Hum. Reprod. Advance Access published online on December 11, 2007
Human Reproduction, doi:10.1093/humrep/dem343
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Activin receptor subunits in normal and dysfunctional adult human testis
1 Monash Institute of Medical Research, 27-31 Wright Street Clayton, Victoria 3168, Australia 2 Prince Henry’s Institute, Clayton, Victoria, Australia 3 Department of Growth and Reproduction, University of Copenhagen (Rigshospitalet), Copenhagen, Denmark 4 Department of Obstetrics and Gynecology, Monash University, Clayton, Victoria, Australia 5 The Australian Research Council Centre of Excellence in Biotechnology and Development, Australia
6 Correspondence address. Tel: +6-13-9594-7418; Fax: +6-13-9594-7111; E-mail: kate.loveland{at}med.monash.edu.au
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Abstract |
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BACKGROUND: The cellular sites of activin action and its regulation in the normal and dysfunctional adult human testis are unknown.
METHODS: Activin type I (ALK2 and ALK4) and type II (ActRIIA and ActRIIB) receptors were detected using immunohistochemistry on Bouins fixed sections of normal, carcinoma in situ (CIS), seminoma, non-seminoma and gonadotropin-deprived human testis. ActRIIA mRNA was localized by in situ hybridization.
RESULTS: ALK2, ALK4 and ActRIIB proteins were observed in Sertoli cells, spermatogonia and some spermatocytes within normal and gonadotropin-suppressed adult human testis; all three receptor subunits were also detected in CIS, seminoma and non-seminoma cells. ActRIIA immunoreactivity was faint to absent in the normal testis and in CIS and non-seminoma cells, whereas some seminoma cells displayed a strong signal. Also in contrast to the normal testis, a majority of spermatogonia and Sertoli cells in gonadotropin-deprived samples exhibited a strong ActRIIA immunohistochemical and in situ hybridization signal.
CONCLUSIONS: Spermatogonia and Sertoli cells appear as the primary targets of activin action in the adult human testis. Changes in testicular function associated with altered hormone levels may enhance ActRIIA mRNA and protein synthesis, thus modifying signalling by activin or other TGFβ ligands within specific cells of the seminiferous epithelium.
Key words: activin receptor/human testis/spermatogonia/seminoma/gonadotropins
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Introduction |
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Members of the transforming growth factor-β (TGFβ) superfamily of growth factors exert a broad range of effects on the differentiation, proliferation and function of numerous cell types including those in the mammalian testis (Loveland and Robertson, 2005
). Among these, activin in particular has been implicated in governing both germ and Sertoli cell function in developing and adult testes (Mather et al., 1990; Boitani et al., 1995; Meehan et al., 2000; Fragale et al., 2001; Itman et al., 2006).
Central to activin signalling is its interaction with two types of transmembrane receptors, type I and type II, each with intrinsic serine/threonine kinase activity. Activin can utilize either one of two type I receptors, ActRIA (ALK2) or ActRIB (ALK4), or two type II receptors, ActRIIA or ActRIIB (Chang et al., 2002; Shi and Massague, 2003). Synthesis of the ActRIIA mRNA is known to be both regulated during, and required for, normal development of the post-natal rodent testis (de Winter et al., 1992; Boitani et al., 1995; Fragale et al., 2001; Wreford et al., 2001). A synergy between activin and follicle stimulating hormone (FSH) actions was identified through in vitro studies of 9 dpp rat testes (Boitani et al., 1995), and this was linked to a transient elevation in ActRIIA mRNA levels between 7 and 11 dpp (Fragale et al., 2001). Mice lacking the ActRIIA gene (Acvr2a) have small testes in adulthood and display a delayed fertility onset (Wreford et al., 2001), potentially due to reduced Sertoli cell proliferation, and decreased germ cell numbers arising from compromised support of germ cells by Sertoli cells.
Neither the function of activin in the human testis nor its cellular sites of action in the adult testis have been defined. However, in the human fetal testis, ActRIIA protein has been localized to interstitial cells and gonocytes, whereas ActRIIB immunostaining has been reported in interstitial cells, gonocytes and Sertoli cells (Anderson et al., 2002). In addition, mRNAs encoding activin receptors and activin/inhibin subunits were detected in testicular carcinomas using RNAase protection assays (van Schaik et al., 1997). The presence of type II activin receptors in human gonocytes (Anderson et al., 2002; our unpublished data) indicates the relevance of activin to germ cell development at a stage when these cells may be sensitive to perturbations that contribute to the development of testicular cancer, a disease understood to arise from a failure of the developmental programming that drives normal germ cell maturation prior to puberty.
Testicular cancer accounts for 1% of neoplasms in men and is the most common malignancy in males aged between 15–35 years (Bergstrom et al., 1996). Testicular germ cell tumors (TGCTs) arise from a common precursor cell known as carcinoma in situ (CIS), first described by Skakkebaek (1972) as atypical spermatogonia in testicular biopsies of patients who subsequently developed testicular cancer. Morphological similarities and comparison of protein expression in CIS and human fetal germ cells indicate CIS originates from gonocytes in early fetal life (Almstrup et al., 2004). The resemblance of CIS cells to gonocytes is supported by shared expression of developmental proteins, including placental-like alkaline phosphatase (PLAP), OCT-3/4, Nanog and the stem cell factor receptor, C-KIT (Jorgensen et al., 1995; Rajpert-De Meyts, 2006). These progress to an overt TGCT after puberty presumably in response to hormonal changes at this time. Histologically and clinically, TGCTs are subdivided into seminomas and non-seminomas (Ulbright, 1993). Seminomas are composed of cells similar to CIS cells, with characteristic lymphocytic infiltration in the supporting stroma, and these are, albeit infrequently, capable of transforming to a non-seminomatous phenotype. Non-seminomas mimic early embryonic development and contain stem cells termed embryonal carcinoma (EC) cells that can differentiate into a broad spectrum of somatic tissues (teratomas) and extra-embryonic derivatives (yolk-sac tumors and choriocarcinomas) (Rajpert-De Meyts, 2006).
Although the precise mechanisms that underpin the transformation of a fetal gonocyte to a CIS cell and its subsequent progression to a TGCT remain poorly characterized, the comparison of gene expression profiles of CIS and TGCTs with fetal germ cells has aided our understanding of the biology of testicular neoplasms. Because studies from several laboratories have implicated activin in the regulation of Sertoli and germ cell growth and function, we hypothesized that activin signaling features in normal human germ cell development and changes when spermatogenesis is disrupted. To test this, we set out to define the cellular sites of activin action by localizing activin receptor subunits within sections of normal and neoplastic human testes. Moreover, because the onset of the transformation of CIS cells into TGCTs is linked to puberty and understood to be driven by associated hormonal changes (Rajpert-De Meyts, 2006), we also examined samples from normal men treated with testosterone (T) and a depot progestin that suppresses both gonadotropin and intra-testicular T levels to <2% baseline and thereby suppresses spermatogenesis (McLachlan et al., 2002). Our findings demonstrate that selected somatic and germ cells are targets for activin action, and identify the potential for hormonal changes to influence activin signaling.
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Samples
Histological analysis was performed on Bouins fixed, paraffin-embedded sections of normal adult human testis, testicular carcinomas and gonadotropin-deprived testes. Testicular carcinoma samples consisting of CIS, seminomas and non-seminomas were obtained from pathology departments in the greater Copenhagen area. All tumor samples were assessed by experienced pathologists and routinely stained with PLAP to identify CIS cells. Permission for their collection and use in immunohistochemical studies was obtained from the Regional Committee for Medical Research Ethics in Denmark.
All other sections were obtained from biopsy samples collected from healthy adult volunteers. Samples of gonadotropin-deprived testis were obtained following treatment with either testosterone (T) or T plus a depot progestin (T+DMPA) for either 2 or 12 weeks (samples from McLachlan et al., 2002). The study was approved by the Human Research and Ethics Committee of the Monash Medical Center and informed consent was obtained from each subject.
Immunohistochemical analyses
Antibodies used for immunohistochemistry recognize the extracellular domain of activin receptors ALK2, ALK4, ActRIIA and ActRIIB (R&D Systems, Minneapolis, USA). These individual antibody samples were each used previously on western blots of human term placenta tissues to verify their selective recognition of proteins bands ranging from 60 to 70 kDa (Manuelpillai et al., 2001; Schneider-Kolsky et al., 2002); each antibody recognized a single band corresponding to the target antigen size. Human term placenta was used in the course of these experiments as a positive control for these immunohistochemistry experiments, yielding reaction patterns identical to those previously reported (Manuelpillai et al., 2001; Schneider-Kolsky et al., 2002). To control for variations in staining intensity between different immunohistochemistry runs, adult human testis samples were included in each run; these consistently demonstrated identical results.
Immunohistochemistry was performed as previously described on dewaxed sections that were blocked with 5% normal rabbit serum diluted in Tris-buffered saline (TBS) with 0.1% BSA before the addition of primary antibody (Loveland et al., 1999). Briefly, antigen retrieval was performed in 50 mM glycine (pH 3.5; >90°C maintained for 8 min), and the primary antibodies were applied at 1.0 µg/ml diluted in TBS with 0.1% BSA for overnight incubation at room temperature. All washes between incubations were performed at room temperature with TBS. Primary antibody binding was detected using a biotinylated anti-rabbit antibody (DAKO; 1:500 dilution in TBS/0.1% BSA, 1 h) and then the Vectastain Elite ABC kit according to the manufacturer’s instructions (Vector Laboratories, Burlingame, CA, USA). Antibody binding was detected as a brown precipitate following development with 3,3'-diaminobenzidine tetrahydrochloride against a Harris Hematoxylin counterstain. Germ cell types were identified primarily on the basis of their nuclear morphology and position within the developing gonad. Immunohistochemistry experiments were performed at least twice on at least four patients from each tested group, with identical results between experiments for each sample. Owing to differences in ActRIIA immunoreactivity seen between different groups compared with the other receptor subunits, a higher number of CIS (9) and seminoma (10) samples were analyzed for expression of ActRIIA protein. Controls for non-specific binding of the secondary antibody were performed in all experiments by omission of primary antibody, and these consistently yielded no signal within the seminiferous epithelium or in the interstitial space. The interstitial staining observed in the presence of the primary antibody was considered to be non-specific as it was not associated with or localized within a particular cell type. Thus staining of interstitial cells was not recorded during analysis of these results.
Histological analysis
Histological analysis was performed to obtain a semi-quantitative assessment of germ cell and Sertoli cell immunohistochemical staining and to record the approximate intracellular localization of all four activin receptors within the normal human testis. Owing to striking differences in immunostaining intensity of ActRIIA observed between 2 and 12 week gonadotropin-deprived testis, analysis was also performed on these samples stained with ActRIIA.
Histological analysis was conducted using a x100 objective lens on a BX-50 microscope (Olympus Corp., Tokyo, Japan) attached to a motorized stage. The image was captured by a Pulinix TMC-6 video camera coupled to a Pentium personal computer using a Screen machine II fast multimedia video adaptor (FAST, Hamburg, Germany). A software package, DH CASTGRID version 1.6 (Olympus Corp., Munich, Germany), was used to superimpose a set of unbiased counting frames on the video image. A set of four counting frames were generated per field (area of each frame 460.5 µm2). Fields counted were selected in an unbiased manner by a systematic uniform random sampling scheme with the use of a motorized stage (Multicontrol 2000, ITK, Lahnau, Germany) and 30 fields were counted per stained section. Cells were included if they fell inside the counting frame and within the acceptance boundaries. For each field, the type of cells stained, the relative intensity of stain [high (+++), medium (++), low (+) and negative (–)], localization of staining (plasma membrane-associated, cytoplasmic and nuclear) and surrounding non-stained cell types were noted. Sertoli and germ cells were identified based on nuclear morphology (Clermont, 1963, 1966a,b). A minimum of 400 cells were scored within each section. Germ cells were grouped as follows using the criteria provided in McLachlan et al. (2002): type A dark spermatogonia, type A pale spermatogonia, type B spermatogonia, pre-leptotene/leptotene spermatocytes, pachytene spermatocytes, steps 1–4 round spermatids and steps 5–8 elongating and elongated spermatids.
In situ hybridization
In situ hybridization with digoxigenenin-labeled cRNA probes (DIG-cRNAs) was used to localize ActRIIA mRNA in dewaxed sections of normal, neoplastic and gonadotropin-deprived human testes, as previously described (Meehan et al., 2000). Primers with specificity for both human (Acc No: NM_001616
[GenBank]
) and mouse (Acc No: NM_007396
[GenBank]
) were designed to amplify the 3'UTR of mouse ActRIIA, a region of high inter-species homology (95% identical between mouse and human; forward primer 5'-GCCCTCTCCAAATCAAGGAT-3', reverse primer 5'-CACAAGCTGTGTCATGTTGG-3'). Selective recognition of 3 and 6 kb mRNAs by this antisense ActRIIA probe was verified by northern blot analysis of mouse tissues (data not shown), in accord with published results (Puglisi et al., 2004).
Hybridization and washing was performed using 100 ng/µl DIG-cRNA with sections incubated overnight at 50°C. Both antisense and sense (negative control) cRNAs were applied under identical conditions on each sample in every experiment.
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Activin receptor proteins within normal adult human testis
Within sections of phenotypically normal adult human testis, the ALK2, ALK4 and ActRIIB receptor proteins were consistently detected as a strong brown staining associated with the membrane and cytoplasm of spermatogonia and Sertoli cells (Fig. 1, Table I). These three receptors were occasionally detected in pre-leptotene/leptotene spermatocytes, but never in more mature germ cell types. Intriguingly, a small subset of spermatogonia and spermatocytes also displayed nuclear staining, in addition to, or independent of cytoplasmic and membrane staining (Fig. 1). In contrast, ActRIIA protein immunostaining was low to absent within the seminiferous epithelium of the normal testis, and only rarely was a faint signal detected in spermatogonia and Sertoli cells. These results identify Sertoli cells and spermatogonia as the cell types containing the three activin receptor subunits, ALK2, ALK4 and ActRIIB, in the normal adult human testis.
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Activin receptors within gonadotropin-deprived testes
To examine the effects of gonadotropin and intra-testicular T levels on activin receptor expression, testis sections were employed from a previous study that investigated the suppression of spermatogenesis by androgen-based contraceptive treatments (McLachlan et al., 2002
). In men treated with either T or with T+DMPA for 2 or 12 weeks, ALK2, ALK4 (not shown) and ActRIIB proteins were consistently detected in spermatogonia, spermatocytes and Sertoli cells, with distinct nuclear localization in some spermatogonia and spermatocytes (Fig. 2, Table I).
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In striking contrast to samples from normal and untreated men, ActRIIA protein was readily detected in all samples from men subjected to 2 weeks of gonadotropin suppression using T or T+DMPA (five samples for each group). Staining was present within spermatogonia, spermatocytes and Sertoli cells. All four activin receptors appeared to be predominantly nuclear in spermatocytes in this group. ActRIIA protein was also detected in the 12 week gonadotropin-suppressed testes (Fig. 2, Table I), but the signal intensity was notably less intense than at week 2 and was more restricted, being limited to a few Sertoli and germ cells. This differential staining of ActRIIA across gonadotropin-deprived groups was documented using histological analysis.
Activin receptor proteins within testicular neoplasms
Identification of activin target sites was performed in samples containing testicular neoplasms, through examination of CIS, seminomas and non-seminomas.
The pre-malignant CIS cells displayed strong membrane signals corresponding to ALK2, ALK4 and ActRIIB (Fig. 3, Table I). Intense nuclear signals were also evident in some CIS cells. Similar to normal testes, ActRIIA protein was undetectable or visualized only as a faint signal of immunoreactivity within the CIS tubules (Fig. 3, Table I). In seminomas, ALK2, ALK4 (not shown) and ActRIIB were present in association with the membrane and nucleus of seminoma cells (Fig. 3). The ActRIIA protein signal was more intense in all seminoma samples when compared with normal and CIS specimens, with immunoreactivity detected on the membrane and in the nucleus of a subset of seminoma cells. Within the morphologically heterogeneous non-seminoma samples, ALK2, ALK4, ActRIIB and ActRIIA proteins were exhibited by a variety of cell types. As the immunostaining pattern of ALK4 within gonadotropin-deprived samples and testicular neoplasms was indistinguishable from those of ALK2 and ActRIIB, as shown in Figs 2 and 3, ALK4 staining is not shown.
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Cellular localization of ActRIIA mRNA
In situ hybridization was utilized to validate the cellular sites of receptor production identified using the antibody to this receptor. This approach also allowed us to determine if ActRIIA mRNA and protein were coordinately regulated.
In situ hybridization analyses of normal human testis revealed no discernable staining, in accordance with the immunohistochemical results (Fig. 2). An ActRIIA mRNA signal was strongly evident in the nucleus and cytoplasm of spermatogonia, the cytoplasm of spermatocytes and at a lesser intensity within Sertoli and peritubular myoid cells of the T and T+DMPA treated testes at both 2 and 12 week time points (Fig. 2).
In contrast to protein localization data, in situ hybridization analysis of CIS samples revealed ActRIIA mRNA transcripts within the nucleus of CIS cells, in the peritubular myoid cells surrounding CIS tubules, and in some immune cells. Interestingly, in a CIS specimen that contained mostly seminiferous tubules with preserved spermatogenesis and had only a few CIS tubules, there was no ActRIIA mRNA signal in either normal or CIS areas (Fig. 4). In contrast to this, a CIS specimen that had relatively equivalent abundance of both normal and CIS tubules, exhibited ActRIIA mRNA in CIS cells, spermatogonia, spermatocytes and Sertoli cells in both normal and CIS tubules (Fig. 4). ActRIIA transcripts were also detected in seminoma cells, in some cells of the inflammatory infiltrate of seminomas and within distinct cell populations in the heterogeneous non-seminomas (Fig. 3).
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Discussion |
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This is the first study to define the cellular sites of activin action within the seminiferous epithelium of normal human testis and its dysfunctional and neoplastic counterparts. Common patterns of receptor expression among the activin receptor subunits identify spermatogonia and Sertoli cells as the primary sites for activin action in all sample groups, and importantly, we provide evidence for hormonal regulation of activin signaling. Our data are in accord with existing knowledge of activin signaling in the rodent testis, where activin influences mitotic germ cells (Mather et al., 1990
; Richards et al., 1999; Meehan et al., 2000), Sertoli cell function (Matzuk et al., 1995; Fragale et al., 2001; Wreford et al., 2001) and the timing of fertility onset (Brown et al., 2000; Wreford et al., 2001). However, the majority of published data relate to developing testes, and our knowledge of a specific role for activin in the adult testis is limited, particularly with regard to its impact within the seminiferous epithelium. The absence of detectable activin receptor protein in haploid germ cells of the adult human testis implies that activin signaling does not directly govern post-meiotic germ cell development, but it may do so indirectly by influencing Sertoli cell function. The intriguing differential expression of ActRIIA mRNA and protein across this variety of adult testis samples highlights the potential for dynamic TGFβ superfamily signaling to influence testicular physiology. Detection of ActRIIA protein only in samples displaying neoplastic transformation of germ cells and following hormonal manipulations highlights the potential of activin signaling to either effect or to be influenced by circumstances leading to spermatogenic disruption. Notably, ActRIIA protein was detected only in cell types bearing the other activin receptor subunits. This raises the prospect that aberrant activin signaling in either spermatogonia or in Sertoli cells may contribute to the progression of testicular dysgenesis.
The administration of T or T+DMPA to suppress gonadotropins in healthy adults was reported to cause significant impairment of type B spermatogonial and early spermatocyte maturation and profound inhibition of sperm release at both 2 and 12 weeks (McLachlan et al., 2002). Thus, the 2 week time point represents an interval when the testis has already undergone a significant loss of its capacity to support spermatogenesis. The marked elevation in detectable ActRIIA protein signal in both sets of 2 week samples (T and T+DMPA) indicates that it is changes in intra-testicular T levels in particular that alter ActRIIA synthesis, with the simplest interpretation being that intratesticular T normally suppresses receptor production.
The progression of CIS cells to form invasive seminomas is commonly associated with puberty, as seen by a sharp rise in the age-specific incidence of germ cell tumors after puberty (Rajpert-De Meyts, 2006). Late puberty has also been identified as a protective factor for testicular cancer (Weir et al., 1998). The marked change in testicular hormone production is a likely trigger for this malignant transformation. Because T may profoundly influence progression of CIS cells into seminoma at the onset of puberty (Rajpert-De Meyts and Skakkebæk, 2000; Rajpert-De Meyts and Skakkebaek, 1993), we speculate that ActRIIA synthesis in seminoma under its influence is central to this transformation (Fig. 5).
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Indeed mRNAs encoding activin receptors and activin/inhibin subunits have been previously demonstrated in TGCTs using RNAase protection assays (van Schaik et al., 1997
). Here, the combined expression of both activin ligand and receptor subunits in tumor germ cells led to the speculation that activin may function as an autocrine factor in tumor progression, enabling independence from Sertoli cell-derived factors. Moreover, immunohistochemical experiments have illustrated seminoma and EC cells as cellular sites of inhibin-
, activin βA and activin βB subunit synthesis, highlighting further the potential for activin to regulate germ cell neoplasms (Cobellis et al., 2001).
In some CIS and seminoma cells, ActRIIA mRNA was observed in the absence of protein. The differential sensitivity of the immunohistochemistry and in situ hybridization detection techniques may underlie our detection of an mRNA signal in the absence of detectable protein, if the protein epitope is in low abundance in spermatogonia and CIS cells within CIS testes. Alternatively, this observation may indicate that ActRIIA mRNA is regulated by a mechanism different to that which controls its translation. We have recently documented this phenomenon for the C-kit tyrosine kinase receptor in mouse spermatogonial stem cells (Prabhu et al., 2006). Thus, we propose a model for some cases of testicular cancer in which ActRIIA mRNA production is linked to the transformation of a fetal gonocyte to a CIS cell, whereas synthesis of ActRIIA protein is controlled by hormonal changes at puberty (Fig. 5). The detection of ActRIIA mRNA in testes of gonadotropin-suppressed men with an apparent transient elevation in ActRIIA protein signal supports this concept.
Detection of activin receptors in association with the nucleus of some spermatogonia and spermatocytes raises the possibility of receptor shuttling between the cell surface and the nucleus. This phenomenon has been reported for a number of growth factors, cytokines and receptors, including receptors for epidermal growth factor, interferon gamma and fibroblast growth factor (Johnson et al., 2004). Furthermore, the activin βA protein itself encodes a nuclear localization signal (Blauer et al., 1999). Alternative pathways and end-points for receptor internalization may regulate cell surface receptor levels or modulate transcription of selected genes, mechanisms that have been recently identified for TGFβ signaling (Di Guglielmo et al., 2003; Zhang et al., 2005). These findings indicate additional pathways by which activin signaling may influence cellular function within the seminiferous epithelium.
Since the molecular events that underpin progression of CIS to testicular germ cell tumors are to date largely unknown, our identification of the potential for activin and TGFβ superfamily ligand to be affected by hormonal changes in the adult seminiferous epithelium provides a clear focus for future research. The investigation of the potential for ActRIIA mRNA and protein to serve as prognostic markers for testicular germ cell tumor progression is now warranted by the findings of this study, which demonstrates that activin signaling through receptors in spermatogonia and Sertoli cells may result from the altered production of an activin receptor subunit.
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This work was supported by funding from the National Health and Medical Research Council of Australia (Grants #334011 and #384108 to K.L. and #169020 to R.M.), the Danish Cancer Society (Grant # DP05113 to E.R.M.), the Australian Research Council (#348239 to K.L.) and Monash University (Postgraduate Scholarship to V.D.).
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Submitted on May 11, 2007; resubmitted on August 31, 2007; accepted on October 1, 2007.
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