: 468–474.
[66]
McClive PJ, Sinclair AH (2001) Rapid DNA extraction and PCR-sexing of mouse embryos. Mol Reprod Dev 60: 225–226.
