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Correspondence to Michail S. Davidoff: davidoff{at}uke.uni-hamburg.de
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Abstract |
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Introduction |
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Results |
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To investigate whether nestin is expressed in testis, we initially performed immunoblot analyses. These studies were capable of demonstrating low levels of nestin, detectable as a protein of 440 kD (Kachinsky et al., 1995), in normal (untreated) 3-mo-old animals. However, the expression in testis of nestin was rapidly and massively enhanced after EDS treatment (Fig. 2 a), and the most significant elevation was observed precisely until the onset of massive Leydig cell regeneration (see the analogous immunoblot for CytP450). Thus, Leydig cell disappearance clearly coincides with elevation, and Leydig cell reappearance with diminution of nestin expression.
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Nestin-expressing VSMCs and PCs protrude from vessel walls and are transformed into Leydig cells
14 d after EDS, nestin-positive cells begin to protrude from the vessel walls (Fig. 3, ac) and to accumulate in their vicinity in form of clusters (Fig. 3 d), strikingly similar to the (CytP450 immunoreactive) Leydig cell clusters observed at that time (Fig. 1 a). Conditions where these cell clusters are still in contact with the blood vessel walls they derive from are detectable (Fig. 3 c). As revealed by double staining, the clusters consist of cells expressing either both nestin and CytP450, or only CytP450 (Fig. 3, e and f), indicating a process in which nestin-immunoreactive cells, during and/or after their displacement from vessel walls, first acquire steroidogenic properties (expression of CytP450) and then loose nestin, finally resulting in typical Leydig cells. This conversion into Leydig cells is accompanied also by a loss of SMA immunoreactivity (not depicted), providing further evidence for a transdifferentiation phenomenon. Approximately 1 wk later, when newly formed Leydig cells are abundant and localized primarily around the tubular compartments, nestin expression is generally reduced but still clearly detectable at larger blood vessels (Fig. 3 g). Newly formed Leydig cells that still display nestin immunoreactivity are only rarely to be found at d 20 (Fig. 3 h) and are nearly undetectable at d 21 (Fig. 3 i), indicating a faster transformation process at that time. In contrast to the situation early after EDS treatment, the rapid and abundant transformation is not accompanied by a pronounced cell division activity. Note that the number of BrdU-labeled nuclei of stem celllike progenitors is several fold higher at d 2 (Fig. 3 j) than at d 21 after EDS (Fig. 3 k). Thus, proliferation of the Leydig cell progenitors predominates during the first week after EDS, whereas their transformation begins at d 14 and accelerates 1 wk later.
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Next, we examined the proteoglycan NG2, known to be expressed in neuronal and glial cell progenitors of the central and peripheral nervous system (Schneider et al., 2001; Chekenya et al., 2002; Belachew et al., 2003) and previously suggested as a marker for PCs (Ozerdem et al., 2001). Like nestin (see Fig. 2 and Fig. 3), NG2 immunoreactivity was found both in VSMCs and PCs (Fig. 4, ik), as well as in newly generated Leydig cells (Fig. 4 k), further indicating a relationship between these cell types. Finally, the selective identification of PDGF receptor-ß (PDGFR-ß; Fig. 4, l and m), known to be involved in development of VSMCs and PCs (Lindahl et al., 1997; Lindahl and Betsholtz, 1998; Hellström et al., 2001; Hoch and Soriano, 2003; Lindblom et al., 2003), and myelin/oligodendrocyte-specific protein (not depicted) immunoreactivity in both newly generated (at d 14 after EDS) Leydig cells and their vascular progenitors, strongly supported their lineage relationship.
Leydig cell generation during postnatal development shows similarities to the Leydig cell repopulation process after EDS treatment
Next, we investigated whether the regeneration of Leydig cells after EDS treatment has similarities to the formation of a mature Leydig cell population in rats during normal postnatal development. Immunoblot analyses of Cyt P450scc revealed a biphasic expression pattern (Fig. 5 a): protein levels most significantly increase during puberty (d 24 and 27) to attain maximum (i.e., adult) values, but there is also an elevated expression at the earliest (postnatal day 5) stage examined. This developmental pattern is consistent with a gradual decrease in the number of fetal-type Leydig cells during initial postnatal development followed by the appearance and plentiful accumulation of adult-type Leydig cells (Ge et al., 1996). Comparative analyses of nestin and NF-H revealed striking similarities to the Leydig cell repopulation process after EDS treatment. Nestin expression is initially high and declines in time with the abundant emergence of (adult) Leydig cells, whereas the postnatal expression of NF-H commences later (between d 5 and 10) and peaks (around d 15) before the switch from nestin to CytP450 expression (Fig. 5 a).
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Analyses of nestin-GFP transgenic mice and human testes
As in rats, a similar pattern of nestin-immunoreactive cells was found in testes of adult mice (unpublished data). To verify the sites of nestin expression by a different approach, we made use of nestin-GFP transgenic mice. These studies, performed with animals expressing GFP under the control of the central nervous system (CNS)specific nestin second intronic enhancer (Lothian and Lendahl, 1997; Mignone et al., 2004), confirmed the expression of nestin in Leydig cells and their vascular progenitors (Fig. 5, eg).
Finally, comparative analyses revealed that this is also valid for the human testis (Fig. 5 h). Remarkably, numerous (still nestin-immunoreactive) newly formed Leydig cells are detectable within areas of Leydig cell hyperplasia in testes of elderly men (Fig. 5 i). Thus, transformation of stem celllike progenitors into Leydig cells can explain (hitherto incomprehensible) increases in the number of Leydig cells (Clegg et al., 1997) despite their lack of mitotic activity (Teerds et al., 1989; Benton et al., 1995).
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Discussion |
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Nestin expression in testis
Although a functional role for nestin is still poorly understood, its specific expression in activated/dynamic cells (e.g., cells that proliferate, migrate, or rapidly change their phenotype) is well established (Sahlgren et al., 2001). Originally identified as a marker for neural progenitor cells during early stages of CNS development (Lendahl et al., 1990), it was meanwhile demonstrated that nestin is also expressed in a variety of tissues outside the CNS (Vaittinen et al., 2001; Zulewski et al., 2001; Li et al., 2003) and in the adult organism during regeneration of injured tissue (Frisén et al., 1995; Okano, 2002). Our present findings demonstrate elevated nestin expression in testis both during development as well as during regeneration after experimentally induced Leydig cell elimination in adult animals. Consistent with observations in other tissues (Lendahl et al., 1990; Sejersen and Lendahl, 1993), nestin was found to be down-regulated and replaced by other intermediate filament proteins upon terminal differentiation. Nestin expression was clearly localized to PCs/VSMCs and newly generated Leydig cells in rat, mouse, and human testes. This characteristic pattern was additionally verified by the analysis of nestin-GFP transgenic animals. It has to be noted that the nestin-GFP mice used in our study direct transgene expression to cell types of neural origin (Mignone et al., 2004), and that GFP was found to be expressed selectively in the adult brain in areas related to continuous neurogenesis (Mignone et al., 2004). Thus, the identification in these animals of GFP in testicular vascular wall (PCs, VSMCs) cells and Leydig cells essentially supports similarity to properties of neural stem/progenitor cells. It is of interest that the mean number of nestin-expressing Leydig cells in mouse (detectable both in the GFP model and by immunohistochemistry in wild-type mice; unpublished data) as well as in human testes is significantly higher than in rat testes, suggesting species-specific differences in the velocity of nestin down-regulation after terminal differentiation.
Lineage relationship between PCs, VSMCs, and Leydig cells
The present paper reveals a close relationship between PCs and VSMCs of the testis microvasculature. Both cell types express SMA and nestin and proliferate in response to EDS treatment. The identification of NG2 and PDGFR-ß immunoreactivity in both cell types corroborates their relationship, and in addition demonstrates properties similar to those of neural progenitors of the CNS (Belachew et al., 2003). These findings are consistent with several previous reports, indicating functional (Allt and Lawrenson, 2001; Hoofnagle et al., 2004; Hu et al., 2004) or lineage (Alliot et al., 1999; Korn et al., 2002) relationship between PCs and VSMCs. Evidence for their transdifferentiation into Leydig cells is based on clear coincidence of marker expression, i.e., transient coexpression of nestin with CytP450 in newly generated Leydig cells. Moreover, the observed connection of newly formed Leydig cell clusters with the blood vessel walls further demonstrates their origin, and previously detected basement membrane fragments at the cell surface of certain Leydig cells (Kuopio and Pelliniemi, 1989; Haider et al., 1995) probably represent remnants of the basal lamina known to surround PCs (Sims, 1991; Nehls and Drenckhahn, 1993). Consistently, immunoreactivity for NG2 and for PDGFR-ß, recently established as reliable markers for activated PCs (Ozerdem and Stallcup, 2003) as well as for myelin/oligodendrocyte-specific protein, was identified in both newly generated Leydig cells and their vascular progenitors. Finally, lineage relationship was significantly supported by analyses of nestin-GFP transgenic mice, showing specific (and selective) expression of the transgene in Leydig cells and their progenitors.
The potential involvement of circulating cells in the process of Leydig cell (re)generation requires attention. However, several lines of evidence argue against such a possibility. The observed immediate induction of proliferation/nestin expression in PCs and VSMCs during the EDS-elicited Leydig cell destruction strongly refers to resident cells, rapidly responding to newly generated and/or disappearing (testosterone) signaling molecules. Furthermore, immunohistochemical analyses, performed with antibodies against the mesodermal/hematopoietic stem cell marker CD34, did not reveal any marker expression in PCs or SMVCs of the testis microvasculature, whereas staining was detectable in endothelial and intertubular connective tissue cells (unpublished data). These findings, indicating an involvement of resident rather than circulating cells, are consistent with cellular activities responsible for increased nestin expression after injury in other tissues (Frisén et al., 1995; Amoh et al., 2004). Remarkably, a recent investigation, identifying so-called side population (SP) cells in testis (Kubota et al., 2003), showed that testis SP cells do not derive from or contain circulating blood cells, but represent resident cells in this tissue.
Considering that many of the properties of Leydig cells and their progenitors are compatible with a neural crest origin (Davidoff et al., 1993, 1999, 2002; Lobo et al., 2004), we started to address this issue experimentally. However, investigations of mice with targeted mutations (see Materials and methods) in genes of the neural crestspecific neuregulin receptors, ErbB2 and ErbB3 (Britsch et al., 1998), did not yet reveal clear-cut results. In this context, it might be of general interest that we recognized during these studies endogenous ß-galactosidase expression in different testicular cell types (unpublished data), which precludes a reliable characterization of testes of transgenic animals expressing ß-galactosidase fusion proteins.
Leydig cell heterogeneity
Furthermore, our observations regarding the different Leydig cell phenotypes are worth mentioning. We noted that the hypertrophied cells arising first in the form of clusters during the Leydig cell repopulation process resemble typical fetal-type cells, whereas the second-arising spindle-shaped cells are similar to the so-called intermediate-type cells that are generated as early stages of adult Leydig cells. Although all phenotypes derive from (the same) nestin-positive vascular progenitor cells, the fetal-type cells strictly emerge at intertubular and the intermediate-/adult-type cells at peritubular sites. This strongly suggests local environmental effects (Kerr et al., 1987; Teerds, 1996; Gnessi et al., 2000), which are assumed to be responsible also for the observed differences in the extent of Leydig cell regeneration between tubules with either high or low recovery of spermatogenesis.
In conclusion, the conversion of VSMCs into neuroendocrine Leydig cells represents a novel example of transdifferentiation (Echeverri and Tanaka, 2002; Tosh and Slack, 2002) in vivo and demonstrates that blood vessels not only provide inductive signals (Lammert et al., 2001) but also cellular components (progenitor cells) other than the mesoangioblasts, a recently characterized class of vessel-associated stem cells (Sampaolesi et al., 2003), for tissue generation and repair. In the testis, the vascular progenitors identified probably contribute to regulation of Leydig cell number during aging and under conditions of Leydig cell hyperplasia/tumorigenesis.
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Materials and methods |
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EDS treatment and proliferation assays
Adult (3-mo-old, 340380 g) male Wistar rats were housed three per cage under standard laboratory conditions. To induce Leydig cell depletion, animals received single i.p. injections (75 mg EDS/kg body weight, dissolved in 25% dimethylsulfoxide). The rats were killed 148 d after EDS injection. For assessment of mitotic cells, EDS-treated animals received i.p. injections of BrdU (150 mg/kg body weight, dissolved in 0.09 M NaOH) 2 h before killing. Tissues were dissected immediately after decapitation of the animals and were either frozen in liquid nitrogen or fixed in Bouin's fluid. The rats were used according to government principles regarding the care and use of animals with permission (G8151/591-00.33) of the local regulatory authority.
Mouse strains
The generation of nestin-GFP transgenic mice, expressing GFP under the control of the nestin gene promoter and a transcriptional enhancer that resides in the second intron of the gene, has been described previously (Mignone et al., 2004). To address certain questions regarding a potential neural crest origin of Leydig cells, we used mice with targeted mutations in genes of tyrosine kinase receptors of the ErbB family (Britsch et al., 1998). The mutants examined either express ErbB2/ß-galactosidase fusion proteins or represent ErbB3 knockouts.
Immunohistochemical and fluorescence analyses
Immunohistochemistry, based on peroxidase activity, and specificity controls on paraffin sections were performed essentially as described previously (Davidoff et al., 1993, 1999, 2002) using commercially available primary antibodies: anti-CytP450 (working dilution 1:300), antirat (MAB 353, 1:500), antihuman (AB 5922, 1:300) nestin, rabbit anti-NG2 (NG2 chondroitin sulfate proteoglycan; 1:300), and anti-MOSP (mouse anti-myelin/oligodendrocyte-specific protein 1:2,000) from CHEMICON International; anti-NF-H (1:200), anti-GFAP (1:1,000), anti-SMA (1:400), anti-GAP43 (1:1,000), anti-GDNF (2 µg/ml), and anti-N-CAM (1:200) from Sigma-Aldrich; anti-TrkA (1:100) as well as anti-PDGFR-ß (P-20; 1:50) from Santa Cruz Biotechnology, Inc.; rat antimouse CD34 (1:100) from BD Biosciences; and mouse antihuman CD34 (1:100) from DakoCytomation.
In case of double staining, the second antigen was detected by alkaline phosphatase activity. We used substrate staining (rather than immunofluorescence) deliberately in these experiments to clearly identify and examine transdifferentiating cells in their normal tissue context (i.e., together with all neighboring cells). For visualization of BrdU, sections were pretreated with 1 M HCl in PBS/0.3% Triton X-100 for 30 min at 37°C followed by 30 min with PBS/2% normal rabbit serum before incubation with anti-BrdU (clone BMC 9318, diluted 1:20 in PBS containing BSA and sodium azide; Roche) for 1824 h at 4°C.
For detection of GFP fluorescence, adult nestin-GFP transgenic male mice were transcardially perfused with 4% PFA in PBS. After additional immersion fixation for 4 h at RT and impregnation with 30% sucrose-PB, the testes were frozen in liquid nitrogen, and cryostat sections (8, 10, and 20 µm) were mounted onto histological slides. After drying for 30 min at RT, the sections were coverslipped with DakoCytomation fluorescent mounting medium. Cell nuclei of certain sections were additionally stained with DAPI.
Immunoblot analyses
Soluble and particulate fractions of tissue homogenates, prepared as described previously (Middendorff et al., 2002), were size fractionated by SDS-PAGE under reducing conditions in 6% (for detection of nestin and NF-H) or 9% (CytP450, GFAP) acrylamide gels and transferred to nitrocellulose membranes. In the immunoblots shown, equal amounts (80 µg protein) of either soluble (nestin, NF-H) or particulate (CytP450, GFAP) fractions were applied, and rat brain proteins were coanalyzed to serve as positive controls in case of the neural antigens examined. The membranes were probed with antibodies against nestin (MAB 353, diluted 1:1,000; CHEMICON International), CytP450 (AB 1244, 1:1,000; CHEMICON International), NF-H (1:750; Biotrend), or GFAP (1:450; Sigma-Aldrich). After incubations with peroxidase-linked antimouse (1:2,000; Pierce Chemical Co., Perbio Science) or antirabbit (1:2,000; Sigma-Aldrich) secondary antibodies, signals were detected using ECL (Amersham Biosciences) on x-ray films.
Photographic documentation
Photographic documentation was made using an Axioskop microscope (Carl Zeiss MicroImaging, Inc.) equipped with the analogous 36 x 24-mm microscopic camera (MC-100; Carl Zeiss MicroImaging, Inc.) and a 9 x 12-cm Polaroid camera. The photographic negatives (black and white) and slides (colored) were subsequently scanned and additionally processed using Adobe Photoshop 7.0 and Adobe Illustrator 10 (Adobe Systems, Inc.).
GFP fluorescence was viewed using a fluorescence microscope (Axioskop-2; Carl Zeiss MicroImaging, Inc.) equipped with appropriate filter combinations. Images were taken with a digital camera (AxioCam HRc; Carl Zeiss MicroImaging, Inc.) and stored at a size of 1300 x 1030 pixels. Images were assembled with Adobe Photoshop and Adobe Illustrator programs (see above).
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Acknowledgments |
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This work was supported by the Deutsche Forschungsgemeinschaft (DA 459/1-1; MI 637/1-1) and Bundesministerium für Bildung und Forschung (01 KY 9103/0 to D. Müller.). The authors declare that they have no competing financial interests.
Submitted: 17 September 2004
Accepted: 20 October 2004
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