Molecular and Cellular Endocrinology 359 (2012) 6677

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Molecular and Cellular Endocrinology

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Review

Activins and inhibins in mammalian testis development: New models, new insights
B. Barakat a, C. Itman b,c, S.H. Mendis a, K.L. Loveland b,c,
a

Monash Institute of Reproduction and Development, Monash University, Clayton, Victoria, Australia Department of Anatomy and Developmental Biology, Monash University, Clayton, Victoria, Australia c Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia
b

a r t i c l e

i n f o

a b s t r a c t
The discovery of activin and inhibins as modulators of the hypothalamic-pituitarygonadal axis has set the foundation for understanding their central importance to many facets of development and disease. This review contains an overview of the processes and cell types that are central to testis development and spermatogenesis and then provides an update focussed on information gathered over the past ve years to address new concepts about how these proteins function to control testis development in fetal and juvenile life. Current knowledge about the interactive nature of the transforming growth factor-b (TGFb) superfamily signalling network is applied to recent ndings about activins and inhibins in the testis. Information about the regulated synthesis of signalling components and signalling regulators in the testis is integrated with new concepts that demonstrate their functional signicance. The importance of activin bioactivity levels or dosage in controlling balanced growth of spermatogonial cells and their niche at different stages of testis development is highlighted. 2012 Elsevier Ireland Ltd. All rights reserved.

Article history: Available online 3 March 2012 Keywords: Spermatogenesis Activin Inhibin Cell signalling Testis development

Contents 1. Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Activins and inhibins: part of a big picture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2. Activins and inhibins: an antagonism essential to male reproductive function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3. Mammalian testis function and development: key events and current questions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3.1. The adult testis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3.2. The fetal testis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3.3. The postnatal testis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Recent discoveries and new questions relating to activins and inhibins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Activin and inhibin in fetal gonadogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Activins and inhibins in the first wave of spermatogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1. Dynamic synthesis of ligands and signalling machinery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.2. Mouse models. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.3. Other TGFb superfamily ligands influence postnatal germ cell development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Regulated cellular responsiveness to activin stimulation as a means to control cell fate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Frontier studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66 66 69 69 69 70 70 71 71 72 72 72 73 73 74 74 74

2.

1. Background 1.1. Activins and inhibins: part of a big picture

Corresponding author. Address: Building 77, Wellington Road, School of Biomedical Sciences, Monash University, Clayton, VIC 3880, Australia. E-mail address: kate.loveland@monash.edu (K.L. Loveland).
0303-7207/$ - see front matter 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.mce.2012.02.018

Activins and inhibins are among the most widely studied members of the transforming growth factor-b (TGF b) superfamily in terms of their impact on reproductive biology. Activin and inhibin

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Fig. 1. Cartoon depicting changes in TGFb and activin signalling in selected mouse models. Ligands of the TGFb superfamily are synthesised as large precursor proteins consisting of a pro-region and a C-terminal mature peptide. These molecules form a disulde-bonded dimer, after which the pro-region is cleaved, releasing a biologically active mature protein, which signals through type I and type II serine/threonine kinase receptors. Activins signal by binding to the type II receptor subunit, which binds to and activates the type I receptor. Inhibins bind to the type III receptor, TGFbRIII, forming a complex with the type II receptor which prevents activin from signalling. TGFb2 requires TGFbRIII for binding and signalling through the type II and I receptors. In the Inhba knockout mouse, loss of the bA subunit results in the absence of both inhibin A and activin A proteins, and the absence of activin A signalling. Activin B signalling is still present, but this binds with lower afnity to the type II receptors and also signals through a different type I receptor to activin A. InhbaBK mice were generated by inserting the bB subunit coding sequence into the bA locus, allowing for the expression of the bB subunit in the same spatiotemporal manner as bA . These mice exhibited a partial rescue of the bA knockout phenotype, and represent a model of reduced activin bioactivity. Inhibin a knockout mice do not make inhibin protein, affecting regulation of activin signalling. The absence of TGFbRIII in TGFbRIII null animals results in diminished TGFb2 signalling and reduced ability for inhibin to inhibit activin signalling.

were rst isolated based on their ability to regulate FSH homeostasis (Franchimont et al., 1979; Ling et al., 1986; Robertson et al., 1985). Activins consist of dimers of activin/inhibin b subunits, four of which have been identied in mammals: bA (Inhba), bB (Inhbb), bC (Inhbc) and bE (Inhbe). The mature proteins arise through cleavage of pre- and propeptides from a single activin b polypeptide; each mature subunit forms 3 intramolecular disulde bonds, and two subunits are joined by a further disulde bond (Fig. 1, lefthand panel). The most widely studied activins are activin A (bA:bA homodimer), activin B (bB:bB homodimer), and activin AB (bA:bB heterodimer). The bC subunit has also been demonstrated to form a homodimer as well as to heterodimerise with bA and bB subunits in vitro (Mellor et al., 2000). It was recently identied as a functional inhibitor of activin A in vivo in an ectopic expression transgenic mouse model (Gold et al., 2009). Inhibins are heterodimers consisting of an a subunit (Inha) and one of the b subunits, so that inhibin A is an a:bA heterodimer and inhibin B is an a:bB heterodimer (Fig. 1, lefthand panel). Inhibins directly compete with activins for receptor binding in the presence of the TGFb type III receptor, betaglycan (Lewis et al., 2000). Activins and inhibins are highly conserved across species, and each subunit is understood to have conserved functions. Of critical importance to revealing these roles has been the development of progressively more specic and sensitive assays that distinguish between individual subunits and monomeric versus dimeric peptides (reviewed in McNeilly, 2011, this volume). This has been further enhanced by the production of mouse models with subunit- and cell-specic genetic modications (reviewed in Loveland et al., 2007), as discussed later in this review. Activin signalling is mediated through formation of a multimeric receptor complex, with subunits that feature intrinsic serine/threonine kinase activity (Massague, 1998). Crosstalk

withother TGFb superfamily ligands at all levels of the signalling pathway is understood to determine the nal response to this stimulus (Fig. 2, lefthand panel). The extent of shared signalling machinery will logically vary between cells in different physiological conditions, so this big picture must be considered in each set of circumstances, with the expectation that synthesis of these signalling components is itself dynamic. However, the canonical pathway for activin action is established to begin when an activin dimer binds the constitutively active Type II receptor subunit, either ActRIIA or ActRIIB, which enables recruitment and then activation through phosphorylation of a Type I receptor subunit, ALK2, ALK4 (by activin A) or ALK7 (by activin B; Tsuchida et al., 2004). The Type 1 receptor kinase activity mediates recruitment and phosphorylation of downstream signalling molecules, typically SMADs 2 and/or 3. The phosphorylated SMADs bind SMAD4 to yield a complex which translocates into the nucleus and affects gene transcription. In general, SMADs 2 and 3 are recruited upon binding of either activins or TGFbs to their distinct cell surface receptors. In contrast, while both activins and BMPs can signal through ActRIIA and ActRIIB receptors, BMPs activate Smads 1, 5 or 8 via phosphorylation of ALK3 or ALK6 Type I receptor subunits (Shi and Massague, 2003). While betaglycan functions to facilitate blockade of activin signalling by inhibin, it is also essential to TGFb2 signalling, as unlike other ligands in this superfamily, TGFb2 cannot directly signal through a TGFb type II receptor (Cheifetz et al., 1990; Lopez-Casillas et al., 1993). Betaglycan is also essential for inhibin antagonism of both activin and BMP signalling capabilities (Wiater and Vale, 2003), enabling inhibin to compete for binding to ActRIIA/IIB receptors without initiating subsequent recruitment of a type I receptor (Wiater and Vale, 2003). Thus the presence of betaglycan can simulataneously curtail signalling by several ligands, including

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Fig. 2. Canonical activin/TGFb signalling pathway is mediated by Smads and activity is modulated by a wide range of intracellular and extracellular proteins. (A): Activin shares Type II and Type I signalling receptor subunits with nodal, BMPs and GDF3, while TGFb signals through a distinct set of receptors, including a third non-signalling subunit, betaglycan (Tgfbr3; shown as a blue transmembrane protein). Activin binding triggers phosphorylation of Smads2 and 3 Inside the cell, signal transducers which are also recruited in response to TGFb binding. In contrast, Smads1, 5 and 8 are recruited following BMP signalling. Recruitment of Smad4 is common to each of these Smads, facilitating their translocation into the nucleus wherein ligand-specic response elements are recognised, leading to transcription of ligand-specic genes. (B): Extracellular (inhibins, follistatin), transmembrane (Bambi) and intracellular signalling modulators (Smad6, Smad7, Smurf1, Smurf2, Man1, Net25, HGS, ZVYFE9) all impact on the signalling pathways. Changing levels of these modulators have been documented throughout testis development and in different cell types.

activin, and selectively facilitate TGFb activity. Further highlighting the complexity of activin signalling regulation and its outcomes, the potency of inhibins A and B to antagonise activins and BMPs can be distinguished. This was shown in a mouse adrenocortical (AC) cell line, with inhibin A a more potent activin antagonist than inhibin B in these cells and the only inhibin that could antagonise BMPs 2, 6 and 7, while inhibin B exhibited a weak antagonist activity against BMP7 but not BMP2 and BMP6 (Farnworth et al., 2006). Such information is relevant to the observation of long-standing interest that, while inhibin B is the major circulating inhibin in adult humans and rodents, only inhibin A is present in rams (reviewed in McNeilly, 2011), and reinforces the value of delineating the cell-specic functions and interactions between these family members. Negative regulation of TGFb superfamily signalling is effected by antagonists which exist both at the cell surface, including inhibin, Bambi, follistatin, noggin, gremlin and chordin, and inside the cell, including inhibitory SMADs 6 and 7 (reviewed in (Itoh and Itoh, 2011) (Fig. 2, righthand panel). Many of these antagonize multiple ligands, while effectively serving simultaneously to enhance signalling from other pathway ligands, as described above for betaglycan. A protein with promiscuous signalling inhibition activity, Bambi was originally characterized as a dominant negative Type I receptor subunit which lacks serine/threonine kinase activity that can inhibit signalling by activins, BMPs and TGFbs (Onichtchouk et al., 1999). Why are there so many different regulators of activin signalling and how do they mediate interactions between the many members of this large superfamily? It appears that the regulated expression of these regulators in the developing male germline is indeed part of the answer, as discussed later. New evidence based on experimental analysis and mathematical modelling has brought to light the capacity for co-expression of multiple negative regulators of TGFb ligands to modulate the dynamic

range of ligand signalling inputs and mediate developmental canalization. In particular, the signalling inhibitors Bambi, SMAD6 and SMAD7 were recently identied as part of a BMP4 synexpression group because they are collectively induced by BMP4 signalling. They have recently been shown to act in vivo to reduce the phenotypic variability arising from environmental changes (Paulsen et al., 2011). The functional intersection of this synexpression group with activin and inhibin signalling events remains to be explored. Nodal is a TGFb-related ligand which also binds to the Type I and Type II activin receptors and is regulated by cell surface antagonists including Lefty and Cerberus. The consequent signal is carried inside the cell by SMAD2 to control downstream target gene activity, mediated by interaction with the FAST1 transcription factor (Schier, 2003; Shen and Schier, 2000). In the presence of its cofactor, Cripto, nodal competes directly with activin for access to the activin Type II receptors (Shen, 2007). Follistatin is a glycosylated monomeric protein which binds activin b subunits to make a stable, inactive complex that effectively blocks activin from interacting with its type II receptors (de Jong, 1997; Thompson et al., 2005). Follistatin can also bind other ligands, including inhibin, BMPs 2, 4, 6, 7, 11, and 15, and myostatin, with lower afnities (Abe et al., 2004; Canalis et al., 2003; Glister et al., 2004; Lin et al., 2003). The regulated synthesis of this protein will have important implications for local integration of signalling by many TGFb superfamily ligands. In addition to canonical TGFb signalling through the Smad transcription factors, mitogen-activated protein kinases (MAPKs) represent another major type of signalling intermediate for TGFb superfamily ligands (Mulder, 2000), comprised of extracellular signal-regulated kinases (Erks), c-Jun-N-terminal kinases (JNKs)/ stress-activated protein kinases (SAPKs), and p38 kinases (Yue and Mulder, 2000).

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Fig. 3. Activin and other TGFb signalling pathway components inuence testis and germline development. (A): Ligands implicated in different stages of development through analysis of genetically modied mice. (B): Gonocytes in the newborn testis are exposed to high levels of activin and inhibin. (C): Levels of activin and its inhibitors, inhibin and follistatin, are highly modulated as the testis develops. Panel C adapted from (Barakat et al., 2008).

The complexities of this signalling system highlight the requirement to delineate which components are present in each cell within every physiological context to fully understand how cells respond to its cues. Guidance for experimental studies in the context of the testis are based on accumulating knowledge of phenotypes and processes which are governed by these proteins, as described in the next sections. 1.2. Activins and inhibins: an antagonism essential to male reproductive function The process of spermatogenesis and all other aspects of male reproductive function depend on the hormones produced by the hypothalamus (gonadotropin-releasing hormone, GnRH), the anterior pituitary (luteinizing hormone, LH, and follicle stimulating hormone, FSH), and the testis (inhibin and testosterone), which collectively comprise the hypothalamic-pituitary-testis (HPT) axis (reviewed in Cooke and Saunders, 2002 and ODonnell et al., 2006). Activin is also important in the HPT axis, serving paracrine and probably also autocrine roles in each organ. Activin stimulates GnRH release from the hypothalamus (Calogero et al., 1998; Lee and Rivier, 1997; MacConell et al., 1999) and FSH release from the pituitary (Carroll et al., 1989, 1991; Weiss et al., 1993, 1995). Activin can also increase the number of GnRH receptors on the surface of gonodatropes and augments the GnRH-mediated transcriptional activation of GnRH receptors to enhance the pituitary

response to GnRH (Braden and Conn, 1992; Norwitz et al., 2002). Activin bioactivity is regulated by the co-ordinated actions of inhibin (endocrine) and follistatin (auto/paracrine) to control the timing, duration and amplitude of FSH secretion (Hurwitz and Santoro, 2004). In the testis, local activin production and control of its signalling pathways are critical for germ and Sertoli cell development and for adult testis function, as detailed in previous reviews (Chang et al., 2002; Itman et al., 2006; Loveland and Robertson, 2005; Xia and Schneyer, 2009). This review focuses on recent developments in the eld. 1.3. Mammalian testis function and development: key events and current questions 1.3.1. The adult testis Spermatozoa form in the adult testis through the highly ordered progression of cellular maturation steps collectively known as spermatogenesis (Clermont, 1966; Kerr, 1989; Kerr et al., 2006). Embedded in the seminiferous tubules of the adult testis, the male gamete transitions between three phases: mitosis (termed spermatogonia), meiosis (spermatocytes) and spermiogenesis (the morphological transformation of haploid round spermatids into elongated spermatids) (Fig. 3A). At the completion of spermatogenesis, spermatozoa are released into the tubule lumen and transit to the excurrent ducts. Spermatogonial stem cells (SSCs), which sustain spermatogenesis throughout adult life, are identied as

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type Asingle (As) spermatogonia in mice and type Apale (Ap) and Adark (Ad) spermatogonia in humans (Clermont, 1966; de Rooij and Russell, 2000; Dym et al., 2009). In adult mice, SSCs transition through an undifferentiated state in which they retain the capacity to function as regenerative stem cells following transplantation into a host testis (Nakagawa et al., 2010),(Brinster, 2002); these cells undergo mitotic nuclear divisions in the absence of complete cytokinesis. Clonal siblings remain linked by cytoplasmic bridges until spermatogenesis is complete (de Rooij and Grootegoed, 1998). The rst maturation steps, from Apr, Aal4-Aal16, are marked by the loss of the essential spermatogonial stem cell receptors for glial cell-derived neurotrophic factor (GDNF), Gfr1a and c-ret (Meng et al., 2000) and by a gradual reduction in neurogenin 3 (Nakagawa et al., 2010; Yoshida et al., 2006). Spermatogonia which have matured sufciently to lose the capacity to function as stem cells are termed differentiated (A1, A2, A3, A4, In and B spermatogonia); these feature the cell surface signalling receptor tyrosine kinase Kit (Schrans-Stassen et al., 1999; Shinohara et al., 1999). The number of spermatogonial mitoses is regulated; there are approximately 9 divisions per clone before spermatocytes form in the mouse (de Rooij and Russell, 2000). The mechanisms underpinning these transitions are of intense interest to stem cell biologists and clinicians in the quest to learn how germline differentiation is controlled and sustained to produce sperm in numbers sufcient for normal fertility. Under the inuence of retinoic acid and mediated by Dmrt1 (Hogarth et al., 2011; Matson et al., 2010), spermatogonia become the primary spermatocytes which undergo sister chromatid exchange and a nuclear division during the rst phase of meiosis (meiosis I) to yield secondary spermatocytes; the second meiotic division (meiosis II) produces 4 haploid round spermatids from each progenitor. Round spermatids undergo major chromosomal remodeling to package their genetic content and elaborate the microtubule-based tail to form mature spermatozoa during spermiogenesis. The architectural and nutritional support required for spermatogenesis is delivered by Sertoli cells, the backbone of the seminiferous epithelium (for review see references: Madara, 1998; Skinner et al., 1991). In adulthood, these FSH-responsive cells are normally non-proliferative and interconnected through the tight junctions which form the blood-testis barrier. This structure blocks passage of macromolecules and cells between the interstitial space and the tubule lumen (Fawcett, 1975), preventing contact between post-mitotic germ cells and immune cells. The seminiferous tubules are ensheathed in a basement membrane, the joint product of Sertoli cells and the contractile peritubular myoid cells which surround the tubules (Skinner and Fritz, 1985). In addition to the mechanical function of contracting to propel spermatozoa through the lumen, peritubular myoid cells also secrete growth factors including TGFa, TGFb, insulin-like growth factor I (IGF-1) and activin A that inuence Sertoli cell development and function, including modulation of Sertoli cell secretion of transferrin, inhibin and androgen binding protein (de Winter et al., 1994; Norton and Skinner, 1989; Skinner et al., 1988, 1989, 1985). The interstitium between these tubules contains blood and lymphatic vessels, Leydig, nerve and broblast cells, macrophages and lymphocytes. The Leydig cells are the site of testosterone production in adults, arising from the conversion of androstenedione via the enzyme 17b hydroxysteroid dehydrogenase 3, a process essential for ongoing spermatogensis in adults (McCarrey, 1993; Russell et al., 1990)). Adult Leydig cells are critically dependent on the pituitary-derived luteinizing hormone (LH) for normal function and full steroidogenesis. 1.3.2. The fetal testis Testis development in the fetal mouse involves several integrated processes which commence after the migration of sexually

indifferent primordial germ cells into the bipotential genital ridge around embryonic day E10.5. Requisite expression of Sry (sexdetermining region of the Y chromosome) by the Sertoli cells directs the gonad to form a testis (Hacker et al., 1995; Koopman et al., 1991); the absence of Sry and expression of Wnt4 in the female gonad results in ovary formation (Vainio et al., 1999). A role for activin in suppressing Wnt4 has been proposed to mediate masculinisation of the gonad (Yao et al., 2006). Somatic cell products attract cells from the adjacent mesonephros into the developing testis (Cool et al., 2008; Martineau et al., 1997; Tilmann and Capel, 1999) to mediate testicular cord formation and enable the coordinated and appropriate cellular maturation of somatic and germ cells. Germ cells become committed to a male fate and are termed gonocytes. Located in the centre of the cords, they are enveloped by Sertoli cells. Gonocytes cease mitotic divisions by E14.5 (Western et al., 2008) and are quiescent until after birth. In contrast, the testicular somatic cell populations expand by mitosis throughout fetal and pre-pubertal life. The mechanisms by which these distinct cell cycle outcomes are effected within the intimate milieu of the fetal testis highlight the context-dependence of growth factor signalling outcomes. Production of androgens by fetal Leydig cells occurs shortly after their differentiation at around E13 in the mouse (Gondos, 1980) and peaks just before birth (E18). These cells secrete androstenedione which is converted to testosterone in the cords (OShaughnessy et al., 2000) and also act to induce testicular descent through production of insulin-like growth factor 3 [(Insl3)/relaxin like factor (RLF)] (Klonisch et al., 2004). Gender-specic and developmentally regulated synthesis of TGFb superfamily ligands, including activins, and their inhibitors are understood to regulate many of these events. 1.3.3. The postnatal testis The quiescent gonocytes resume mitosis and migrate to the basement membrane, becoming spermatogonia with distinct transcriptional proles (McCarrey, 1993; Wang et al., 2001). Gonocyte migration and proliferation occur as independent events in rodents (McGuinness and Orth, 1992; Nagano et al., 2000), with gonocytes beginning migration toward the basement membrane 2 days before they resume mitosis in mice (Nagano et al., 2000). Most gonocytes have relocated to the cord basement membrane by 4.5 day post-partum (dpp) (Nagano et al., 2000), coinciding with appearance of the rst spermatogonial stem cells (McLean et al., 2003). In humans, these events occur in early fetal life; gonocytes which fail to migrate and/or mature during fetal life are considered to be able to form adult germ cell tumour precursors, termed carcinoma in situ (CIS) cells, partly because of their similar morphological and protein expression proles (Loveland et al., 2010). CIS cells progress to form seminomas and non-seminomas at or following puberty, with testicular germ cell tumours typically diagnosed in young men. The incidence of this disease is increasing in prevalence worldwide (Bray et al., 2006; Skakkebaek et al., 2001), highlighting the need for animal models to identify its underlying aetiology. In rodents, the rst sperm mature directly from gonocytes during rst wave of spermatogenesis, while in adults, sperm arise from SSCs. There is much interest in understanding how the onset of spermatogenesis is controlled in both circumstances, as this knowledge would inform efforts to diagnose and treat cases of infertility due to spermatogenic arrest where SSCs remain, including after radiotherapy. Of central importance is the Kit receptor tyrosine kinase, which is present in differentiated spermatogonia and in primary spermatocytes in rodents (Manova et al., 1990; Schrans-Stassen et al., 1999). Interestingly, while traditional histological methods rst identied differentiated spermatogonia at 8 dpp in mice, recent studies using in situ hybridisation (Yoshida et al., 2006) and ow cytometry (Ohbo et al., 2003) have shown that

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Kit positive (Kit+) cells are present at 3 dpp. Van Haaster and de Rooij rst proposed that some gonocytes convert directly into differentiated spermatogonia while others generate SSC (van Haaster and de Rooij, 1993). This was conrmed using the transcription factor neurogenin 3 (Ngn3) as a marker exclusive to SSC and undifferentiated spermatogonia (Yoshida et al., 2006). Gonocytes, which are Ngn3, give rise to Kit+ spermatogonia detectable at 3 dpp using in situ hybridisation, and these cells progress through spermatogenesis to make the rst sperm. The Ngn3+/Kit SSC population rst observed at 45 dpp is also derived from gonocytes and corresponds to the stem cell pool from which subsequent spermatogenic cycles begin. Because of their importance in governing this transition of stem cells into the differentiation pathway, there has been long-standing interest in the identication of factors which regulate production of Kit in germ cells and its ligand (named Kit ligand, KL, or stem cell factor, SCF) in Sertoli cells. A summary of such factors and the underlying transcriptional mediators has been recently published in relationship to spermatogenesis in the mammalian testis (Mithraprabhu et al., 2010), and amongst these are factors relating to TGFb superfamily function, as described below. The number and function of Sertoli cells, the population of which is formed prior to puberty, are the critical determinants of the adult testis sperm output, as each Sertoli cell supports the maturation of a nite number of germ cells at any one time (Orth et al., 1988; Sharpe, 1994). Murine Sertoli cells are mitotically active during the rst two postnatal weeks (Vergouwen et al., 1991) with no signicant change in Sertoli cell numbers occurring after 18 dpp (Vergouwen et al., 1993). In these rst weeks after birth, rodent Sertoli cells exhibit a plethora of morphological and functional changes as they reach their mature, non-proliferative phenotype. This includes progressive reduction in synthesis of the TGFb super} llerian hormone and activin A, emergence family ligands, anti-Mu of androgen responsiveness, transient upregulation of the Type IIA activin receptor, formation of the blood-testis barrier and polarized secretion to form an epithelium with a lumen (Fragale et al., 2001; Sharpe et al., 2003). Each of these changes represents the acquisition by the seminiferous epithelium of the capacity to support spermatogonial progression through meiosis and then spermiogenesis, enabling mature spermatozoa to be released as individual cells into the lumen. 2. Recent discoveries and new questions relating to activins and inhibins Several experimental approaches have identied roles served by activins and inhibins in testis development, including the production and analysis of mice with genetic modications that alter activin signalling, testis cell or organ cultures with addition of activin and other TGFb superfamily ligands (including inhibin) and unbiased transcriptome screens. The following sections survey relatively new ndings in the context of previous investigations, beginning with the fetal testis. 2.1. Activin and inhibin in fetal gonadogenesis Until recently, little new information had emerged regarding the functional involvement of these proteins in fetal development since Toppari and colleagues provided the rst evidence of a sexually dimorphic response to activin and its inhibitors in the gonad. This early study provided evidence that activin suppressed cellular proliferation in both testes and mesonephroi of E14 rat testis cultures but not at E15 or E18 (Kaipia et al., 1994), with the latter times corresponding to the period of gonocyte quiescence. Subsequently, activin receptors were identied by immunohistochemistry in human fetal gonocytes (Anderson et al., 2002),

demonstrating the potential for activin to inuence their development. Transcript analyses using Affymetrix microarrays and real time PCR showed Inhba transcripts become progressively elevated in the testis, but not the ovary, immediately after sex determination (Mendis et al., 2011; Small et al., 2005), while a differential display approach showed the activin antagonist, follistatin, is selectively upregulated in the fetal ovary (Menke and Page, 2002). Two independent reports recently described the fetal testes phenotype of mice lacking activin A coding sequence, either as a complete knockout (Inhba-/- strain; Fig. 1; Matzuk et al., 1992) or following conditional deletion of the Inhba coding sequence from Leydig cells (Amhr2cre/+;Inhba/) providing direct evidence that activin A drives Sertoli cell proliferation (Archambeault and Yao, 2010; Mendis et al., 2011), with proliferation reduced to 50% of wild type values in both models. Evidence that somatic cells, but not germline cells, produce the activin A which mediates this has been obtained using a somatic cell-specic cre (Sf-1) to delete this subunit (Archambeault et al., 2011). Fetal Leydig cells were identied as the source of activin A essential for Sertoli cell proliferation and seminiferous cord coiling during fetal life using the anti-Mllerian hormone type 2 receptor-cre mouse strain to delete Inhba specically in fetal Leydig cells, while Sertoli cell-specic deletion of Smad4 (rendering these cells unresponsive to activin A and other TGFb ligands) yielded reduced fetal Sertoli cell proliferation in these mice (Archambeault and Yao, 2010). The authors have reasoned that the Smad4 deciency most likely related to loss of activin signalling because no similar phenotype was reported in mice lacking other family ligands TGFb13, anti-Mllerian hormone or activin B. Importantly, the fetal Leydig cell-specic deletion of Smad4 reproduced the fetal Sertoli cell phenotype and smaller testes of the complete activin A null (Inhba-/-) mouse (Mendis et al., 2011), highlighting the central importance of Leydig cells as a site of fetal activin production. In addition to this somatic cell phenotype, the cords of newborn Inbha-/- mice were noted to have considerably more gonocytes (Loveland et al., 2005), and two distinct proliferation measures demonstrated that Inhba-/- testes contain proliferating germ cells at E15.5 and E17.5, when these cells are quiescent in wild type littermates (Mendis et al., 2011). Other TGFb superfamily signals affect both germ and somatic cell development in the fetal testis, and these would be functioning simultaneously with activin. In particular, TGFb has been implicated in controlling germline survival and passage through the cell cycle. Early cell organ culture work (Olaso et al., 1998) demonstrated that TGFb ligands induce apoptosis of proliferating, but not quiescent, fetal rat gonocytes. More recently, the selective absence of the TGFbRII receptor subunit in germ cells was linked to a delay in gonocyte entry into quiescence, while TGFb2 addition increased the duration of gonocyte quiescence in cultured gonads (Moreno et al., 2010). In murine models of chronic TGFb ligand depletion, including TGFb2-/- and TGFb1-/- strains, reduced gonocyte number at birth and decreased seminiferous cord numbers at E15 were described (Memon et al., 2008). Because TGFb2, inhibin a and activin A protein subunits are made by gonocytes during their period of fetal quiescence (Meehan et al., 2000; Mendis et al., 2011; Moreno et al., 2010; Fig. 3B), it is logical to hypothesize that the combined inuence of these factors determines gonocyte cell cycle status through autocrine and/or paracrine signalling. Importantly, signalling by TGFb2 and activin A each appears to affect growth of both germline and somatic cells, with normal levels of activin A shown to create a balance between gonocyte number and niche size (Mendis et al., 2011), an observation also seen in the early postpartum testis (Mithraprabhu et al., 2010) and discussed below. Forming a functional link between activin/inhibin and TGFb is betaglycan; mice lacking Tgfbr3 produce no betaglycan and have reduced or absent TGFB2 signalling (Stenvers et al., 2003; Fig. 1). While these mice do not usually survive to birth, formation of

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testicular cords is abnormal when examined at E12.5 and E13.5, and several Sertoli cell transcripts are reduced while those of germ cells are not (Sarraj et al., 2010). The Tgfbr3 transcript was identied as exclusively interstitial prior to birth (Sarraj et al., 2007) and Tgfbr3-/- mice showed evidence of reduced functional development of Leydig cells, with no change in Leydig cell number (Sarraj et al., 2010). Adding to the importance of Leydig cell-derived activin for cord growth noted above (Archambeault and Yao, 2010), this information provides a new mechanistic grounding for understanding the importance of TGFb pathway crosstalk between testicular compartments that is required for normal fetal development. 2.2. Activins and inhibins in the rst wave of spermatogenesis 2.2.1. Dynamic synthesis of ligands and signalling machinery Roles for activin in stimulating Sertoli cell proliferation and regulating germ cell proliferation in the postnatal testis were identied well over a decade ago using various cell and organ culture approaches (Boitani et al., 1995; Buzzard et al., 2003; Fragale et al., 2001; Mather et al., 1990; Meehan et al., 2000). However, our understanding of their in vivo relevance was limited by a lack of precise knowledge of the cellular sites of ligand and receptor synthesis. A report from the Boitani laboratory (Fragale et al., 2001) demonstrated that, while all activin receptor subunits were present in the developing rat testis, the 6 kb transcript encoding the ActRIIA subunit was selectively upregulated in Sertoli cells at the developmental age (79 dpp) when they undergo a nal wave of in vitro proliferation in response to the combination of FSH plus activin. This result suggested that activin signalling is highly modulated as Sertoli cells are entering the phase of terminal differentiation, highlighting the potential for regulated production of signalling machinery to contribute to their altered responsiveness to activin. This point is addressed further below. A recent study from our laboratory documented the highly dynamic cellular localisation patterns and transcript levels of activin bA, activin bB, activin bC and inhibin a subunits in the mouse testis from birth to adulthood (Barakat et al., 2008). The Inhba transcript and its encoded activin bA subunit was detected in Sertoli and Leydig cells at all ages. The presence of activin A protein, but not Inhba transcript in gonocytes at 0 dpp was similar to an early report of the newborn rat testis (Meehan et al., 2000). This observation supports the hypothesis that the activin A subunit or dimeric protein is stored for a period of days then either secreted or degraded as germ cells commence the rst wave of spermatogenesis. Both Inhba transcript and protein were detected in germ cells at all other ages, spanning from mitotic spermatogonia through to round spermatids. The concentration of activin A in whole mouse testis lysates was highly modulated during the rst postpartum week, signicantly decreasing between birth (0 dpp) and 1 dpp, then signicantly increasing to peak at 3 dpp before decreasing again to reach levels below that present at birth by 6 dpp. Such observations indicate that ne control of activin levels is occurring as gonocytes re-enter the cell cycle and migrate to the cord basement membrane at the onset of spermatogenesis, again reinforcing an earlier report showing that rat testis cords cultured with follistatin plus FSH exhibited increased gonocyte migration to the basement membrane (Meehan et al., 2000). The Inhbb transcript and its encoded activin bB subunit protein were also detected in Sertoli, Leydig and germ cells in a pattern nearly identical to Inhba and activin A, suggesting that activin A, activin AB and activin B may all be contributing to the control of cellular differentiation at this time (Barakat et al., 2008). The activin bC protein was also detected in the postnatal testis in somatic and germ cells, and it was clearly observed in the nucleus of gonocytes (at 0 dpp), spermatogonia (at 3 dpp) and in round spermatids (Barakat et al., 2008). The

signicance of this is not known, however the bA protein has also been identied in the nucleus of primary spermatocytes (Barakat et al., 2008; Blauer et al., 1999) and the mature coding region shown to contain a sequence sufcient to direct nuclear localisation (Blauer et al., 1999). Inhibin a protein was not detected in germ cells but was present in Sertoli and Leydig cells at all ages analysed. The signal intensity variation between adjacent Sertoli cells in 0 dpp, 15 dpp and adult testes (see Fig. 3C) provides a very clear indication that Sertoli cells are functionally heterogeneous at all ages. This observation is yet to be linked to differences in Sertoli cell responsiveness to stimuli or other functional parameters but provides an intriguing clue regarding the focal nature of Sertoli cell tumours in Inha mice (Matzuk et al., 1992). 2.2.2. Mouse models The establishment of mouse strains lacking activin A (Inhba-/-; Matzuk et al., 1992) and activin B (Inhbb-/-; Matzuk et al., 1995) led to the initial diagnosis that activin A was essential for postnatal life and activin B was not. These have also led to the understanding that inhibin a is tumour suppressor and revealed many roles of physiological importance for activin, including in the mediation of cancer cachexia (Vanchieri, 2010; Zhou et al., 2010). Inha-/- males develop gonadal tumours comprised of stromal, Sertoli-like cells; at 4 weeks of age the tumours are typically focal, but by 6 weeks of age they have spread to invade blood vessels, disrupt the seminiferous epithelium architecture and impair spermatogenesis. These tumours secrete activin A and B, and Inha-/mice succumb to cachexia, which can be reversed by interfering with activin signalling (Q. Li et al., 2007). With the creation of an activin A hypomorph, the Inhba BK/BK strain (herein referred to as BK; Fig. 1), in which the activin bA mature subunit coding sequence was replaced with the activin bB coding sequence (Brown et al., 2000), the rst in vivo demonstration of a role for activin A in male fertility was produced. These mice survive to adulthood, demonstrating the capacity for activin B to replace activin A to a certain extent, including alleviation of the craniofacial defects that caused neonatal lethality in Inbha mice. However, these mice have perturbed hair and eye development, and the onset of fertility in BK males is delayed by just over one week. This observation of delayed fertility made it apparent that activin inuenced more than just Sertoli cell proliferation in the developing testis. Functional differences between the bA and bB subunits are attributed to their distinct afnities for the activin type II receptors, with activin A having higher afnity than activin B for ActRIIA (Mathews and Vale, 1991) and activin B having higher afnity for ALK7 (Tsuchida et al., 2004). Hence, the InhbaBK/BK mouse represents a model of reduced total activin bioactivity from which some roles of activin A in postnatal testicular development can be deduced. The number and relative proportion of Sertoli cells and germ cell subtypes have been determined in InhbaBK/BK mice at day 7, when Sertoli cells are immature and proliferating, and at day 14, when they have ceased mitosis and adopt mature features (Mithraprabhu et al., 2010). Sertoli cell numbers are signicantly lower in InhbaBK/BK mice at day 7, tting with the known role of activin in promoting their proliferation, while in contrast, the ratio of germ to Sertoli cells was signicantly higher, indicating that germ cell proliferation is not tightly linked to the pace of epithelium development at this time. This was reinforced by the elevated ratio of germ cell:Sertoli cell transcripts (encoding Kit and clusterin, respectively) and signicantly higher levels of meiotic markers in InhbaBK/BK testes. However, the total Kit transcript was signicantly elevated only in the whole testes of heterozygote animals, suggesting that the pace and nature of germline maturation is dictated by the dose of activin in a non-linear manner. Remarkably, ow cytometry measurements indicate that the relative proportion of Kit + germ cells is higher in InhbaBK/BK testes, and the Kit transcript

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was elevated in germ cell/Sertoli cell cultures following exposure to activin treatment in samples of 8 day, but not 4 day, testis cells. These ndings support the growing understanding that activin bioactivity affects the testicular maturation at many levels, and that responsiveness to this factor changes as the testis matures (Mithraprabhu et al., 2010). 2.2.3. Other TGFb superfamily ligands inuence postnatal germ cell development Glial cell line-derived neurotropic factor, GDNF, is a noncanonical member of the TGFb superfamily that signals through a different set of receptors, GFRa1 and c-ret. GDNF is made by Sertoli cells and its synthesis at an appropriate level is essential for maintenance of SSCs, as shown through analysis of mice either haplo-insufcient or over-producing GDNF (Meng et al., 2000). The transition of SSCs into differentiating spermatogonia is characterized by the loss of GDNF receptors and acquisition of the Kit receptor (Nakagawa, 2010), reinforcing the importance of identifying the factors, such as activins, certain BMPs and retinoic acid, which may directly impact on Kit expression (Mithraprabhu and Loveland, 2009; Mithraprabhu et al., 2010; Pellegrini et al., 2003; Young et al., 2011). Exposure of spermatogonia from juvenile mice to BMP4 appears to elevate Kit protein and signalling activity (Pelligrini et al., 2003), while both activin A and BMP4 reduce the stem cell potential of such juvenile spermatogonia (KanatsuShinohara et al., 2003). Of additional relevance to the roles played by activin in directly contolling germline development is the potential for nodal to affect SSCs. Evidence of nodal and its receptors in mouse SSCs and in a mouse gonocyte-like cell line has been reported (He et al., 2009), suggestive of its potential paracine and/or autocine effects on these primitive cell types. However this nding could not be conrmed by a different group (Wu et al., 2010), leaving the potential for a balance between nodal, activin and BMP signalling to coordinate spermatogonial maturation as an important issue to address in future studies. 2.3. Regulated cellular responsiveness to activin stimulation as a means to control cell fate The large collection of in vivo and in vitro evidence discussed so far illustrates the complex nature by which ligands of the TGFb superfamily control testis development and adult fertility. That testicular somatic and germ cells display different responses to ligands, despite residing within the same microenvironment, and that ligand production in the developing and adult testis is regulated (Barakat et al., 2008; Buzzard et al., 2003; Loveland and Robertson, 2005), implies that there exist mechanisms to selectively regulate the responsiveness of specic cell-types to ligands, as discussed above. In a bid to understand how cell-specic responses to ligands are effected in the testis, we and others have documented the expression patterns of signal-transducing Smad2 and Smad3 in the developing and adult mouse testis. All testicular cells in the neonatal, juvenile and peripubertal mouse testis, and all somatic cells and germ cells from spermatogonia through to round spermatids in the adult testis, have nuclear-localized phosphorylated Smad2/3 (Itman et al., 2009a). Surprisingly, this has also been documented in the fetal testis in the absence of activin A (Mendis et al., 2011). Because these and other data discussed earlier in this review indicate simultaneous transduction by several TGFb superfamily members, we and others proposed that differential responsiveness of cells to these ligands may be achieved by regulated production of Smad2 versus Smad3 (Itman and Loveland, 2008; Kano et al., 2001; Wang and Zhao, 1999; Xu et al., 2003) and by modulators of Smad activity. This is likely to include the BMP4 synexpression group of signalling

inhibitors, Bambi, Smad6 and Smad7, as the levels of these proteins determine the dynamic range of BMP4 signals within individual cells (Paulsen et al., 2011). Several studies have identied highly regulated expression patterns of these signalling molecules and modulators in the testis, the relevance of which is now beginning to be understood. Smad3 has been recently identied as a fundamental modulator of Sertoli cell responses to activin A. As previously described, activin is essential for normal proliferation of immature Sertoli cells (Boitani et al., 1995; Buzzard et al., 2003; Fragale et al., 2001; Mendis et al., 2011), as well as later potentially inuencing Sertoli cell maturation (Itman et al., 2009b, 2011a). Developmentally regulated responses of Sertoli cells to activin correspond to a switch in activin signal transduction, from preferential utilization of Smad3 by immature, proliferative Sertoli cells to utilization of both Smad2 and Smad3 by maturing Sertoli cells (Itman et al., 2009b). The physiological relevance of these ndings is illustrated by mice with excess activin production (Inha-/- mice) and mice with altered Smad3 dosage. In Inha-/- mice, excessive activin production results in Sertoli cell tumours due to their inability to cease proliferating and lack of maturation. This phenotype is Smad3- dependent; Smad3-Inha double knockout mice do not develop tumours (Q Li et al., 2007; Looyenga and Hammer, 2007) showing that tight regulation of Smad3 production and/or activity is necessary for normal Sertoli cell maturation. This hypothesis has been supported by the phenotypic difference between Smad3-/- and Smad3+/ mice; mice lacking Smad3 entirely have delayed maturation, whereas both Sertoli cell maturation and spermatogenesis are advanced in mice haploinsufcient for Smad3 (Itman et al., 2011a). The importance of identifying factors that regulate the expression of Smad2 versus Smad3 and which modulate Smad activity and stability are key outcomes from these studies. A shift in the ratio of Smad2 versus Smad3 occurs around puberty, when testicular production of Smad3 is declining and when onset of Smad2-utilization in response to activin occurs (Itman et al., 2009b, 2011a). Our initial approach to identify regulators of Smad2 and Smad3 expression established that this may be driven by testosterone, which selectively upregulates Smad2, but not Smad3 in a mouse Sertoli cell line (Itman et al., 2011a). Regulators of TGFb signalling, which may either promote or down-regulate Smad activity, also exhibit dynamic expression patterns in the developing and adult mouse testis (Itman and Loveland, 2008; Itman et al., 2009a, 2011b). Of particular relevance to Smad2/Smad3 activity in immature Sertoli cells is the regulated expression of Zfyve9 (mouse homologue of human SARA, Smad Anchor for Receptor Activation) and Hgs, two molecules which interact with activin receptors to promote Smad phosphorylation. SARA is necessary for maximal activation of Smad2, but not Smad3, (Runyan et al., 2005) whereas HGS can inhibit Smad3-mediated signalling when co-expressed with SARA (Goto et al., 2001). Hgs transcripts are absent from immature Sertoli cells, including when Zfyve9 transcripts are observed, but both are present in Sertoli cells of the adult. This suggests that is one mechanism by which immature Sertoli cells selectively transduce activin A signals via Smad3 is through the regulated production of Smad activity modulators (Itman et al., 2009a). Regulated synthesis of Smads and signalling regulators in gonocytes and spermatogonia has also been reported, providing some insight into how TGFb superfamily ligands may inuence establishment of the SSC population. As described above, gonocyte migration to the basement membrane, resumption of mitosis and transition into spermatogonia occur in an environment of high activin levels (Barakat et al., 2008). Whereas activin increases gonocyte numbers and impairs their differentiation (Meehan et al., 2000), upon differentiation into spermatogonia, activin promotes germ cell proliferation (Mather et al., 1990). These opposing effects of activin, at the particular

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time when the SSC population is being established and the rst differentiating spermatogonia appear, necessitate tight regulation of activin responsiveness and signalling outcomes. Differential production of positive and negative regulators of activin/TGFb superfamily signalling may determine the outcome of gonocyte/SSC responsiveness to activin at this time. In support of this hypothesis, the inhibitory Smad6 protein was detected in only a subset of spermatogonia of the 5 dpp mouse testis (Itman and Loveland, 2008), with a shift from the expression of signalinhibitory factors (SMURF2, MAN1) to signal-promoting factors (Zfyve9) observed as gonocytes differentiate into spermatogonia. While up-regulation of the transcript encoding follistatin is restricted to the murine female gonad in fetal life (Menke and Page, 2002), measurable levels of protein are present in the newborn testis (Barakat et al., 2008). The concentration of testicular follistatin rises signicantly shortly after birth, as does activin, but reaches its highest level at day 8, after testicular activin A levels have signicantly declined. Follistatin has been identied in spermatogonia as they rst form from gonocytes at the onset of spermatogenesis (Meehan et al., 2000). Understanding the role of this extracellular protein in ne-tuning the outcomes of integrated TGFb superfamily signals will be challenging due to the presence of both circulating and cell-associated forms. Local production appears not to be essential for grossly normal testis development, as fetal testes from Fst-/- mice, when transplanted to the ear of an immunocompromised host developed as normally and exhibited full spermatogenesis (Lin et al., 2006). Ectopic expression of follistatin and the related FST-like 3 (FSTL3), resulted in reduced testicular size and spermatogenic impairment in mice (Guo et al., 1998; Xia et al., 2004), perhaps related to increase in Leydig cell numbers reported in these models. However, the lack of a consistent correlation between Leydig cell numbers and testosterone measurements in juvenile FSTL3 transgenic over-expressor mice indicates that some other aspect of Leydig cell function besides steroidogenesis is affected when activin signalling is chronically impeded. Much remains to be learned about how these and other somatic cells respond to activin. 2.4. Frontier studies Studies of the developing testis and of spermatogenesis provide a wealth of opportunity with which to interrogate the fundamental principles that underpin cellular transitions. Spermatogenic cells range from a state of pluripotency to those that are terminally differentiated. These differentiate within a niche provided rst by several expanding immature somatic cell populations in fetal and juvenile life. In contrast, adult spermatogenesis relies on a stable, essentially terminally differentiated, yet highly functionally dynamic, set of somatic cells. Understanding the difference between rst wave and adult spermatogenesis is of great importance for learning how and when the germline may be sensitive to injury or disruption. This review has considered recent discoveries that highlight new ways of thinking about activin and inhibin functions, and it remains to be considered what additional insights can be gleaned from future work in this eld, particularly as tools for understanding cellular signalling interactions improve. An emerging depth of understanding of the mechanism by which signalling pathways are integrated will most certainly be relevant to spermatogenesis and testis development. The recent identication of interactions between nodal and activin in embryonic cell fate determination (Lee et al., 2007) provides a relevant example that demonstrates how Smad2 function can be governed by competing ligand actions. This information is highly relevant to the discovery that Smad utilization is dramatically different between proliferating, immature Sertoli cells and those which are post-mitotic (Itman et al., 2009b). There is currently limited infor-

mation about the importance of non-Smad-mediated signalling by activin in the testis. Whereas p38 activation by TGFb regulates cyclic formation and disassembly of the Sertoli cell blood-testis barrier (Lui et al., 2003) and GDNF acts via the MAPK pathway to maintain spermatogonial stem cell capacity (Meng et al., 2000), there have been no reports to date of activation of non-Smad intermediaries in response to activin in testicular cells. Collectively, these observations highlight the wealth of insights into activin and TGFb superfamily signalling biology that can be gained from examining events during testis development. This requires attention to a multiplicity of interdependent cellular differentiation events, now known to be balanced by the levels of activin signalling. These appear to be nely tuned to enable the potential for full fertility to be realized during development, and perhaps also through adulthood. Acknowledgements Funding was provided by the NHMRC (ID545917 and ID 545916 to KL, GNT1011192 to CI) and Monash University (postgraduate scholarships to BB and SM). References
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