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Reproduction (2007) 134 757-765
DOI: 10.1530/REP-07-0161
Copyright © 2007 Society for Reproduction and Fertility
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RESEARCH

Stem cell factor affects fate determination of human gonocytes in vitro

Jiongjiong Tu1,2, Liqing Fan1,2, Ke Tao2, Wenbing Zhu2, Jianjun Li3 and Guangxiu Lu1,2

1 National Center of Human Stem Cell Research and Engineering, 2 Institute of Human Reproduction and Stem Cell Engineering, Central South University, Changsha, Hunan 410078, China and 3 Changsha Maternity and Child Health Hospital, Changsha, Hunan 410007, China

Correspondence should be addressed to G Lu; Email: lugxdirector{at}yahoo.com.cn


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
The stem cell factor (SCF), binding its tyrosine kinase receptor c-Kit, has been shown to play essential roles in the proliferation, differentiation, and survival of germline cells. However, few reports are available about the effect of SCF on the development of human gonocytes within the fetal testis. The objective of this study was to investigate whether SCF affects the biological behaviors of human gonocytes before or after they enter the mitotic arrest stage. Employing an organ culture system, we observed that addition of exogenous SCF could influence the morphology of human gonocytes in vitro. Moreover, SCF was able to trigger the colony formation of round gonocytes, which were characterized positive for alkaline phosphatase activity, Oct-4, SSEA-4, and c-Kit as well. We found that SCF exerted actions in a dose- and age-dependent manner, although the stimulatory effect lasted no more than 14 days. We also showed that SCF played a role in suppressing the apoptosis of human gonocytes. Blocking of SCF signaling with either phosphatidylinositol 3-kinase or mitogen-activated protein kinase inhibitor resulted in similar apoptotic features as well as the SCF-withdrawal cultures. Taken together, we report that SCF acts as a potent regulator in the fate determination of human gonocytes. Our studies should form the basis for in vitro studies and facilitate investigation of the molecular mechanisms underlying this unique stage.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
Male germline development is a highly ordered and complex process. In early gametogenesis, human gonocytes arise when their precursors, primordial germ cells (PGCs), migrate into the embryonic genital ridge and become enclosed in the primitive seminiferous cords by 7 weeks of gestation (Wartenberg 1981). The gonocytes gradually reduce their mitotic cycles and enter the mitotic arrest stage by 16–18 weeks of gestation (Goto et al. 1999). Thereafter, a large number of gonocytes are committed to undergo apoptosis and the survivors differentiate to spermatogonial stem cells, which will initiate the wave of spermatogenesis in puberty (Helal et al. 2002). However, the precise mechanisms underlying these complex events are poorly understood.

In mouse embryos, stem cell factor (SCF) is expressed in somatic tissues in a gradient pattern along the pathway of PGC migration, with the highest levels in the genital ridges (Keshet et al. 1991, Matsui et al. 1991), whereas c-Kit is expressed on PGCs through their migratory phase and until shortly after they cease proliferation in the embryonic gonads (Manova & Bachvarova 1991). Preliminary studies have reported that mutation of either SCF (Steel locus) or its receptor c-Kit (W locus) results in reduced numbers of PGCs (McCoshen & McCallion 1975, Brannan et al. 1992, Buehr et al. 1993), and the interactions of SCF/c-Kit increase the proliferation and suppress apoptosis of PGCs and differentiated spermatogonia cells as well (Dolci et al. 1991, 2001, Godin et al. 1991, Pesce et al. 1993, Yan et al. 2000). Further studies have revealed that phosphatidylinositol 3-kinase (PI-3K) and MEK are involved in the intracellular signaling of SCF in male germline development (Dolci et al. 2001, De Miguel et al. 2002).

In humans, SCF has also been found expressed in the surrounding tissues of PGCs, and c-Kit is confined to PGCs as well (Sandlow et al. 1997, Hoyer et al. 2005). When PGCs migrate into the embryonic gonad and differentiate to gonocytes, the Sertoli cells within the primitive seminiferous cords could compensate for the secretion of SCF as well as the tissues along PGCs migration pathway (Strohmeyer et al. 1995). However, the expression of c-Kit is reduced with the developmental age consistent with the low mitotic activity of human gonocytes in vivo (Gaskell et al. 2004, Pauls et al. 2006). Based on this evidence, we proposed that SCF signaling might play an undefined role in the development of human gonocytes within the fetal testis.

In the present paper, we developed an organ culture system to study human gonocytes in vitro. We tested different doses of SCF on the morphological change and proliferation activity of human gonocytes derived from 12- to 14-week, 16- to 18-week, and 20- to 22-week fetal testes. The SCF-induced gonocyte colonies were characterized by detection of alkaline phosphatase (AP) activity and pluripotent markers. In addition, PI-3K and MEK inhibitors were used to examine the intracellular signaling of SCF in human gonocytes.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 Acknowledgements
 References
 
SCF affects the morphology of human gonocytes in vitro
The dissociated seminiferous cords, which were mainly composed of gonocytes and primitive Sertoli cells, were cultured in gelatin-coated four-well tissue culture plates with or without SCF. In this organ culture system, different doses of SCF (10 ng/ml, 100 ng/ml and 500 ng/ml) were tested on human gonocytes derived from 12- to 14-week, 16- to 18-week, and 20- to 22-week fetal testes. After 2 days of culture, fibroblast-like Sertoli cells began to firmly attach to the bottom of the plates, while the gonocytes were exposed and anchored on the somatic monolayer. In the SCF-absent controls, most gonocytes developed cytoplasmic extensions and converted to spindly shape (Fig. 1AGo). In contrast, many gonocytes remained in round shape with the addition of SCF (Fig. 1BGo). In contrast to somatic cells, both the spindly and round gonocytes were positive for AP activity (Fig. 1C and DGo), which can be easily scored. The statistical data showed that the index of round gonocytes was significantly higher in the SCF-treated cultures, whereas treatments with 10, 100, and 500 ng/ml SCF resulted in no significant differences. The result also showed a less profound effect of SCF on the late gonocytes than on the early gonocytes (Fig. 1EGo).


Figure 1
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  		Figure 1 Morphology of human gonocytes after 2 days of culture in vitro. (A and B) Representation of spindly gonocytes in the absence of SCF (A, arrows) and round gonocytes in the presence of SCF (B, arrowheads). (C and D) Representation of AP activity of spindly gonocytes (C, arrows) and round gonocytes (D, arrowheads). (E) Quantification of the round gonocytes with untreated controls versus stimulation with 10, 100, and 500 ng/ml SCF from 12–14 week, 16–18 week, and 20–22 week age groups. Means ± S.E.M. for three independent experiments are shown. Note that there was no significant difference within the treatments of 10, 100, and 500 ng/ml SCF and less profound effect of SCF on late gonocytes. ***P < 0.001 when compared with controls. Scale bar = 50 µm.

 
Identification of the gonocyte-derived colonies with the stimulation of SCF
After 3 days of stimulation with SCF, colonies could be induced from round gonocytes, and they displayed a broad size distribution of > 2–4 cells, > 4–8 cells, > 8–16 cells, and > 16 cells by AP staining (Fig. 2Go). In contrast, most spindly gonocytes remained in single or pair pattern and few colonies were observed, suggesting that the round gonocytes retain stronger clonogenic capacity than the spindly ones. The phenotypic characteristics of these gonocyte-derived colonies were examined by immunofluorescence staining. As shown in Fig. 3Go, the gonocyte colonies were stained positive for Oct-4, SSEA-4, and c-Kit, suggesting that these colonies share similar pluripotent markers as well as embryonic germ cells (EGCs) that were cultured from PGCs (Shamblott et al. 1998, Turnpenny et al. 2003, Liu et al. 2004, Park et al. 2004, Pan et al. 2005). The result also showed that the cultured gonocyte colonies were only dotted stained with c-Kit, suggesting that the c-Kit might be down-regulated in the present culture system. Nevertheless, the results indicated that these gonocyte colonies have an undifferentiated phenotype.


Figure 2
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  		Figure 2 AP staining of the gonocyte-derived colonies ranged from > 2 to 4 cells (A), > 4 to 8 cells (B), > 8 to 16 cells (C), and > 16 cells (D) after 3 days of stimulation with SCF. Scale bar = 20 µm.

 

Figure 3
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  		Figure 3 Phenotype of the gonocyte-derived colonies induced by SCF. Immunofluorescence staining showed that Oct-4 and SSEA-4 were strongly positive whereas c-Kit was dotted stained on the membrane of human gonocytes (arrowheads). Scale bar = 50 µm.

 
SCF promotes formation of gonocyte colonies in a dose- and age-dependent manner
To examine the dose effect of SCF on the proliferation activity of human gonocytes, the colony number and size was evaluated at days 7 and 14 in the SCF-treated cultures as well as the controls. As shown in Fig. 4Go, few colonies were observed in the SCF-absent controls. The colony number was increased at 10 ng/ml SCF but mainly peaked at > 2–4 cells. In contrast, there was a dramatic increase in the colony number and size at 100 or 500 ng/ml SCF in three tested age groups. The maximum dose of 500 ng/ml resulted in no further enhancement, suggesting that 100 ng/ml may be the saturated dose to enhance the proliferation of gonocytes. The statistical data showed significant differences in response to SCF among 12–14 week, 16–18 week, and 20–22 week cultures. The increased colonies, especially those with > 8–16 cells and > 16 cells, were obviously confined to the early cultures. In addition, the colonies were significantly reduced after day 14 when compared with those at day 7, suggesting that SCF could not support the consistent growth of gonocytes in the present culture system.


Figure 4
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  		Figure 4 Effect of SCF on the formation of gonocyte colonies in vitro. Quantification of the colonies with untreated controls versus stimulation with 10, 100, and 500 ng/ml SCF from 12–14 week, 16–18 week, and 20–22 week age groups. Cumulative colony numbers were evaluated from 100 randomly selected colonies from at least three independent experiments and scored as four size groups of > 2–4 cells, > 4–8 cells, > 8–16 cells, and > 16 cells. Colonies with 1–2 cells were considered to be quiescent and excluded from the figure. Means ± S.E.M. for three independent experiments are shown. **P < 0.01, ***P < 0.001 when compared with controls. ###P < 0.001 when compared with 10 ng/ml groups. There was no significant difference between 100 and 500 ng/ml groups. Note that the maximal effect of SCF was achieved on early gonocytes, and the colony number was reduced after 14 days of culture in vitro.

 
We further determined the proliferation activity of human gonocytes using BrdU incorporation tests and 100 ng/ml SCF was added to stimulate colony formation. Consistent with the colony distribution profile, the proliferation index was significantly higher in the SCF-treated cultures, and the early cultures showed a relatively higher incorporation index as well. In contrast to day 7 cultures, the proliferation index was dramatically reduced at day 14 in three tested age groups, suggesting that the proliferation activity of human gonocytes is impaired during the long-term cultures (Fig. 5Go).


Figure 5
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  		Figure 5 Quantification of BrdU-positive gonocytes at 7 and 14 days with the stimulation of 100 ng/ml SCF. Means ± S.E.M. for three independent experiments are shown. In the left panels, ***P < 0.001 when compared with controls. In the right panels, no significant differences were observed. Note that the higher incorporation index was confined to early gonocytes and the index of BrdU-positive gonocytes was reduced after 14 days culture in vitro.

 
SCF suppresses apoptosis of human gonocytes in vitro
We next examined the anti-apoptotic effect of SCF in vitro. The gonocyte colonies were induced with the addition of 100 ng/ml. At day 7, SCF was removed and the cells were cultured without SCF for 24 h. As shown in Fig. 6AGo, the colonies began to detach from the somatic monolayer and dissociated into single cells with typical apoptotic features such as shrunken membranes. These dissociated cells were confirmed to be apoptotic by TUNEL staining. In contrast, the gonocyte colonies showed intact and integrated appearances in the SCF-present controls. To block the effect of SCF, PI-3K inhibitor (LY294002) or MEK inhibitor (U0126) was added with SCF in the culture medium. After 24 h incubation, the gonocyte colonies showed similar apoptotic features as well as the SCF-withdrawal conditions (Fig. 6AGo), suggesting that these two kinases are both involved in downstream SCF signaling in human gonocytes. The statistical data showed that the apoptotic index was dramatically increased in the SCF-withdrawal and inhibitor-treated cultures in contrast to the SCF-present controls in three tested age groups (Fig. 6BGo).


Figure 6
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  		Figure 6 Effect of SCF on suppressing apoptosis of human gonocytes in vitro. (A) Representation of apoptotic features of gonocytes with the treatments of SCF (100 ng/ml), SCF-withdrawal, SCF plus LY294002, and SCF plus U0126 for 24 h. Note that the single cells dissociated from the colonies were stained with TUNEL-positive signals (arrowheads). (B) Quantification of TUNEL signals showed significant apoptosis in the SCF-withdrawal, SCF plus LY294002, and SCF plus U0126 cultures. Means ± S.E.M. for three independent experiments are shown. ***P < 0.001 when compared with SCF-present controls. Scale bar = 50 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 Acknowledgements
 References
 
The study of human gonocytes and their niches is of paramount importance to understand the mechanisms of self-renewal and differentiation of these unique germ stem cells. In the present study, we report that SCF maintained round gonocytes and promoted their colony formation in a dose- and age-dependent manner. SCF also acted as an anti-apoptotic factor in the cultures. To our knowledge, this is the first report describing the regulatory mechanism underlying the development of human gonocytes.

The relative number of spindly versus round gonocytes is difficult to assess in vivo, but in vitro studies found that there were two obvious distinct morphological categories with the influence of exogenous SCF. The minimum dose of SCF (10 ng/ml) seems to be sufficient to rescue the round gonocytes, whereas no such effect was observed in the controls, although Sertoli cells produce endogenous SCF in a paracrine manner (Strohmeyer et al. 1995). It is possible that these two subpopulations are analogous to the migrating and degenerating gonocytes similar to previous observations in rodents (Orth & Boehm 1990, Hasthorpe et al. 1999). Orwig et al. (2002) have reported that it is essential for gonocytes to change their appearance to acquire migration ability, whereas the rest would undergo apoptosis. In the present study, addition of exogenous SCF may prevent this transformation and keep gonocytes in a relatively stationary status, and the low expression of endogenous SCF by Sertoli cells may account for their morphological change in vitro. However, the effect of exogenous SCF was less profound on late gonocytes, as we have shown. A possible explanation could be that most late gonocytes have already been committed to migration fate prior to culture, and SCF merely works on the residual round ones.

Interestingly, the rescued round gonocytes, rather than the spindly ones, formed colonies with the stimulation of SCF. We found that these gonocyte-derived colonies expressed pluripotent markers similar to EGCs, which show a well-known pluripotency for the embryonic body formation and differentiation to other cell lineages (Turnpenny et al. 2006). Thus, to some degree, SCF contributes to the maintenance of the pluripotency of human gonocytes rather than triggering differentiation as reported in late spermatogonia cells (Feng et al. 2002), suggesting that SCF plays different roles in specific stages. In addition, there are two potential explanations for the reduced c-Kit on the cultured gonocytes. First, the organ culture system that mimics physiology niches may recapitulate the programmed down-regulation of c-Kit as reported in vivo (Gaskell et al. 2004, Pauls et al. 2006). Secondly, a negative feedback loop model may account for this in that Cbl proteins, a newly established family as components of the ubiquitin ligation machinery, could induce phosphorylation by SCF and, in turn, lead to the degradation of c-Kit receptors (Joazeiro et al. 1999, Zeng et al. 2005).

On the other hand, we found that SCF was able to activate the proliferation of the less active or quiescent gonocytes beyond 16–18 week of gestation (Goto et al. 1999), suggesting that SCF is a potent mitogen in mediating the mitotic cycles of human gonocytes. The fact that SCF functions in a dose-dependent manner indicates that the low expression of endogenous SCF in the local niches, as well as the effect on morphological determination of human gonocytes, may also be responsible for the occurrence of mitotic arrest in vivo. According to the immunohistochemistry analysis, the early fetal testis houses more c-Kit-positive gonocytes when compared with the late fetal testis (Gaskell et al. 2004, Pauls et al. 2006), which agrees well with their age-dependent growth in response to SCF. Thus, the higher expression of c-Kit could explain the maximum effect of SCF on the early cultures due to the activation of SCF signaling via SCF/c-Kit complexes. Consistent with this idea, studies in mice have shown that late gonocytes are almost c-Kit negative, which leads to the redundant effect of SCF on their colony formation (Yoshinaga et al. 1991, Vincent et al. 1998, Ohta et al. 2000, Kanatsu-Shinohara et al. 2003, 2004). Although SCF acts as a potent mitogen in the first few days, the overcrowded Sertoli cells associated with gonocytes may release negative factors and mask the effect of SCF during long-term cultures. It has been reported that Sertoli cells not only favor the survival of gonocytes, but also inhibit their colony formation in vitro (Griswold 1995, Hasthorpe et al. 1999). The strict control of germ cell numbers in the fetal testis may be a protection mechanism to prevent the formation of seminomas derived from the unlimited duplication of germ stem cells, which could be induced by the c-Kit mutations (Kemmer et al. 2004).

Our results also showed that SCF acts as a survival factor in the culture of human gonocytes. Instead of undergoing apoptosis, the round gonocytes can survive and proliferate with the addition of SCF, suggesting that SCF may have a role in mediating the programmed death of human gonocytes within the developing testis (Helal et al. 2002). It has been reported that SCF signaling up-regulates the expression of Bcl-2, a prosurvival protein, thereby preventing apoptosis in a number of cell types (Carson et al. 1994, Jin et al. 1998, Zeuner et al. 2003, Dhandapani et al. 2005, Kimura et al. 2005). In this study, we noted that PI-3 and MEK kinase pathway are also involved in the anti-apoptotic mechanism of SCF signaling, which shares a relatively conserved pathway as well as the previous observations in mouse germline cells (Dolci et al. 1991, 2001, Pesce et al. 1993, Yan et al. 2000).

In conclusion, in this paper, we report that SCF is a potent factor that affects the fate determination of human gonocytes, which provides a new insight into the regulatory mechanism underlying this specific stage. However, the present culture conditions are not optimal for long-term maintenance, and many improvements need to be addressed. Although we demonstrate here that SCF acts as both mitogen and survival factor for human gonocytes, much remains to be studied on the intrinsic regulation of SCF in the fetal testis, which would favor our further understanding of early gametogenesis in humans.


   Materials and Methods
Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
Acknowledgements
 References
 
Testis collection
Human fetal testes were obtained as a result of therapeutic termination of pregnancy using a protocol approved by the ethics committee at Central South University of China, and the patients gave their written informed consent according to the national guidelines (Medical Research Council 2001). The testes were collected from fetuses of 12–14 weeks (n = 9), 16–18 weeks (n = 7), and 20–22 weeks (n = 7) of gestation. All terminations were performed in the Gynecology and Obstetric Clinics, Changsha Maternity & Child Health Hospital. The developmental age of the fetuses was determined by the date of the last menstrual bleeding. Weight and length measurements evaluated at the autopsy were used to assure proper gestational development.

Organ culture
The fetal testes were decapsulated under a stereomicroscope in Hank’s balanced salt solution (HBSS, Gibco) and mechanically dissociated with fine scissors. The spliced tissues were exposed to 1 mg/ml collagenase (type IV, Sigma) and 1 mg/ml DNase I (Sigma) at 37 °C for 20 min, followed by two washes in Dulbecco’s PBS (DPBS, Gibco) at 40 g centrifugation for 5 min to remove redundant interstitial cells. The fragmented seminiferous cords were collected at 300 g centrifugation for 5 min and resuspended in culture medium. The resulting seminiferous cords were aliquoted into four-well tissue culture plates (Nunclon; Nunc, Roskide, Denmark) pre-coated with 0.1% gelatin. The culture medium was changed every other day in all experiments. Basic culture medium used in this study was Dulbecco’s modified Eagle’s medium (D-MEM) supplemented with 10% fetal bovine serum (FBS), 1 x non-essential amino acids (NEAA), 50 µM 2-mercaptoethanol, 2 mM glutamine, and 1 mM sodium pyruvate (all from Gibco). To investigate the dose effect of SCF, human recombinant stem cell factor (SCF; R & D Systems, Minneapolis, MN, USA) was added at 0–500 ng/ml in the culture medium. For blocking experiments, PI-3K and MEK inhibitors (Cell Signaling Technology, Beverly, MA, USA) were used at conventional concentration of 10 µM as reported (Dolci et al. 2001). All cultures were maintained at 37 °C in a humidified 5% CO2 atmosphere.

AP staining and colony counting
To examine endogenous AP activity, cultured cells were fixed with 4% paraformaldehyde and stained with Fast Red Substrate Pack (Zymed) by following the manufacturer’s instructions. According to the AP staining, the gonocyte-derived colonies were counted and grouped as > 2–4 cells, > 4–8 cells, > 8–16 cells, and > 16 cells. Single or paired gonocytes were excluded for being considered quiescent.

Immunofluorescence staining
Cultured cells were fixed in 4% paraformaldehyde in PBS at room temperature for 20 min and blocked with PBS containing 0.1% Triton X-100, 4% normal goat serum, and 1% BSA at room temperature for 1 h. The cells were incubated with primary antibodies at 4 °C overnight. The primary antibodies against human Oct-4 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and SSEA-4 (Chemicon International, Temecula, CA, USA) were diluted at 1:100, and antibody against human c-Kit (DAKO Corporation, Carpinteria, CA, USA) was diluted at 1:300. After washing thrice with PBS, fluorescein isothiocyanate (FITC)- or rhodamine-conjugated secondary antibody (both from Santa Cruz Biotechnology) diluted at 1:100 was added and incubated at 37 °C for 1 h. Negative controls incubated with PBS instead of primary antibodies revealed no positive staining.

5-Bromodeoxyuridine (BrdU) labeling
To determine cell proliferation, BrdU (Sigma) was added to the culture medium at a final concentration of 10 µM. After 24 h incubation, cultured cells were fixed in Bouin’s solution and kept in 70% alcohol. The cells were then denatured with HCl (2 M) for 10 min at room temperature followed by immediate wash with borate buffer (0.1 M) for another 20 min. Unspecific site binding was prevented by blocking with 5% BSA and 1% Triton X-100 in PBS for 1 h. BrdU mouse monoclonal antibody (1:500; Sigma) was used as primary antibody, and anti-mouse FITC (1:100; Santa Cruz Biotechnology) was used as secondary antibody. Nuclear staining was performed by adding 300 nM 4,6-diamidino-2-phenylindole (DAPI; Sigma) for 5 min at room temperature before visualization. The percentage of BrdU/DAPI-labeled nuclei within the colonies was assessed as the proliferation index of gonocytes.

TUNEL assay
TUNEL assay was performed to detect the amount of apoptotic cells within the gonocyte colonies. To induce apoptosis, SCF was removed or co-added with specific kinase inhibitors in the culture medium for 24 h. Cultured cells with different treatments were fixed with 4% paraformaldehyde for 10 min at room temperature and then washed three times in PBS. Staining was performed using a fluorescein-based cell death detection kit (Roche Applied Science) according to the manufacturer’s recommended protocol to quantify apoptotic cells. Counterstaining with DAPI was used to quantitate the total cell numbers of the colonies. The percentage of TUNEL/DAPI-labeled nuclei within the colonies was assessed as the index of apoptosis.

Statistical analysis
All experiments were repeated independently at least thrice in duplicate. The values from all experiments were used for calculation of the means and their respective standard errors of the mean (S.E.M). Statistical tests of one-way ANOVA followed by the non-parametric test of Kruskal–Wallis were used to determine the significant differences between different experimental groups and the controls. The P values < 0.05 were considered statistically significant.


   Acknowledgements
Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
References
 
We thank Profs Guangying Lu and Liansheng Chen for their helpful suggestions. Supported by National Nature Science Foundation of China (No. 30170480 and No. 30470884). The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.


   Footnotes
 
J Tu and L Fan contributed equally to this work

Received 8 April 2007
First decision 11 May 2007
Revised manuscript received 11 June 2007
Accepted 29 August 2007


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A. J. Childs, P. T.K. Saunders, and R. A. Anderson
Modelling germ cell development in vitro
Mol. Hum. Reprod., September 1, 2008; 14(9): 501 - 511.
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