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Reproduction (2008) 136 459-469
DOI: 10.1530/REP-08-0241
Copyright © 2008 Society for Reproduction and Fertility
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RESEARCH

Initiation of testicular tubulogenesis is controlled by neurotrophic tyrosine receptor kinases in a three-dimensional Sertoli cell aggregation assay

Kathrin Gassei1, Jens Ehmcke1,2 and Stefan Schlatt1,2

1 Department of Cell Biology and Physiology, Center for Research in Reproductive Physiology, University of Pittsburgh School of Medicine, 3500 Terrace Street, Pittsburgh, Pennsylvania 15261, USA2 Institute of Reproductive and Regenerative Biology, Centre of Reproductive Medicine and Andrology, Domagkstraße 11, 48149 Münster, Germany

Correspondence should be addressed to S Schlatt; Email: stefan.schlatt{at}ukmuenster.de


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 Declaration of interest
 Funding
 References
 
The first morphological sign of testicular differentiation is the formation of testis cords. Prior to cord formation, newly specified Sertoli cells establish adhesive junctions, and condensation of somatic cells along the surface epithelium of the genital ridge occurs. Here, we show that Sertoli cell aggregation is necessary for subsequent testis cord formation, and that neurotrophic tyrosine kinase receptors (NTRKs) regulate this process. In a three-dimensional cell culture assay, immature rat Sertoli cells aggregate to form large spherical aggregates (81.36±7.34 µm in diameter) in a highly organized, hexagonal arrangement (376.95±21.93 µm average distance between spherical aggregates). Exposure to NTRK inhibitors K252a and AG879 significantly disrupted Sertoli cell aggregation in a dose-dependent manner. Sertoli cells were prevented from establishing cell–cell contacts and from forming spherical aggregates. In vitro-derived spherical aggregates were xenografted into immunodeficient nude mice to investigate their developmental potential. In controls, seminiferous tubule-like structures showing polarized single-layered Sertoli cell epithelia, basement membranes, peritubular myoid cells surrounding the tubules, and lumen were observed in histological sections. By contrast, grafts from treatment groups were devoid of tubules and only few single Sertoli cells were present in xenografts after 4 weeks. Furthermore, the grafts were significantly smaller when Sertoli cell aggregation was disrupted by K252a in vitro (20.87 vs 6.63 mg; P<0.05). We conclude from these results that NTRK-regulated Sertoli–Sertoli cell contact is essential to the period of extensive growth and remodeling that occurs during testicular tubulogenesis, and our data indicate its potential function in fetal and prepubertal testis differentiation.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 Declaration of interest
 Funding
 References
 
The development of the testis during ontogenesis presents one of the earliest events of sex determination and is dependent on the sex-specific expression of Sry in XY primodial gonads (Gubbay et al. 1990, Sinclair et al. 1990). Initially, pre-Sertoli cells in the undifferentiated genital ridge aggregate and establish tight contacts with each other, increase in size, and gradually deposit basal laminar components around these cell aggregates (Magre & Jost 1991, Merchant-Larios & Taketo 1991). Primordial germ cells become enclosed within these aggregates during the process of cell aggregation. Subsequently, the migration of precursor cells of other somatic testicular cell lineages such as myoid peritubular, endothelial, and interstitial cells from the mesonephros is essential for the formation of testis cords (Capel 2000). Migration of mesonephric cells into the gonad is exclusive to XY gonads and does not occur in XX gonads (Buehr et al. 1993). Regulation of the morphogenetic events leading to testis cord formation upon specification of Sertoli cells in the male gonad remains poorly understood.

The family of neurotrophic growth factors has been proposed to be involved in testicular development (Wheeler & Bothwell 1992, Levine et al. 2000). Nerve growth factor (NGF) was the first member of the family to be found, followed by neurotrophin 3 (NTF3), neurotrophin 5 (NTF5), and brain-derived neurotrophic factor (BDNF; Reichardt 2006). The factors act through the specific high-affinity neurotrophic tyrosine kinase receptors NTRK1 (for NGF, formerly known as trkA), NTRK2 (for NTF5 and BDNF, formerly known as trkB), and NTRK3 (for NTF3, formerly known as trkC). Additionally, all factors share the common low-affinity neurotrophic growth factor receptor p75/LNGFR. Recently, neurotrophic growths factors were reported to participate in the development of various non-neural organs, such as the dermatome, kidney, and ovary (Sainio et al. 1994, Brill et al. 1995, Dissen et al. 1995). In the testis, Ngf mRNA was first detected in spermatocytes and early spermatids in the adult rodent testis where it acts as a survival factor for maturing spermatozoa (Olson et al. 1987, Ayer-LeLievre et al. 1988). At the onset of meiosis, DNA synthesis in stage VIII and IX preleptotene spermatocytes is thought to be under autocrine control through NGF (Parvinen et al. 1992). By contrast, stage-dependent secretion of androgen-binding protein by Sertoli cells is also regulated by NGF (Lönnerberg et al. 1992), suggesting a paracrine role of neurotrophins in the testis. Neurotrophin receptors were localized to Sertoli cells in the prepubertal testis (Djakiew et al. 1994), whereas transient p75/LNGFR receptor expression in late-meiotic spermatocytes and early spermatids was found in the adult testis (Seidl et al. 1996). NGF was also found in the embryoinc testis and it was suggested that neurotrophins might be involved in mesenchymal–epithelial transitions that occur during morphogenesis (Wheeler & Bothwell 1992, Russo et al. 1995). On the other hand, NTF3 secreted by Sertoli cells was suggested to act as a paracrine factor that stimulates mesonephric cell migration at this stage of gonad development (Levine et al. 2000). This is in contrast to reports that show NTF3 expression exclusive to peritubular cells during embryonic development and early postnatal life (Russo et al. 1995).

Members of the transforming growth factor β 1 (TGFB1) superfamily of morphogens have been shown to orchestrate testicular development (for review, see Itman et al. 2006). Members of the superfamily include the TGFB1 subfamily, the activin subfamily, the bone morphogenetic proteins subfamily, and the more distantly related superfamily members, glial-derived neurotrophic factor and anti-Mullerian hormone (AMH), which act as key factors during testicular development (Griswold 1998, Buageaw et al. 2005, Fouchecourt et al. 2006). In particular, activin stimulates Sertoli cell proliferation during early postnatal life (Boitani et al. 1995, Schlatt et al. 1999). Activin action in the reproductive system is modulated by its antagonist follistatin, which is known to be present in the rat testis at this age (Buzzard et al. 2004). Activin thereby contributes to the establishment of the proper testicular size that is directly depending on Sertoli cell proliferation (Atanassova et al. 2005). In turn, Sertoli cell mitotic activity is modulated by follicle-stimulating hormone (FSH)-mediated contact inhibition (Schlatt et al. 1996). Thus, the regulatory network of activin, follistatin and FSH is a center piece of testicular development and function. At the cellular level, FSH activates different signal transduction cascades in Sertoli cells, including cAMP-mediated activation of protein kinase A (PRKACA), the MAP kinase pathway, and the phosphatidylinositol 3-kinase, C2 domain containing, {alpha} polypeptide (PIK3C2A) pathway (Walker & Cheng 2005).

During postnatal testis development, Sertoli cells proliferate and maintain a general undifferentiated state. Up to postnatal day 10, these immature Sertoli cells retain their potential to reorganize into testis cords in vitro (Hadley et al. 1985, 1990), and to partially mature when cultured on extracellular matrix components (Suarez-Quian et al. 1984, Tung & Fritz 1987). We recently showed that three-dimensional extracellular matrix gel in combination with xenografting stimulates in vitro morphogenesis of seminiferous tubule-like structures from immature Sertoli cells (Gassei et al. 2006). Using this novel approach to mimic testicular morphogenesis, we conducted preliminary investigations to explore the relevance of neurotrophins, FSH and activin on aggregation of early postnatal Sertoli cells. We then focused our investigations on neurotrophin signaling as a key element of Sertoli–Sertoli cell contact and tubulogenesis.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 Declaration of interest
 Funding
 References
 
Characterization of testicular cell types after enzymatic digestion
Testicular single cells were isolated from 7-day-old rats and subjected to immunostaining in order to identify different cell types in the preparation (Fig. 1). In cell suspensions from three consecutive isolation procedures, Sertoli (AMH, 88.47%±3.5 positive cells), peritubular myoid (IGHMBP2, 16.44%±2.1), and Leydig cells (CYP11A1, 0.27%±0.1) were identified and quantitatively assessed. An additional experiment included a germ cell-specific marker (DDX4, 25.04%±2.74). Peritubular myoid (15.8%±1.31) and Leydig cells (0.32%±0.12) were present in quantities similar to those observed before. Negative controls where the primary antibody was omitted prior to incubation with the fluorescent-labeled secondary antibody did not show specific staining (data not shown).


Figure 1
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  		Figure 1 Characterization of the single-cell suspension. (A) After sequential enzymatic digestion, Sertoli cells represented the main cell type in the cell suspension, as confirmed by positive staining for anti-Mullerian hormone (AMH). Scale bar=100 µm. (B) A small fraction of peritubular myoid cells was observed (IGHMBP2). Scale bar=10 µm. (C) Only very few Leydig cells remained in the cell suspension (CYP11A1). Scale bar=100 µm. (D) Some germ cells were present in the cell suspension as identified by DDX4 immunofluorescence staining. Scale bar=50 µm.

 
Formation of Sertoli cell spheres in three-dimensional culture
The morphological cascade of Sertoli cell aggregation in three-dimensional culture has been described by us before (Gassei et al. 2006). We observed rapid cell migration within hours after plating (Fig. 2). Live-imaging experiments showed that 4 h after plating, single cells assembled into cord-like structures (Fig. 2C). The cascade was completed after 72 h when cultures were terminated for quantitative analysis. Sertoli cell aggregation led to the formation of large spherical aggregates (Fig. 2G). These spheres showed clear edges due to refraction and could be easily distinguished in the Matrigel in a typical hexagonal arrangement (Fig. 2H). No differences between cultures using standard Matrigel or growth factor-reduced Matrigel were observed (data not shown).


Figure 2
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  		Figure 2 Morphological cascade of Sertoli cell aggregation in three-dimensional culture. (A) After 1.5 h of plating, Sertoli cells are attached to the culture matrix. Scale bar=100 µm. (B) After 2.5 h, Sertoli cells start to aggregate. Scale bar=100 µm. (C) Sertoli cells form a network of cord-like structures after 3.5 h in culture. Scale bar=100 µm. (D) The network of cord-like structures is not present after 16 h, when Sertoli cells further aggregate to form clusters. Scale bar=100 µm. (E) The migration of Sertoli cells through the Matrigel is terminated after 23 h. Spherical aggregates show cytoplasmic processes. Scale bar=100 µm. (F) Aggregation and compaction of Sertoli cell spheres is mainly completed after 44 h. Scale bar=100 µm. (G) The morphological cascade is completed after 72 h. Sertoli cell aggregates form compact spherical aggregates that do not change over longer culture periods (16x). Scale bar=100 µm. (H) Spherical aggregates show a highly organized distribution within the Matrigel after finalizing migration and aggregation (4x). Scale bar=500 µm.

 
Two characteristics of spherical aggregates were measured to quantify Sertoli cell aggregation in control and treatment groups. As a measure of Sertoli cell aggregation, we first determined the diameters of spherical aggregates to gain information on aggregate size. Second, the distance between spherical aggregates was considered a measure for Sertoli cell migration through the Matrigel. The spatial arrangement of Sertoli cell aggregates provides information about the gradual attractive or repulsive signals established within the three-dimensional culture matrix.

Overall, the three-dimensional culture system and the applied computer-based analysis of Sertoli cell clusters proved to be applicable to assess reliable parameters. Small areas of the micrograph were out of focus due to the concave meniscus of the three-dimensional Matrigel and were excluded from analysis.

Data derived for both parameters were statistically tested in order to evaluate the validity and the repeatability of the assay. The coefficient of variation (CV) for data derived from five independent control experiments (no treatment) ranged from 5.5 to 7.9% (diameter) and 0.5 to 5.4% (distance). The low CV values obtained from this experiment showed that the assay could be repeated with high reliability and validity with only little dispersion of data.

Antagonizing neurotrophin signaling, but not activin or FSH action, disturbs Sertoli cell aggregation
In qualitative preliminary experiments inhibition of NTRK signaling caused interruption of the morphological cascade of Sertoli cells. In addition to complete cell aggregation in control experiments, two different patterns of Sertoli cell aggregation were observed in this experiment. Partially inhibited Sertoli cell aggregation led to the formation of loose Sertoli clusters that were smaller than spherical aggregates but still showed some degree of organization (Fig. 3C). By contrast, high doses of inhibitor completely disturbed Sertoli cell aggregation. Sertoli cells attached to the Matrigel but remained as single cells throughout the culture period (Fig. 3D).


Figure 3
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  		Figure 3 Disruption of Sertoli cell migration and aggregation by the NTRK inhibitors K252a and AG879. (A) No-treatment control. Sertoli cell aggregates after 3 days of culture form large spherical aggregates. (B) Vehicle control with 0.5% DMSO. No influence on Sertoli cell aggregation was observed. (C) Incubation with 1 nM K252a partially inhibits Sertoli cell aggregation. (D) Incubation with 5 nM K252a has a dramatic inhibitory effect on Sertoli cell aggregation. No spherical aggregates were observed after 3 days of culture. (E) Similarly, Sertoli cell aggregation was partially inhibited by 5 µM AG879. (F) Twenty micro molar AG879 disrupted the morphogenetic cascade of Sertoli cell cord formation in vitro. Occasionally, small clusters were observed after 3 days of culture (All scale bars=50 µm).

 
Addition of exogenous NTF3 did not enhance Sertoli cell aggregation. Furthermore, exogenous FSH did not demonstrate a morphogenic effect, and antagonizing FSH action could not disturb Sertoli cell aggregation. Similarly, antagonizing activin action by supplementing cultures with follistatin did not prevent Sertoli cells from aggregating or stimulate enhanced aggregation (Table 1). We therefore decided to further investigate the role of neurotrophins during Sertoli cell aggregation.


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  		Table 1 Qualification and quantification of Sertoli cell aggregation.

 
NTRK3 involvement in Sertoli cell aggregation
Application of the NTRK inhibitor K252a caused a dose-dependent disruption of Sertoli cell aggregation (Table 1, panel B). In cultures treated with 1 nM K252a, cell migration was partially inhibited. In these cultures, Sertoli cell clusters were predominantly observed after 3 days of culture (Fig. 3C). Higher doses of the inhibitor (5 and 10 nM) completely inhibited Sertoli cell aggregation and only single cells could be observed (Fig. 3D). For quantitative analysis, samples that showed spherical aggregates or Sertoli cell clusters in more than 40% of all captured frames were measured, whereas samples with mainly single cells present in the culture were qualitatively recorded and not subjected to quantitative analysis (Table 2). Accordingly, the cultures supplemented with 1 nM K252a were analyzed and compared with no-treatment and vehicle controls (Fig. 4A).


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  		Table 2 Evaluation of dose-dependent inhibition of Sertoli cell morphological cascade.

 

Figure 4
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  		Figure 4 Quantitative analysis of aggregate size and distances between Sertoli cell aggregates upon K252a treatment. (A) Effect of K252a on Sertoli cell aggregate size (diameter) and distance. At a concentration of 1 nM K252a, both parameters were significantly decreased when compared with no-treatment and vehicle controls (DMSO). (B) The inhibitory effect of 1 nM K252a could not be rescued by simultaneously incubating cells with 100 ng/ml NTF3. (C) Neutralizing anti-NTF3 antibody did not show an effect on Sertoli cell aggregation when added to three-dimensional cultures in low, medium, or high concentrations. Spherical aggregates were observed in all cultures. (D) Supplementation of cultures with anti-NTRK3 antibody alone or in combination with NTF3 did not affect the formation of spherical aggregates (Different lettering indicates significant statistical differences between treatment groups as revealed by one-way ANOVA or Kruskal–Wallis one-way ANOVA on ranks analysis).

 
Aggregate size averaged to 81.36 µm (±7.34 µm S.E.M.) diameter in controls, and the distance between Sertoli cell spheres averaged to 376.95 µm (±21.93 µm S.E.M.). By contrast, K252a in concentrations below the IC50 value (3 nM) caused partial Sertoli cell inhibition and the formation of cell clusters with a reduced diameter of 39.93 µm (±3.31 µm S.E.M.), representing a decrease of 50.92%. Similarly, Sertoli cell clusters also showed a highly significant decrease in distance (68.57%) and averaged to 118.47 µm (±6.29 µm S.E.M.; P<0.001). The overall hexagonal arrangement of Sertoli cell clusters was still established in the cultures containing 1 nM K252a, but was not observed in the cultures incubated with above IC50 concentration (5 and 10 nM; Table 2).

Recent studies have suggested a role of NTF3 in testicular cord formation in organ cultures (Levine, Cupp et al. 2000). We therefore tested whether the effect of K252a in vitro was due to the suppression of neurotrophin 3 signaling. We investigated the influence of exogenous NTF3 in the cultures and the effects of neutralizing anti-NTF3 antibodies on Sertoli cell aggregation in vitro. To attempt a higher degree of Sertoli cell aggregation, the cultures were supplemented with 10, 50, or 100 ng/ml neurotrophin 3 (NTF3). We did not find a stimulating effect of NTF3 on Sertoli cell aggregation, and the appearance of spherical aggregates was similar to no-treatment controls. Furthermore, exogenous NTF3 was not able to rescue Sertoli cell aggregation upon treatment with K252a (Fig. 4B). In addition, incubating cell cultures with a neutralizing anti-NTF3 antibody or neutralizing anti-NTRK3 antibody did not affect cell migration and aggregation (Fig. 4C and D).

NTRK1 signaling is ivolved in Sertoli cell aggregation
Although K252a is a competent inhibitor of high-affinity neurotrophin receptors, it acts in a non-specific manner and has been shown to inhibit NTRK3 as well as NTRK1 and NTRK2. In a second set of experiments, we therefore examined the action of NGF on Sertoli cell aggregation. Similar to the results obtained for exogenous NTF3, supplementation of cultures with NGF did not stimulate Sertoli cell aggregation (Fig. 5B). However, when Sertoli cells were cultured in the presence of the specific NTRK1 antagonist tyrphostin AG879, the same dose-dependent effect on Sertoli cell inhibition was observed as described for K252a (Fig. 3E and F). Quantitative analysis revealed a highly significant decrease in spherical aggregate size by 46.69% (32.21 µm±2.86 vs 60.42 µm±3.62; P<0.001) in cultures treated with 5 µM AG879 (Fig. 5A). In addition, the cultures appeared increasingly unorganized and distances between cell clusters were reduced from 318.10 µm±17.87 in controls to 116.56 µm±11.45 after treatment with the inhibitor (P<0.001). These results present a significant decrease in cell cluster distance by 63.36%. In cultures treated with concentrations equal or above the IC50 value (10 µM) of tyrphostin AG879 (10 µM and 20 µM), single Sertoli cells were mainly present as a result of a complete inhibition of Sertoli cell migration and aggregation (Table 2). Although these results point to the involvement of NTRK1 in Sertoli cell aggregation, experiments using neutralizing anti-NGF antibody surprisingly did not affect Sertoli cell aggregation in this assay (Fig. 5B).


Figure 5
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  		Figure 5 Quantification of the inhibitory effect of tyrphostin AG879 on Sertoli cell aggregation. (A) Tyrphostin AG879 at a concentration of 5 µM significantly inhibited Sertoli cell migration and aggregation within the three-dimensional culture system. (B) Neutralizing anti-NBF antibody did not show an effect on Sertoli cell aggregation when added to three-dimensional cultures in low or high concentrations. Spherical aggregates were observed in all cultures (Different lettering indicates significant statistical differences between treatment groups as revealed by one-way ANOVA).

 
Tubule formation in xenografts depends on NTRK-mediated Sertoli cell aggregation
Sertoli cell aggregates from three-dimensional cell cultures treated with K252a were xenografted to host mice in order to test the necessity of Sertoli cell aggregation for testicular differentiation and the development of seminferous tubule-like structures. As controls, Sertoli cell aggregates treated with DMSO were grafted. The survival rate of hosts was 100%. Xenografts could be recovered from all hosts (n=24 for controls, n=23 for 1 nM K252a, n=24 for 5 nM K252a) and graft weights were significantly decreased in treatment groups (P<0.05). The grafts from the control group (DMSO) averaged to 20.87 mg (±2.16 S.E.M.), whereas the grafts from the low-concentration treatment group (1 nM) averaged to 9.92 mg (±1.82 S.E.M). Grafts from both groups were vascularized. By contrast, the grafts from the high-concentration group (5 nM; 6.63 mg ±0.75 S.E.M.) showed fewer blood vessels and appeared clear (Fig. 6).


Figure 6
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  		Figure 6 Distribution of graft weights after 4 weeks in the three treatment groups. The range of graft weights is shown as dot blot for each group (blue circles). Mean and S.D. are denoted on the right (yellow circles). Statistically significant differences are indicated by different letters (a and b) and were identified by Kruskal–Wallis one-way ANOVA on ranks analysis.

 
Histological sections of the grafts revealed that Sertoli cell aggregates developed into seminiferous tubule-like structures after 4 weeks in the host (Fig. 7A, control). Polarized epithelial Sertoli cells were observed. Furthermore, peritubular myoid cells were present on the outside of tubule-like structures. A basement membrane surrounding tubule-like structures was present, as well as lumen formation (Fig. 7B). The grafts derived from Sertoli cells treated with 1 nM K252a showed similar tubular organization, but the grafts were smaller and contained fewer tubule-like structures (data not shown). In contrast, complete inhibition of Sertoli cell aggregation by 5 nM K252a prior to xenografting caused complete absence of seminiferous tubule-like structures in xenografts (Fig. 7C). Grafts were encapsulated similar to controls, but Matrigel patches contained only few single cells that were mainly aligned along the host-derived capsule (Fig. 7D).


Figure 7
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  		Figure 7 Development of xenografts from K252a-treated Sertoli cell cultures. Resin-embedded semi-thin sections (4 µm) were stained with the periodic acid/Schiff's reagent method. (A) Low-power micrograph of a xenograft from the control group. Scale bar=100 µm. (B) High-power micrograph of the xenograft shown in (A). Sertoli cells (black arrows) differentiated to form seminiferous cord-like structures with lumen (asterisk) and basement membrane. Peritubular myoid cells are aligned along tubules (arrowheads). Vascularization (v) is observed close to the tubules. Scale bar=50 µm. (C) Xenografts did not contain tubule-like structures when Sertoli cells were treated with 5 nM K252a. Scale bar=100 µm. (D) High-power micrograph of the xenograft in (C). Sertoli (black arrow) and peritubular cells (arrowhead) are present in Matrigel residues but remain unorganized and as single cells. Scale bar=50 µm.

 
At the time of graft removal, body weights and seminal vesicle weights from the castrated recipients were recorded. All hosts appeared healthy and we did not find differences in body weight between treatment groups. Notably, seminal vesicles were significantly larger in control animals than in animals carrying grafts after in vitro inhibitor treatment (Table 3), thus indicating steroidogenic activity in controls, but not in the treatment group.


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  		Table 3 Xenografting after treatment with K252a.

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 Declaration of interest
 Funding
 References
 
In this study, we developed a quantifiable cell culture assay for Sertoli cell aggregation that models early testicular organogenesis. We then used this assay to examine the relevance of NGF, NTF3, FSH, and activin on Sertoli cell aggregation.

Our results indicate a regulatory role of high-affinity neurotrophic tyrosine kinase receptors during Sertoli cell aggregation. Recent studies on explants of the undifferentiated gonadal primordium have suggested a potential role of neurotrophin-mediated signaling during testicular development (Levine et al. 2000). In accordance with this, we observed a compelling dose-dependent anti-aggregation effect on Sertoli cells of neurotrophic tyrosine kinase receptor inhibitors (K252a and AG879), indicating that Sertoli cell aggregation underlies regulation via NTRK signaling.

By contrast, Sertoli cell aggregation could not be inhibited by follistatin, the naturally occuring binding protein for activin. From this result, we conclude that signaling through activin-specific receptor subunits does not contribute to Sertoli cell aggregation. Similarly, addition of FSH or blocking of FSH receptor binding did not interfere with Sertoli cell aggregation in vitro, indicating that Sertoli cells aggregate independent of FSH signaling via cAMP and PRKACA. This result is surprising because of the known regulatory effects of activin and FSH on postnatal Sertoli cell proliferation and tubular outgrowth. One possible explanation could be our preliminary observation that Sertoli cells do not proliferate in three-dimensional Matrigel culture and only resume mitosis upon xenografting.

On the contrary, we show that NTRK-mediated aggregation of immature Sertoli cells is crucial for subsequent differentiation of Sertoli cells and tubule formation in xenografts. Sertoli cells that were prevented from aggregating in vitro by K252a supplementation did not develop into seminiferous tubule-like structures in xenografts. As previously shown, although three-dimensional culture does support Sertoli cell in vitro morphogenesis, differentiation is arrested after short-term culture. However, Sertoli cells resume maturation upon xenografting to an immunodeficient host, leading to the formation of seminiferous tubule-like structures (Gassei et al. 2006). Similarly, Sertoli cell aggregates from control groups showed a mature phenotype in xenografts and were arranged in tubule-like structures. By contrast, tubule-like structures were absent in xenografts after exposure of Sertoli cells to NTRK inhibitors. We conclude from these results that Sertoli–Sertoli cell adhesive contacts within aggregates are mandatory for subsequent testis cord formation and tubulogenesis.

K252a and AG879 have been used in previous studies reporting a role of NTF3 in testis cord formation (Levine et al. 2000, Cupp et al. 2003). To examine whether NTF3 could be accounted for the inhibition of in vitro Sertoli cell aggregation by K252a, some Sertoli cell cultures were supplemented with a combination of K252a and NTF3 at the time of plating. NTF3 did not rescue the inhibitory effect of K252a. This is in correspondence to the lack of a stimulatory effect of NTF3 alone. To more specifically inhibit NTF3 action, we then used neutralizing antibodies against NTF3 and NTRK3 to inhibit Sertoli cells in culture. In these experiments, we did not see an inhibitory effect, and no differences in aggregation patterns could be observed between treated cells and untreated controls. However, since K252a and AG879 also have been suggested to exert inhibitory effects on NTRK1, we tested NGF and neutralizing anti-NGF antibody in our cultures, but did not detect a measurable effect on Sertoli cell behavior.

K252a has been shown to act as a competitive inhibitor for PRKACA and PRKCC, and thereby might inhibit Sertoli cell aggregation via various signaling pathways. To rule out the inhibitory effects other than those on NTRKs, we used K252a at concentrations lower than the Ki values of 18–24 nM for PRKACA, PRKCC, and PKG inhibition. In addition, previous studies using organ cultures to investigate the chemotactic role of neurotrophins during testis cord formation were performed with 100 nM K252a, and in these studies, no inhibitory effect on PRKACA by K252a was observed (Levine et al. 2000). K252a and AG879 had strikingly similar, dose-dependent inhibitory effects on Sertoli cell aggregation when using two different concentrations close to the respective IC50 values (3 nM for K252a and 10 µM for AG879). Partial inhibition of Sertoli cell aggregation was observed at concentrations below this threshold, whereas higher concentrations completely inhibited Sertoli cell aggregation. This implies that the morphological effects observed for K252a and AG879 are due to the inhibition of NTRKs.

In summary, the combined in vitro/in situ strategy to recapitulate testicular tubulogenesis allows for the detailed study of the morphogenesis of the somatic component of the testis in a reliable and quantifiable fashion. We demonstrate that neurotrophic receptor tyrosine kinases NTRK1 and NTRK3 are important for the in vitro aggregation of immature rat Sertoli cells, which occurs in a manner similar to that observed in E13–E13.5 embryonic gonads (Magre & Jost 1991). It remains unclear whether this action is mediated by NGF or NTF3 specifically or independent of the ligands.

In conclusion, we have shown that Sertoli cell aggregation is a crucial event during early testicular development and an indespensable prerequisite for subsequent testis cord formation and testicular development. Failure of Sertoli cells to make contact with one another results in the disturbance of testis cord formation.


   Materials and Methods
Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
Declaration of interest
 Funding
 References
 
Isolation of primary Sertoli cells
Seven-day-old CD rats were used as donors in order to yield a single-cell suspension of undifferentiated Sertoli cells. At this age, only a relatively small fraction of premeiotic germ cells is present in the testis, which greatly facilitates Sertoli cell isolation. Rat pups were provided by Charles River Laboratories, Inc. (Wilmington, MA, USA). All procedures were in compliance with the University of Pittsburgh Guidelines for the Care and Use of Laboratory Animals.

A sequential enzymatic digestion protocol was used to obtain testicular single-cell suspensions enriched for Sertoli cells (Schlatt et al. 1996). Upon dissection of the testes, the tunica albuginea was removed and the tissue digested to small tubule fragments with 1 mg/ml collagenase I (no. C2674; Sigma) and 5 µg/ml DNAse (15 U/ml, no. 104132; Roche Applied Science) in digestion medium. Digestion medium consisted of Dulbecco's Minimum Essential Medium (DMEM, 4.5 g glucose/ml) mixed 1:1 with Ham's F12 (Sigma N6658) and supplemented with 1% MEM non-essential amino acids (100x; BioWhittaker 13-114E), 100 IU/ml penicillin, and 100 µg/ml streptomycin (100x; Cellgro 30-002-CI). To remove interstitial and peritubular cells, tubule fragments were allowed to settle by unit gravity for 5 min before discarding the supernatant. A second digestion step was performed similarly by incubating tubule fragments with 1 mg/ml collagenase I and 5 µg/ml DNAse in combination with 1 mg/ml hyaluronidase (Sigma H-3506). The resulting cell suspension was centrifuged at 200 g for 10 min and the pellet was resuspended in culture medium consisting of low-glucose DMEM (1 g/ml), 1% MEM nonessential amino acids, 100 IU/ml penicillin, and 100 µg/ml streptomycin solution. Cell numbers were determined using a Bright-Line hematocytometer (Hausser Scientific no. 3100).

Identification of different cell types in the cell suspension was assessed by immunocytochemistry. The cells were plated onto poly-L-lysine-coated cover slips for 15 min and formalin fixed (4% in DPBS) for 15 min. After washing with 1x DPBS, the cells were incubated with 10% goat serum diluted in dilution buffer (1x TBS with 0.1% BSA) for 30 min at room temperature. The cells were immunostained with antibodies raised against AMH (Sertoli cell specific; cat. no. sc-6886; 1:50 dilution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), {alpha}- immunoglobulin mu-binding protein 2 (IGHMBP2; peritubular myoid cell marker; Sigma no. A2547; 1:2000 dilution), cytochrome P450, family 11, subfamily a, polypeptide 1 (CYP11A1, Leydig cell marker; Chemicon no. AB1244; 1:500; Millipore, Billerica, MA, USA), and DEAD (Asp-Glu-Ala-Asp) box polypeptide 4 (DDX4; germ cell marker; Abcam no. ab-13840; 1:100; Abcam Inc., Cambridge, MA, USA). Antibodies were incubated over night at 4 °C. For the anti-DDX4 antibody, heat-mediated antigen retrieval in citrate buffer (pH 6) prior to antibody incubation was necessary to obtain optimal immunostaining. Negative controls were performed in all experiments by omitting primary antibodies to ensure antibody specificity. The cover slips were washed thoroughly with 1x TBS (3x5 min) and incubated with goat anti-mouse IgG (for IGHMBP2 detection), goat anti-rabbit IgG (DDX4 and CYP11A1 detection), or chicken anti-goat IgG (for AMH detection) at a 1:100 dilution for 60 min at room temperature. Secondary antibodies were conjugated to AlexaFlour488 chromophor. Cover slips were then washed thrice for 5 min with TBS and nuclei were stained with 0.5 µg/ml DAPI stain for 5 min. After washing, cover slips were mounted with Vectashield medium for fluorescence microscopy. For each marker, ~1000 cells from three independent experiments were counted to determine the percentage of positively stained cells.

Three-dimensional cell culture
Approximately, 1x106 cells were plated onto 300 µl reconstituted extracellular matrix gel (Matrigel, BD Biosciences, Bedford, MA, USA; no. 354234, diluted 1:1 with culture medium) on 24-well culture plates. The cells were cultivated in a humidified incubator at 35 °C in an atmosphere containing 5% CO2. In pilot experiments, growth factor-reduced Matrigel (BD Biosciences) was used to test whether growth factors that naturally occur in Matrigel might influence Sertoli cell behavior in vitro.

Qualitative and quantitative experiments using a variety of supplements were performed as depicted in Table 1. The cells were treated at the time of plating. Compounds used in this study included human recombinant neurotrophin-3 (Sigma no. N1905), neutralizing anti-NTRK3 antibody (Sigma no. T2450), anti-neurotrophin 3 antibody (Chemicon AB1532), receptor tyrosine kinase inhibitors K252a (Sigma no. K1639), tyrphostin AG879 (Sigma T2067), recombinant NGF (Sigma no. N6009), and neutralizing anti-NGF antibody (Sigma no. N6655). For control experiments, the cells were either cultured with culture medium alone (no-treatment control) or DMSO was added to the medium in comparable concentrations to the treatment groups (vehicle control). Additionally, cultures were also supplemented with recombinant human follicle stimulating hormone (FSH, from the National Hormone Program; kindly provided by Dr A F Parlow, Torrance, CA, USA), anti-FSHR antibody (clone H-90), and activin-antagonizing follistatin (Sigma no. F2177) to control whether signaling via cAMP second messenger (through FSH) or through activin specific TGFB1 receptor subunits might interfere with Sertoli cell migration and aggregation. All compounds were used at concentrations recommended by the supplier or as suggested by data from previous research to ensure optimal stimulation of Sertoli cells in culture. For clarity reasons, amounts of growth factors, reagents, and gonadotrophins are compiled in Table 1.

Live imaging of cell cultures in Matrigel and quantitative analysis
Phase contrast micrographs of spherical Sertoli cell aggregates in Matrigel were captured with an Olympus IX71 inverted microscope equipped with a Retiga 4000R digital camera (QImaging, Surrey BC, Canada) and Northern Eclipse imaging software (MVIA Inc., Monaca, PA, USA).

The spatial arrangement of spherical cell aggregates was analyzed after 72 h of culture. Phase contrast micrographs were taken at 16x magnification of four to six randomly selected fields from each culture. In samples that showed few clearly defined aggregates, all structures were analyzed. For samples with a more scattered appearance, cell clusters were randomly selected using a grid overlay. Ten grid points in the center of the micrograph were used for analysis and clusters at these grid points were assigned to measurements.

Data were collected for two qualitative parameters. First, we determined the diameters of spherical aggregates and clusters. The diameter was measured using the straight line measurement of the Northern Eclipse software at the largest point of the aggregate. The second parameter that was quantitatively evaluated was the distance from each aggregate subjected for diameter determination to the nearest, similarly identified spherical aggregate. The straight line measurement tool was used by measuring the distance between the midpoints of the neighboring aggregates. All absolute values obtained for each parameter were combined and averaged for each culture (see Table 1 for treatment groups and repeats). Data were expressed as means±S.E.M., and data sets were statistically analyzed with SigmaStat software using one-way ANOVA and, if applicable, a subsequent pairwise multiple comparison post hoc test. The CV was determined for both parameters (diameter of aggregates and distance between aggregates) in the case of negative controls (no treatment) as a measure of dispersion and to validate the cell culture-based assay.

Xenografting
Adult male nude mice (strain: nu/nu) were obtained from Charles River Laboratories and served as hosts for cell culture grafts. Castration was performed at the time of grafting to assess androgen levels in hosts upon graft development by means of seminal vesicle weights. Sertoli cells were prepared as described above and three treatment groups were defined. As vehicle controls, the cells were incubated with 0.25% DMSO. The low-concentration group was supplemented with 1 nM K252a inhibitor, in contrast to the high-concentration group that was treated with 5 nM K252a at the time of cell plating. Sertoli cells were cultured for 9 days in extracellular matrix gel and were then injected s.c. in the back of hosts using an 18 G injection needle. Each host received six injections of 250 µl extracellular matrix gel. For each treatment group, three hosts were grafted (18 grafts total per treatment group). The grafts were allowed to develop for 4 weeks. Hosts were killed by exsanguinations under deep anesthesia and the grafts were removed from the inner surface of the back skin. Grafts were fixed in Bouin's fixative overnight and then transferred to 70% ethanol for resin embedding. Body weights, seminal vesicle weights, as well as graft numbers and weights were recorded for each animal.

Histology
Xenografts were embedded in resin (Technovit 7100; Heraeus Kulzer, Hanau, Germany) and 4 µm semi-thin sections were prepared. The sections were stained with the periodic acid/Schiff's reagent method followed by hematoxylin counterstaining for histological evaluation.


   Declaration of interest
Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Declaration of interest
Funding
 References
 
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.


   Funding
Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Declaration of interest
 Funding
References
 
This work was financially supported by the start-up funds from the University of Pittsburgh School of Medicine, and NIH grant U54 HD 008610, Center grant, project 1 (S S), and a doctoral scholarship from the Ernst Schering Research Foundation (K G).

Received 22 February 2008
First decision 27 March 2008
Revised manuscript received 2 June 2008
Accepted 24 July 2008

   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Declaration of interest
 Funding
 References
 

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