Histochem Cell Biol DOI 10.1007/s00418-012-0991-7
ORIGINAL PAPER
Fibroblast growth factor receptor 3 is highly expressed in rarely dividing human type A spermatogonia Kathrein von Kopylow • Hannah Staege • Wolfgang Schulze • Hans Will • Christiane Kirchhoff
Accepted: 10 June 2012 Ó Springer-Verlag 2012
Abstract Human spermatogonia (Spg) and their fetal precursors express fibroblast growth factor receptor 3 (FGFR3). To further elucidate the role of FGFR3 in the control of Spg self-renewal, proliferation, and/or differentiation, and to narrow down the FGFR3-positive cell type(s) in the normal adult human testis, tissue sections and whole mount preparations of seminiferous tubules were analyzed combining immunofluorescence and confocal fluorescence microscopy. FGFR3 protein was chiefly observed in cellular membranes and cytoplasmic vesicles of a subpopulation of type A Spg, which comprised the chromatin rarefaction zone-containing type Adark. Cytoplasmic expression of FGFR3 and nuclear expression of proliferation-associated antigen KI-67 were mutually exclusive. Similarly, FGFR3positive Spg were negative for Doublesex and Mab-3 related transcription factor 1 (DMRT1). By contrast, undifferentiated embryonic cell transcription factor 1 (UTF1) and survival time-associated PHD finger in ovarian cancer 1 protein (SPOC1) were co-expressed in the nuclei of FGFR3-positive Spg. Whole mounted seminiferous tubules illustrated the clonogenic arrangement of the FGFR3/UTF1 double-positive Spg, which mainly occurred as pairs or quadruplets and, different from the KIT-positive Spg, showed no overlap with KI-67 labeled clusters. Taken together, in the adult human testis, FGFR3 expression is a feature of small clones of rarely dividing type A Spg which resemble ‘‘undifferentiated’’ Spg, including the spermatogonial stem cells. K. von Kopylow W. Schulze C. Kirchhoff (&) Department of Andrology, University Hospital Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany e-mail:
[email protected] H. Staege H. Will Heinrich-Pette-Institut, Leibniz-Institut fu¨r Experimentelle Virologie, Hamburg, Germany
Keywords FGFR3 UTF1 Human Spermatogonia Whole mount
Introduction Fibroblast growth factors (FGFs) control pluripotency and lineage specification in various stem cell systems (for review, see Lanner and Rossant 2010). They also seem to have a role in the regulation of spermatogonial proliferation in vivo (Mayerhofer et al. 1991; Steger et al. 1998a; Gonzalez-Herrera et al. 2006) and in vitro (Kubota et al. 2004; Kanatsu-Shinohara et al. 2005; Aponte et al. 2008; Wu et al. 2009; Ebata et al. 2011). Further, primordial germ cells of the mouse are reprogrammed in vitro by FGF-2 to form pluripotent cells (Durcova-Hills et al. 2006). FGF effects are mediated through high-affinity receptor tyrosine kinases, FGFRs 1–4, which have different binding affinities for each of the ligands (for review, see Cotton et al. 2008). Immunolocalization studies in the human testis suggested that the autocrine and paracrine regulation of Spg occurred via these receptors (Steger et al. 1998a). Supporting evidence comes from the observation of a strong ‘paternal age effect’ of activating mutations in genes of the growth factor receptor pathway, including FGFR3, where initially rare mutations become enriched over time because of a selective advantage (Goriely et al. 2009). Apparently, in the pathological situation, Spg in which the FGFR3 mutation originally arose are positively selected for and expand clonally within the testis, leading to a relative enrichment of mutant sperm (for review, see Goriely and Wilkie 2010, 2012). More recently, FGFR3 was shown to be specifically expressed by human Spg and their fetal precursors (Juul et al. 2007; Goriely et al. 2009; von Kopylow et al. 2010,
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2012). The identification of a whole regulatory network for FGFR3 (von Kopylow et al. 2010) suggests a central role for this receptor. To further elucidate its role, we performed co-expression studies with various markers, i.e., C23/nucleolin, the proliferation-related antigen KI-67, DMRT1, UTF1 and SPOC1 (PHF13). The C23 antibody was employed here to distinguish the peripherally and centrally localized nucleoli of human type A and type B Spg, respectively (Hartung et al. 1990). Ki-67 is strictly associated with cell proliferation (for review, see Scholzen and Gerdes 2000; Beresford et al. 2006). Cattoretti et al. (1992) described the monoclonal antibody MIB-1, which after antigen retrieval, recognized the KI-67 nuclear antigen also in paraffin-embedded human testis (Davidoff et al. 1993; Steger et al. 1998b). DMRT1 is a bifunctional transcriptional regulator of male differentiation expressed in both germ cells and Sertoli cells (Lei et al. 2007, 2009; von Kopylow et al. 2012). Conditional gene targeting demonstrated its importance for the development of c-Kit-positive, i.e., ‘‘differentiated’’ Spg, promoting differentiation-associated mitosis rather than meiosis (Matson et al. 2010; Matson and Zarkower 2012). Undifferentiated embryonic cell transcription factor 1, first described as a pluripotency associated protein in human embryonic stem cells (Okuda et al. 1998), is a candidate gene in the regulation of spermatogonial stem cell (SSC) pluripotency (Guan et al. 2006). In rat testis, Utf1 expression is restricted to a subpopulation of ‘‘undifferentiated’’ type A Spg (van Bragt et al. 2008). The human homolog is expressed in fetal germ cells and testicular germ cell cancer, and also in normal adult Spg (Kristensen et al. 2008; von Kopylow et al. 2010; Wang et al. 2010) where it shows only marginal overlap with DMRT1 and KIT (von Kopylow et al. 2012). SPOC1 is also a marker of spermatogonial nuclei (Kinkley et al.
Fig. 1 FGFR3-related immunofluorescence in Spg of normal adult c human testis. a Cell surface and cytoplasmic vesicles of Spg without (left panel) and with chromatin rarefaction zone (right panel, arrow) are immunopositive for FGFR3; chromatin was labeled by SPOC1 antibody. Dashed lines mark positions of basement membrane. b pAb FGFR3 antibody shows prevailing cytoplasmic pattern (upper panel) and occasionally a weak nuclear staining (lower panel). c Different FGFR3 antibodies yield congruent staining patterns. Single confocal channels (left and center), merged with DNA binding dye DRAQ5 (right). d FGFR3/C23 nucleolin co-immunofluorescence. Overview of DRAQ5-counterstained tubular cross section (left) shows FGFR3 immunofluorescence in Spg with peripheral nucleoli. Apale and Adark Spg, Sertoli cell nuclei (showing large central nucleolus) and lamina propria are marked. M = mitotic figure. Right upper panel (FGFR3/ C23 overlay) and right lower panel (C23/DRAQ5 overlay) show enlargement of boxed area with two unequivocally recognized FGFR3-positive type A Spg. Arrowheads (right panel below) highlight the nucleoli; dashed lines mark the position of the basement membrane
2009; Bo¨rdlein et al. 2011; von Kopylow et al. 2012). In the mouse, it is co-expressed with PLZF and is a prerequisite for sustained spermatogenesis and normal male fertility (Bo¨rdlein et al. 2011). Combining these markers, we show that the FGFR3-positive cells of the normal adult human testis represent a subpopulation of KI-67- and DMRT1-negative, but UTF1- and SPOC1-positive type A Spg which are organized in small cohorts.
Materials and methods Patients and testicular biopsies Human testis tissue was obtained from patients presenting at the Department of Andrology, University Hospital Hamburg-Eppendorf, Germany, between August 2008 and December 2011. The Ethic Committee Approval (OB/X/
Table 1 Primary antibodies and control sera Antigen
Company
Code
Species
Clonality
Dilution
C23
Santa Cruz Biotechnology, USA
sc-8031
Mouse
Monoclonal
1:100
References
c-Kit
Dako, Germany
A4502
Rabbit
Polyclonal
1:200
DMRT1
Atlas Antibodies AB, Sweden
HPA027850
Rabbit
Polyclonal
1:1200
Looijenga et al. (2006)
FGFR3
Santa Cruz Biotechnology, USA
sc-13121
Mouse
Monoclonal
1:5/1:10
Juul et al. (2007); von Kopylow et al. (2010) von Kopylow et al. (2012)
FGFR3
Cell Signaling Technology, USA
#4574
Rabbit
Monoclonal
1:100
FGFR3
Abcam, UK
ab10649
Rabbit
Polyclonal
1:100
Schrans-Stassen et al. (1999)
Ki-67 (Clone MIB-1)
Dako, Germany
M7240
Mouse
Monoclonal
1:10
Cattoretti et al. (1992)
PGP9.5
Dako, Germany
Z5116
Rabbit
Polyclonal
1:500
Positive control
Rabbit Ig Fraction
Dako, Germany
X0936
Rabbit
n.a.
1:500
Negative control
SPOC1
Dr. Elisabeth Kremmer, Munich
n.a.
Rat
Monoclonal
Undiluted to 1:10
Kinkley et al. (2009); Bo¨rdlein et al. (2011)
UTF1
Chemicon, USA
MAB4337
Mouse
Monoclonal
1:200
Kristensen et al. (2008)
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Histochem Cell Biol
FGFR3 SPOC1
sc-13121 (mAb mouse)
ab10649
10µm
10µm a
FGFR3 DRAQ5
b
sc-13121 (mAb mouse)
ab10649 (pAb rabbit)
FGFR3 FGFR3 DRAQ5
10µm
c
FGFR3 (#4574 mAb rabbit) DRAQ5 C23
Apale
Adark
A
Sertoli cell nuclei
M
Lamina propria
50µm
10µm
d 2000 and WF-007/11) and complete written informed consent of all persons were obtained prior to their inclusion in the study, and the study was conducted in accordance
with the ethical principles laid down in the 1964 Declaration of Helsinki and its later amendments. Tissue samples were taken simultaneously at surgery for therapeutic
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Histochem Cell Biol
FGFR3 KI-67 DRAQ5
a
b
Nuclei of peritubular cells
Spermatogonia
50µm
25µm c
d
Mitotic figures
Interstitium
e
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25µm f
Histochem Cell Biol b Fig. 2 FGFR3/KI-67 co-immunofluorescence in tissue sections of normal adult testis. a–c Overviews of DRAQ5 counterstained sections reveal cytoplasmic FGFR3 and KI-67-specific MIB-1 nuclear staining related to different Spg subtypes. Spg with cytoplasmic FGFR3 reside immediately at the basement membrane. Multiple nuclear staining patterns in FGFR3-negative Spg are seen with the KI-67 antibody (a, arrows). d Enlargement of the boxed area in c includes a typical example of alternating FGFR3-positive and KI-67-positive Spg, i.e., Spg with FGFR3-positive cytoplasm containing a flattened KI-67negative nucleus, and round KI-67-positive nuclei with dense clumps of stained material in FGFR3-negative cytoplasm. e, f FGFR3 antibody labels membranes and cytoplasmic vesicles of KI-67negative Spg, while KI-67 antibody labels nucleoli and mitotic figures of nuclei in FGFR3-negative Spg. Cluster of mitotic figures (encircled) is seen on the right. Dashed lines mark the positions of basement membrane
1:300; Invitrogen) in combination with DNA stain DRAQ5 (Alexis Biochemicals, San Diego, USA). The specificity of immunostainings was additionally confirmed by omission of primary antibodies and staining of parallel sections with antibodies directed against an irrelevant antigen. The staining patterns described below were only observed when primary and secondary antibodies were used together, and were absent when negative control sera were employed. In addition, potential cross-reactivity of secondary antibodies with primary antibodies from different species was excluded by appropriate staining experiments.
testicular sperm extraction (TESE) and diagnostic purposes as previously described (Schulze et al. 1999). For this study, tissue samples from a total of 14 normogonadotropic patients, which showed full spermatogenesis in the entire biopsy and the presence of a normal inter-tubular tissue, were used (compare McLachlan et al. 2007).
Five tissue samples obtained at surgery were placed in 1 ml of pre-warmed DMEM/F-12 and digested at 32 °C with collagenase D (1 mg/ml; Roche Diagnostics GmbH, Mannheim, Germany). Digestion was conducted for 60–90 min under visual inspection using a microscope to obtain isolated seminiferous tubules. Suitable fragments of isolated tubules were fixed in mDF for 4 h at 4 °C. Subsequently, whole mounts of tubules were prepared following the protocol of Ehmcke and Schlatt (2008).
Tissue fixation, embedding and sectioning for immunofluorescence For tissue sections, small fragments of nine different testes were immediately immersed in modified Davidson’s fluid, which was shown to optimally preserve the morphology of the adult testis (Latendresse et al. 2002; von Kopylow et al. 2010, 2012). The fixative volume was at least 109 tissue volume; fixation time was 6–8 h. Paraffin embedding was performed using a Thermo Fisher Scientific Shandon Excelsior tissue processor (Thermo Scientific, Karlsruhe, Germany); sectioning (5 lm) was performed on a Leica RM2255 automated rotary microtome (Leica Microsystems, Bensheim, Germany). Immunofluorescence staining Deparaffinization of tissue sections, antigen retrieval and blocking of unspecific binding sites were performed using standard procedures as described (von Kopylow et al. 2010, 2012). Primary antibodies (mouse monoclonal, rat monoclonal, rabbit monoclonal and rabbit polyclonal) and their optimum dilutions are listed in Table 1. Each primary antibody was tested on deparaffinized and rehydrated tissue sections or on whole mounted tubules (see next chapter) from at least three different patients. Rabbit polyclonal PGP9.5 (Z5116, Dako, Hamburg, Germany) served as positive control and rabbit immunoglobulin fraction (X0936 Dako, Hamburg, Germany) as a negative control. Primary antibodies were applied overnight and detected employing the appropriate combination of Alexa Fluor 488, 555 and 647 secondary antibodies (dilution 1:200 or
Whole mount preparations
Confocal laser scanning fluorescence microscopy Tissue sections and whole mounted segments of tubules were evaluated on a Zeiss Confocor 2 confocal scanning system based on the Axiovert 200 M inverted microscope, using the LSM/FCS software (Carl Zeiss, Jena, Germany). Visualization of green fluorophore (Alexa Fluor 488) was achieved by using an Argon/2 laser (LASOS Lasertechnik GmbH) of red fluorophore (Alexa Fluor 555) by a helium/ neon 1 laser (543 nm; LASOS) and of blue fluorophore (Alexa Fluor 647 and DNA dye DRAQ5) by a helium/neon 2 laser (633 nm; LASOS). The pinhole for each channel and the respective objective was set at 1 airy unit for captured confocal images. For controls, identical photomultiplier and pinhole settings were used. Confocal images were captured with a Zeiss Axiocam digital camera. Signals described below were confirmed not to be due to bleed-through between channels.
Results FGFR3 is expressed in type A spermatogonia Validating and extending upon earlier studies, three FGFR3 antibody preparations were tested, directed against different epitopes (compare Table 1), i.e., two monoclonal antibody preparations generated against the N-terminal and C-terminal parts of the receptor protein, respectively
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Histochem Cell Biol
FGFR3 DMRT1 SPOC1
c
Sertoli cell nuclei
a
d e
50µm
10µm b
Fig. 3 Triple immunofluorescence of FGFR3, DMRT1 and SPOC1 in sections of normal adult testis. a, b Overviews of tubular cross sections show small groups of Spg with robust cytoplasmic FGFR3 expression (green). Nuclei of these cells are invariably negative for DMRT1 and are only stained by SPOC1 (red). Simultaneous staining of nuclei by DMRT1 (blue) and SPOC1 (red) resulted in a granular or
speckled magenta color. Weak DMRT1 staining of Sertoli cell nuclei is indicated. Boxed areas of a and b are enlarged in c and d, respectively. c–e Image details emphasize alternate cytoplasmic FGFR3 and DMRT1 nuclear staining of Spg, the SPOC1-positive Spg exhibiting mutually exclusive expression of these markers
(compare von Kopylow et al. 2012), and a polyclonal serum raised against a 14-mer peptide of the extreme C-terminus. FGFR3-related immunoreactivity was restricted to germ cells residing immediately adjacent to the basement membrane of the seminiferous tubules (Fig. 1),
i.e., Spg. DNA counterstaining by DRAQ5 or SPOC1 nuclear staining confirmed that the FGFR3-positive Spg comprised the classically described Adark subtype with a clearly recognizable nuclear rarefaction zone (compare also with von Kopylow et al. 2012). Regarding the
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subcellular distribution of FGFR3, the antibodies produced largely superimposed patterns, a considerable proportion of the receptor protein residing within cytoplasmic vesicles. In addition to the prevailing cytoplasmic pattern, the polyclonal antiserum induced a weak nuclear staining (Fig. 1b, lower panel). To further characterize the FGFR3-expressing cell type by the position of the nucleoli, the C23 mAb was employed in combination with the FGFR3 rabbit mAb. Strongly FGFR3-positive Spg with many cytoplasmic vesicles in which the nucleoli could be discerned typically had ovoid nuclei with a homogenous, fine-granular chromatin and one or more distinct C23-positive nucleoli attached at the nuclear periphery (Fig. 1d), well-known characteristics of human type A Spg. By comparison, the nuclei of overtly FGFR3-negative germ cells were less uniform in their nuclear appearance. Frequently, they showed characteristics of type B Spg and/or (preleptotene) spermatocytes, i.e., round nuclei with aggregates of chromatin and C23-stained nucleoli away from the nuclear membrane (not shown). FGFR3 and KI-67 are expressed in non-overlapping Spg subpopulations Fibroblast growth factor receptor 3 (mouse mAb) and KI-67 antibodies were combined to monitor the FGFR3positive and proliferating cell fractions within the same tissue sections (Fig. 2). Different from FGFR3, KI-67related immunoreactivity was located in the nuclei of Spg. Within the nuclei, KI-67 appeared to be associated with multiple structures, showing either speckled staining or prominent foci which partially corresponded to the nucleoli (Fig. 2c–e). FGFR3 and KI-67 immunofluorescence not only affected different cellular compartments, but also different cell populations. Apparently, the FGFR3-positive and KI-67-positive cells represented non-overlapping Spg subpopulations, which differed in their morphology. Regarding the FGFR3-positive Spg, contact to the basement membrane was immediate and broad, and their DRAQ5-counterstained nuclei often appeared ovoid or flattened. Compared to the latter, Spg with KI-67-positive nuclei were often positioned more centrally within the seminiferous tubules, and the nuclei were round (Fig. 2d, e), characteristics of type B Spg. The distribution on the basement membrane of FGFR3and KI-67-expressing Spg subpopulations was irregular, possibly indicative of a low degree of spermatogonial synchronization in a given tubular segment. Only rarely the KI-67-positive nuclei in a tubular segment appeared synchronized, supported by the occurrence of clustered mitotic figures (Fig. 2e, f) where most of the KI-67 protein seemed to be attached to the surface of the condensed chromosomes (Fig. 2e). From their positions within the seminiferous
epithelium, their rounded nuclei and the centrally located KI-67-stained foci, it was assumed that a considerable proportion of the KI-67-positive cells belonged to the B subtype of human Spg. Still, KI-67-positive nuclei of different nuclear appearance were occasionally seen (Fig. 2a, arrows). Weak KI-67-related staining was observed also during the later stages of spermatogenesis, up to the earliest spermatid stage (early round spermatids; compare Fig. 2), corresponding to an earlier report in mice (Traut et al. 2002). DMRT1 and FGFR3 expressions show an inverse relationship In the following triple immunofluorescence study, FGFR3 (mouse mAb) was combined with DMRT1 and SPOC1 antibodies. FGFR3 immunoreactivity was predominantly cytoplasmic (see above), whereas both DMRT1 and SPOC1 were chromatin associated (Fig. 3). While DMRT1 showed homogenous chromatin staining, SPOC1 nuclear staining often showed a fine granular or speckled appearance, which seemed not to be associated with the nucleoli. In addition to the nuclei of Spg, DMRT1 also labeled the nuclei of Sertoli cells (Fig. 3; compare von Kopylow et al. 2012). SPOC1, in comparison, was only seen in the nuclei of Spg and was introduced here to unambiguously discern these cells. Similar to the above-described FGFR3/KI-67 staining pattern, nuclear DMRT1 and cytoplasmic FGFR3 staining showed an inverse relationship, i.e., the Spg subfraction with DMRT1-positive nuclei was consistently negative for FGFR3. Conversely, all robustly FGFR3positive Spg had DMRT1-negative nuclei (Fig. 3). In summary, while the nuclei of Spg were distinct from the Sertoli cell nuclei by their SPOC1 immnunoreactivity, DMRT1 and FGFR3 represented markers of non-overlapping subpopulations of Spg, comparable to the abovedescribed mutually exclusive expression of FGFR3 and KI-67. FGFR3-positive Spg are a subpopulation of UTF1-expressing cells To further characterize the FGFR3-expressing Spg, triple immunofluorescence was performed combining FGFR3 (rabbit mAb) with UTF1 and SPOC1. FGFR3 cytoplasmic staining was associated with UTF1/SPOC1 double-stained nuclei (Fig. 4), the most robustly FGFR3-expressing Spg characterized by an intense UTF1 nuclear staining which exceeded their SPOC1 nuclear staining. In comparison, Spg, which lacked UTF1 or showed only weak UTF1 staining as compared to SPOC1, were negative for FGFR3 (compare single channel images of Fig. 4). A closer examination of the nuclear morphology confirmed that the FGFR3-positive Spg comprised nuclei with and without
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Histochem Cell Biol
FGFR3 UTF1 SPOC1
50µm
25µm
a
b
FGFR3 UTF1 SPOC1
Interstitium
c
25µm
chromatin rarefaction zones. This was substantiated by fluorescent DNA staining using DRAQ5 (data not shown). Occasionally, peripheral nucleoli became apparent as an
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unstained area, again indicating that the robustly FGFR3expressing Spg belonged to the A type. This confirmed and extended our earlier findings that FGFR3 and UTF1 were
Histochem Cell Biol b Fig. 4 Triple immunofluorescence of FGFR3, UTF1 and SPOC1 in sections of normal adult testis. Overlays show confocal images of tubular cross section (a) and of planar section (b). Lower panels show separate channels of confocal images. FGFR3, blue; UTF1, red; SPOC1, green. Strong nuclear co-staining of UTF1 and SPOC1 goes along with cytoplasmic FGFR3 staining, while SPOC1-only nuclei are negative for cytoplasmic FGFR3 (arrows). c Details of Spg with varying nuclear staining intensities of UTF1 and SPOC1 reveal that strong UTF1 staining compared to SPOC1 staining goes along with cytoplasmic FGFR3. Spg with chromatin rarefaction zones are among these cells. Arrow points at SPG with SPOC1-positive, but UTF1negative nucleus
expressed by both, human type Apale and Adark Spg. By comparison, no chromatin rarefaction zone or peripheral nucleoli were ever discerned in the nuclei of SPOC1positive, but FGFR3- and UTF1-negative Spg. FGFR3-positive Spg are organized in small KI-67-negative cell clusters To reveal the spatial organization of the FGFR3/UTF1positive Spg, we performed immunofluorescence on whole mounted segments of human seminiferous tubules. In conjunction with DRAQ5 staining and confocal microscopy, the topographical arrangements of interconnected Spg could be visualized (Fig. 5). Congruent to the results on tissue sections (see above), the robustly FGFR3-positive cells represented a subpopulation of the UTF1-positive Spg. Chromatin rarefaction zones were regularly discerned, confirming that the FGFR3-positive Spg comprised the Adark subtype (see above). Accumulation in vesicles of much of the FGFR3 protein rendered the position of cytoplasmic bridges blurred. Still, in selected tubular segments, groups of UTF1-positive nuclei, with internuclear distances of less than 25 lm, which were connected by FGFR3-stained bridges, became apparent suggesting that a majority of the FGFR3/UTF1-positive clusters consisted of pairs or quadruplets of Spg (Fig. 5a, b); larger doublepositive clusters were rare (not shown). At the same time, neighboring unstained clusters, which were negative for both markers and which often comprised cohorts of eight or more aligned Spg, became apparent (compare Fig. 5b, right panel). In certain optical sections, isolated FGFR3/ UTF1-positive Spg were occasionally observed (Fig. 5, arrows). However, interconnected cells seemed to exist in a different layer of the Z-stack suggesting that they may not represent single cells. Different from the FGFR3/UTF1 labelings, FGFR3/KI67 double immunofluorescence on whole mounted seminiferous tubules confirmed that these markers stained separate subpopulations of Spg (Fig. 5c). Robustly FGFR3positive Spg always contained KI-67-negative nuclei, while in adjacent regions of the same tubular segments
FGFR3-negative cells with strongly KI-67-positive nuclei were observed. In parallel experiments, KIT/KI-67 double immunofluorescence on whole mounted seminiferous tubules revealed broad co-staining of both markers, i.e., Spg which were positive for the receptor tyrosine kinase KIT had KI-67-positive nuclei in their vast majority (not shown).
Discussion The present study characterized the FGFR3-expressing cells of the adult human testis as a subpopulation of UTF1/ SPOC1-positive, but KI-67 and DMRT1-negative type A Spg. The FGFR3-expressing Spg comprised the classical Adark subtype with visible chromatin rarefaction zones (compare von Kopylow et al. 2012) which divide only rarely. FGFR3-related immunostaining was predominantly seen in the cytoplasm of Spg, in part located on the cell surface, but largely accumulating in cytoplasmic vesicles. Application of three different antibodies produced superimposed results (compare also with von Kopylow et al. 2012) arguing for the specificity of the observed staining pattern. A weak nuclear staining occasionally seen with the polyclonal serum was reminiscent of the results described by an earlier study, which located FGFR3 to the nuclei of Spg (Steger et al. 1998a). Growth factor receptors like FGFR3 are generally thought to carry out their role in signal transduction at the cell surface. Still, similar to other receptor tyrosine kinases (RTKs; for review, see Schlessinger and Lemmon 2006), FGFR3 has recently been reported to undergo regulated intra-membrane proteolysis in response to activation at the cell surface (Degnin et al. 2011). Unlike the other RTKs, however, ligand-induced cleavage of FGFR3 requires endocytosis of the intact receptor protein to an endosomal compartment where a two-step cleavage takes place, finally generating a soluble C-terminal fragment that can traffic to the nucleus (Degnin et al. 2011). Different from other markers, the expression of KI-67 is strictly associated with cell proliferation (for review, see Scholzen and Gerdes 2000; Beresford et al. 2006), immunostaining techniques yielding labeling indices comparable to nucleotide incorporation (Muskhelishvili et al. 2003). The protein is present during the S-, G2-, M- and late G1phases of the cell cycle (Gerdes et al. 1984; Verheijen et al. 1989a, b), but is absent during G0 and most of G1. As described, KI-67 appeared to be associated with multiple nuclear structures, probably depending on cell cycle stages (for review, see Scholzen and Gerdes 2000), showing either speckled staining or prominent foci which partially corresponded to the nucleoli (compare Kill 1996). From the overtly KI-67-negative phenotype of the FGFR3-positive
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Histochem Cell Biol
FGFR3
FGFR3 UTF1
FGFR3
FGFR3 UTF1
a
* *
*
* * * * *
b
c
123
FGFR3 KI-67
DRAQ5 KI-67
100µm
Histochem Cell Biol b Fig. 5 Double Immunofluorescence of whole mounted human seminiferous tubules. a, b FGFR3/UTF1 co-immunofluorescence. Confocal images of different segments show cytoplasmic FGFR3 immunofluorescence (green), delineating cellular boundaries of Spg clusters (left panels). A majority of the FGFR3-positive Spg occurs as pairs and quadruplets as indicated by dashed lines. In the corresponding overlays (right panels), UTF1 (red) labels the nuclei of Spg confirming that FGFR3-positive Spg (green) represent a subpopulation of UTF1-positive Spg. Arrows point at apparently single FGFR3/ UTF1-positive Spg. In the right panel of b, a chain of eight unlabeled cells is highlighted by asterisks. c FGFR3/KI-67 co-immunofluorescence, confocal images showing mutually exclusive labeling of Spg. Corresponding nuclei are seen in right panel showing KI-67/DRAQ5 nuclear counterstaining. Note a quadruplet of FGFR3-positive and KI-67-negative Spg (dashed line) adjacent to the groups of KI-67 labeled nuclei
Spg, it may be concluded that they were in G0 or G1, i.e., quiescent or at least long cycling. This applies specifically to the Adark subtype which was regarded as quiescent or rarely dividing on the basis of a low [3H] thymidine labeling index (Chowdhury and Steinberger 1973). Indeed, the nuclei of human Spg, in which chromatin rarefaction zones were discerned, were consistently negative for KI-67 (compare also with von Kopylow et al. 2012). Similarly, the ‘‘undifferentiated’’ type A Spg of the rodent testis show a much longer cell cycle than the other types of Spg (Huckins 1971). It could be that FGFR3 expression restrained the proliferation of human type A Spg, and the switch from a quiescent to a proliferating status was accompanied by loss of the receptor protein. This speculation is consistent with the anti-proliferative effects associated with FGFR3 expression in other cellular contexts (for review, see Dailey et al. 2005). Likewise, FGFR3 and DMRT1 expression in human Spg seemed mutually exclusive. In mice, Dmrt1 promotes differentiation-associated mitosis of Spg and controls the mitosis versus meiosis decision; loss of the Dmrt1 gene caused Spg to precociously enter meiosis (Matson et al. 2010). A comparable role may be assumed in humans where DMRT1 gene mutations are implicated in male infertility, testicular dysgenesis and spermatocytic seminomas (Ottolenghi and McElreavey 2000; Looijenga et al. 2006; Krentz et al. 2009; Turnbull et al. 2010). Nuclear DMRT1 expression was previously detected in a subpopulation of human KI-67-positive Spg (von Kopylow et al. 2012) and also in pre-meiotic spermatocytes (Looijenga et al. 2006). Also, Looijenga et al. (2006) presented supportive evidence that DMRT1-positive tumor cells may undergo partial meiosis, followed by re-entry into the mitotic cycle (Looijenga et al. 2006). Thus, DMRT1 could have a role in the mitosis versus meiosis decision also in the human testis. The inverse relationship of FGFR3 expression, on the one hand, and of KI-67 and DMRT1 expression, on the other, may indicate that the FGFR3positive Spg of the human testis do not participate in this
decision. Rather, they may represent a more primitive Spg subtype which divides only rarely, reminiscent of the classically described ‘‘undifferentiated’’ Spg of the rodent testis. A functional antagonism may also be suspected. In the mouse testis, DMRT1 directly represses the pluripotency regulator UTF1, and indirectly activates the glial cell line-derived neurotrophic factor (GDNF) co-receptor RET (Krentz et al. 2009). Cytoplasmic FGFR3 expression was a feature of a subpopulation of UTF1-positive human Spg. UTF1 is a known marker of many stem cell types, including the SSCs (Guan et al. 2006) and also a supporting factor in in vitro pluripotency induction (Zhao et al. 2008). In human embryonic SCs, UTF1 is a direct target gene of POU5F1 (OCT3/4; Nishimoto et al. 2005) and SOX2. In the normal adult human testis, although it does not express POU5F1 and SOX2, UTF1 is strongly expressed in Spg (Kristensen et al. 2008; von Kopylow et al. 2010, 2012). In the rat testis, UTF1 expression is confined to a subpopulation of the ZBTB16 (PLZF)-positive early type A Spg (van Bragt et al. 2008; Mok et al. 2012). Furthermore, the pattern of UTF1-positive cells over the different stages of the cycle in rat seminiferous epithelium suggested that it is restricted to Asingle, Apaired, and short chains of Aaligned Spg, which are in the ‘‘undifferentiated’’ state. In analogy, the pairs and quadruplets of strongly UTF1- and FGFR3-positive Spg in the human testis may also represent ‘‘undifferentiated’’ type A Spg, including the SSCs. The transition from ‘‘undifferentiated’’ to ‘‘differentiated’’ Spg coincides with the gain of the c-Kit receptor. In the mouse, ‘‘undifferentiated’’ type A Spg are negative for c-Kit, while survival and/or proliferation of the ‘‘differentiating’’ type A Spg requires c-Kit expression (Yoshinaga et al. 1991; Schrans-Stassen et al. 1999; Feng et al. 2000). In line with this, human repopulating Spg, including the SSCs, have been reported to be KITneg/low (Izadyar et al. 2011). Unfortunately, co-staining of FGFR3 and KIT was not successful in our study. Indeed, KIT immunostaining of human Spg has proven difficult, and its expression in the normal adult human testis is still controversial (compare Rajpert-De Meyts and Skakkebak 1994; Bokemeyer et al. 1996; Dym et al. 2009; Steiner et al. 2011). We recently observed KIT labeling in the UTF1-negative subfraction of human Spg (von Kopylow et al. 2012). Moreover, KIT protein was largely observed in clusters of KI-67-positive, i.e., proliferating Spg, contrary to FGFR3 which labeled pairs and quadruplets of UTF1-positive but KI-67-negative, i.e., largely quiescent Spg. Frequent co-occurrence of KIT cytoplasmic staining and KI-67 nuclear staining suggested that the KIT-positive cell types were mitotically active progenitor type A and type B Spg, at least in their majority (compare also with von Kopylow et al. 2012). This is different from the pairs and quadruplets of FGFR3/
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UTF1-positive A type Spg which appeared to be quiescent or at least long cycling, characteristics of the SSCs. In the normal adult testis, to support lifelong spermatogenesis, SSCs must undergo regular self-renewal, but their number should neither increase nor decrease. Uncontrolled proliferation may lead to testis cancer, while an unbalanced differentiation will lead to spermatogonial depletion and finally infertility; hence, the decision of proliferation and/or differentiation has to be tightly controlled. While gain-of-function mutations in the FGFR3 gene seem to promote uncontrolled clonal expansion of human Spg (for review, Goriely and Wilkie 2010, 2012), wild-type FGFR3 may be an important factor involved in the control of SSC self-renewal and differentiation. Unequivocal SSC identification and enrichment of these cells has remained difficult, which may be blamed at least in part on the lack of specific surface markers. FGFR3 is a novel candidate surface marker, possibly facilitating the enrichment and isolation of ‘‘undifferentiated’’ human Spg, including the SSCs. Acknowledgments We thank Professor Dr. med. Volker Steinkraus, Dermatologikum Hamburg, for his continued support in histology and Dr. Elisabeth Kremmer, Helmholtz Centre, Munich, for generously providing the SPOC1 antibody. We are grateful to Stefan Ficke and Dr. R. Reimer, Heinrich-Pette-Institut, Hamburg, for technical support. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (contract SCH 587/3-2), and grants from the Deutsche Krebshilfe and the Mu¨ggenburg Stiftung to HW.
References Aponte PM, Soda T, Teerds KJ, Mizrak SC, van de Kant HJ, de Rooij DG (2008) Propagation of bovine spermatogonial stem cells in vitro. Reproduction 136(5):543–557 Beresford MJ, Wilson GD, Makris A (2006) Measuring proliferation in breast cancer: practicalities and applications. Breast Cancer Res 8(6):216–226 Bokemeyer C, Kuczyk MA, Dunn T, Serth J, Hartmann K, Jonasson J, Pietsch T, Jonas U, Schmoll HJ (1996) Expression of stem-cell factor and its receptor c-kit protein in normal testicular tissue and malignant germ-cell tumours. J Cancer Res Clin Oncol 122(5):301–306 Bo¨rdlein A, Scherthan H, Nelkenbrecher C, Molter T, Bo¨sl MR, Dippold C, Birke K, Kinkley S, Staege H, Will H, Winterpacht A (2011) SPOC1 (PHF13) is required for spermatogonial stem cell differentiation and sustained spermatogenesis. J Cell Sci 124(Pt 18):3137–3148 Cattoretti G, Becker MH, Key G, Duchrow M, Schlu¨ter C, Galle J, Gerdes J (1992) Monoclonal antibodies against recombinant parts of the Ki-67 antigen (MIB 1 and MIB 3) detect proliferating cells in microwave-processed formalin-fixed paraffin sections. J Pathol 168(4):357–363 Chowdhury AK, Steinberger E (1973) Radioautography of histologic sections of human testicular tissue. Stain Technol 48(6):305–309 Cotton LM, O’Bryan MK, Hinton BT (2008) Cellular signaling by fibroblast growth factors (FGFs) and their receptors (FGFRs) in male reproduction. Endocr Rev 29(2):193–216
123
Dailey L, Ambrosetti D, Mansukhani A, Basilico C (2005) Mechanisms underlying differential responses to FGF signaling. Cytokine Growth Factor Rev 16(2):233–247 Davidoff MS, Schulze W, Middendorff R, Holstein AF (1993) The Leydig cell of the human testis—a new member of the diffuse neuroendocrine system. Cell Tissue Res 271(3):429–439 Degnin CR, Laederich MB, Horton WA (2011) Ligand activation leads to regulated intramembrane proteolysis of fibroblast growth factor receptor 3. Mol Biol Cell 22(20):3861–3873 Durcova-Hills G, Adams IR, Barton SC, Surani MA, Mc Laren A (2006) The role of exogenous fibroblast growth factor-2 on the reprogramming of primordial germ cells into pluripotent stem cells. Stem Cells 24(6):1441–1449 Dym M, Kokkinaki M, He Z (2009) Spermatogonial stem cells: mouse and human comparisons. Birth Defects Res C Embryo Today 87(1):27–34 Ebata KT, Yeh JR, Zhang X, Nagano MC (2011) Soluble growth factors stimulate spermatogonial stem cell divisions that maintain a stem cell pool and produce progenitors in vitro. Exp Cell Res 317(10):1319–1329 Ehmcke J, Schlatt S (2008) Identification and characterization of spermatogonial subtypes and their expansion in whole mounts and tissue sections from primate testes. Methods Mol Biol 450:109–118 Feng LX, Ravindranath N, Dym M (2000) Stem cell factor/c-kit upregulates cyclin D3 and promotes cell cycle progression via the phosphoinositide 3-kinase/p70 S6 kinase pathway in spermatogonia. J Biol Chem 275(33):25572–25576 Gerdes J, Lemke H, Baisch H, Wacker HH, Schwab U, Stein H (1984) Cell cycle analysis of a cell proliferation-associated human nuclear antigen defined by the monoclonal antibody Ki67. J Immunol 133(4):1710–1715 Gonzalez-Herrera IG, Prado-Lourenco L, Pileur F, Conte C, Morin A, Cabon F, Prats H, Vagner S, Bayard F, Audigier S, Prats AC (2006) Testosterone regulates FGF-2 expression during testis maturation by an IRES-dependent translational mechanism. FASEB J 20(3):476–478 Goriely A, Wilkie AO (2010) Missing heritability: paternal age effect mutations and selfish spermatogonia. Nat Rev Genet 11(8):589 Goriely A, Wilkie AO (2012) Paternal age effect mutations and selfish spermatogonial selection: causes and consequences for human disease. Am J Hum Genet 90(2):175–200 Goriely A, Hansen RM, Taylor IB, Olesen IA, Jacobsen GK, McGowan SJ, Pfeifer SP, McVean GA, Rajpert-De Meyts E, Wilkie AO (2009) Activating mutations in FGFR3 and HRAS reveal a shared genetic origin for congenital disorders and testicular tumors. Nat Genet 41(11):1247–1252 Guan K, Nayernia K, Maier LS, Wagner S, Dressel R, Lee JH, Nolte J, Wolf F, Li M, Engel W, Hasenfuss G (2006) Pluripotency of spermatogonial stem cells from adult mouse testis. Nature 440(7088):1199–1203 Hartung M, Wachtler F, de Lanversin A, Fouet C, Schwarzacher HG, Stahl A (1990) Sequential changes in the nucleoli of human spermatogonia with special reference to rDNA location and transcription. Tissue Cell 22(1):25–37 Huckins C (1971) The spermatogonial stem cell population in adult rats. II. A radioautographic analysis of their cell cycle properties. Cell Tissue Kinet 4(4):313–334 Izadyar F, Wong J, Maki C, Pacchiarotti J, Ramos T, Howerton K, Yuen C, Greilach S, Zhao HH, Chow M, Chow YC, Rao J, Barritt J, Bar-Chama N, Copperman A (2011) Identification and characterization of repopulating spermatogonial stem cells from the adult human testis. Hum Reprod 26:1296–1306 Juul A, Aksglaede L, Lund AM, Duno M, Skakkebaek NE, RajpertDe Meyts E (2007) Preserved fertility in a non-mosaic
Histochem Cell Biol Klinefelter patient with a mutation in the fibroblast growth factor receptor 3 gene: case report. Hum Reprod 22(7):1907–1911 Kanatsu-Shinohara M, Miki H, Inoue K, Ogonuki N, Toyokuni S, Ogura A, Shinohara T (2005) Long-term culture of mouse male germline stem cells under serum-or feeder-free conditions. Biol Reprod 72(4):985–991 Kill IR (1996) Localisation of the Ki-67 antigen within the nucleolus. Evidence for a fibrillarin-deficient region of the dense fibrillar component. J Cell Sci 109(6):1253–1263 Kinkley S, Staege H, Mohrmann G, Rohaly G, Schaub T, Kremmer E, Winterpacht A, Will H (2009) SPOC1: a novel PHD containing protein modulating chromatin structure and mitotic chromosome condensation. J Cell Sci 122(Pt 16):2946–2956 Krentz AD, Murphy MW, Kim S, Cook MS, Capel B, Zhu R, Matin A, Sarver AL, Parker KL, Griswold MD, Looijenga LH, Bardwell VJ, Zarkower D (2009) The DM domain protein DMRT1 is a dose-sensitive regulator of fetal germ cell proliferation and pluripotency. Proc Natl Acad Sci USA 106(52): 22323–22328 Kristensen DM, Nielsen JE, Skakkebaek NE, Graem N, Jacobsen GK, Rajpert-De Meyts E, Leffers H (2008) Presumed pluripotency markers UTF-1 and REX-1 are expressed in human adult testes and germ cell neoplasms. Hum Reprod 23(4):775–782 Kubota H, Avarbock MR, Brinster RL (2004) Growth factors essential for self-renewal and expansion of mouse spermatogonial stem cells. Proc Natl Acad Sci USA 101(47):16489–16494 Lanner F, Rossant J (2010) The role of FGF/Erk signaling in pluripotent cells. Development 137(20):3351–3360 Latendresse JR, Warbrittion AR, Jonassen H, Creasy DM (2002) Fixation of testes and eyes using a modified Davidson’s fluid: comparison with Bouin’s fluid and conventional Davidson’s fluid. Toxicol Pathol 30(4):524–533 Lei N, Hornbaker KI, Rice DA, Karpova T, Agbor VA, Heckert LL (2007) Sex-specific differences in mouse DMRT1 expression are both cell type- and stage-dependent during gonad development. Biol Reprod 77(3):466–475 Lei N, Karpova T, Hornbaker KI, Rice DA, Heckert LL (2009) Distinct transcriptional mechanisms direct expression of the rat Dmrt1 promoter in Sertoli cells and germ cells of transgenic mice. Biol Reprod 81(1):118–125 Looijenga LH, Hersmus R, Gillis AJ, Pfundt R, Stoop HJ, van Gurp RJ, Veltman J, Beverloo HB, van Drunen E, van Kessel AG, Pera RR, Schneider DT, Summersgill B, Shipley J, McIntyre A, van der Spek P, Schoenmakers E, Oosterhuis JW (2006) Genomic and expression profiling of human spermatocytic seminomas: primary spermatocyte as tumorigenic precursor and DMRT1 as candidate chromosome 9 gene. Cancer Res 66(1):290–302 Matson CK, Zarkower D (2012) Sex and the singular DM domain: insights into sexual regulation, evolution and plasticity. Nat Rev Genet 13(3):163–174 Matson CK, Murphy MW, Griswold MD, Yoshida S, Bardwell VJ, Zarkower D (2010) The mammalian doublesex homolog DMRT1 is a transcriptional gatekeeper that controls the mitosis versus meiosis decision in male germ cells. Dev Cell 19(4):612– 624 Mayerhofer A, Russell LD, Grothe C, Rudolf M, Gratzl M (1991) Presence and localization of a 30-kDa basic fibroblast growth factor-like protein in rodent testes. Endocrinology 129(2):921– 924 McLachlan RI, Rajpert-De Meyts E, Hoei-Hansen CE, de Kretser DM, Skakkebaek NE (2007) Histological evaluation of the human testis–approaches to optimizing the clinical value of the assessment: mini review. Hum Reprod 22(1):2–16 Mok KW, Mruk DD, Lee WM, Cheng CY (2012) Spermatogonial stem cells alone are not sufficient to re-initiate spermatogenesis
in the rat testis following adjudin-induced infertility. Int J Androl 35(1):86–101 Muskhelishvili L, Latendresse JR, Kodell RL, Henderson EB (2003) Evaluation of cell proliferation in rat tissues with BrdU, PCNA, Ki-67(MIB-5) immunohistochemistry and in situ hybridization for histone mRNA. J Histochem Cytochem 51(12):1681–1688 Nishimoto M, Miyagi S, Yamagishi T, Sakaguchi T, Niwa H, Muramatsu M, Okuda A (2005) Oct-3/4 maintains the proliferative embryonic stem cell state via specific binding to a variant octamer sequence in the regulatory region of the UTF1 locus. Mol Cell Biol 25(12):5084–5094 Okuda A, Fukushima A, Nishimoto M, Orimo A, Yamagishi T, Nabeshima Y, Kuro-o M, Nabeshima Y, Boon K, Keaveney M, Stunnenberg HG, Muramatsu M (1998) UTF1, a novel transcriptional coactivator expressed in pluripotent embryonic stem cells and extra-embryonic cells. EMBO J 17(7):2019–2032 Ottolenghi C, McElreavey K (2000) Deletions of 9p and the quest for a conserved mechanism of sex determination. Mol Genet Metab 71(1–2):397–404 Rajpert-De Meyts E, Skakkebaek NE (1994) Expression of the c-kit protein product in carcinoma-in situ and invasive testicular germ cell tumours. Int J Androl 17(2):85–92 Schlessinger J, Lemmon MA (2006) Nuclear signaling by receptor tyrosine kinases: the first robin of spring. Cell 127(1):45–48 Scholzen T, Gerdes J (2000) The Ki-67 protein: from the known and the unknown. J Cell Physiol 182:311–322 Schrans-Stassen BH, van de Kant HJ, de Rooij DG, van Pelt AM (1999) Differential expression of c-kit in mouse undifferentiated and differentiating type A spermatogonia. Endocrinology 140(12):5894–5900 Schulze W, Thoms F, Knuth UA (1999) Testicular sperm extraction: comprehensive analysis with simultaneously performed histology in 1418 biopsies from 766 subfertile men. Hum Reprod 14(1):82–96 Steger K, Tetens F, Seitz J, Grothe C, Bergmann M (1998a) Localization of fibroblast growth factor 2 (FGF-2) protein and the receptors FGFR 1–4 in normal human seminiferous epithelium. Histochem Cell Biol 110(1):57–62 Steger K, Aleithe I, Behre H, Bergmann M (1998b) The proliferation of spermatogonia in normal and pathological human seminiferous epithelium: an immunohistochemical study using monoclonal antibodies against Ki-67 protein and proliferating cell nuclear antigen. Mol Hum Reprod 4(3):227–233 Steiner M, Weipoltshammer K, Viehberger G, Meixner EM, Lunglmayr G, Scho¨fer C (2011) Immunohistochemical expression analysis of Cx43, Cx26, c-KIT and PlAP in contralateral testis biopsies of patients with non-seminomatous testicular germ cell tumor. Histochem Cell Biol 135(1):73–81 Traut W, Endl E, Scholzen T, Gerdes J, Winking H (2002) The temporal and spatial distribution of the proliferation associated Ki-67 protein during female and male meiosis. Chromosoma 111(3):156–164 Turnbull C, Rapley EA, Seal S, Pernet D, Renwick A, Hughes D, Ricketts M, Linger R, Nsengimana J, Deloukas P, Huddart RA, Bishop DT, Easton DF, Stratton MR, Rahman N (2010) Variants near DMRT1, TERT and ATF7IP are associated with testicular germ cell cancer. Nat Genet 42:604–607 van Bragt MP, Roepers-Gajadien HL, Korver CM, Bogerd J, Okuda A, Eggen BJ, de Rooij DG, van Pelt AM (2008) Expression of the pluripotency marker UTF1 is restricted to a subpopulation of early A spermatogonia in rat testis. Reproduction 136(1):33–40 Verheijen R, Kuijpers HJ, Schlingemann RO, Boehmer AL, van Driel R, Brakenhoff GJ, Ramaekers FC (1989a) Ki-67 detects a nuclear matrix-associated proliferation-related antigen. I. Intracellular localization during interphase. J Cell Sci 92(Pt 1): 123–130
123
Histochem Cell Biol Verheijen R, Kuijpers HJ, van Driel R, Beck JL, van Dierendonck JH, Brakenhoff GJ, Ramaekers FC (1989b) Ki-67 detects a nuclear matrix-associated proliferation-related antigen. II. Localization in mitotic cells and association with chromosomes. J Cell Sci 92(Pt 4):531–540 von Kopylow K, Kirchhoff C, Jezek D, Schulze W, Feig C, Primig M, Steinkraus V, Spiess AN (2010) Screening for biomarkers of spermatogonia within the human testis: a whole genome approach. Hum Reprod 25(5):1104–1112 von Kopylow K, Staege H, Spiess AN, Schulze W, Will H, Primig M, Kirchhoff C (2012) Differential marker protein expression specifies rarefaction zone-containing human Adark spermatogonia. Reproduction 143(1):45–57 Wang P, Li J, Allan RW, Guo CC, Peng Y, Cao D (2010) Expression of UTF1 in primary and metastatic testicular germ cell tumors. Am J Clin Pathol 134(4):604–612
123
Wu Z, Falciatori I, Molyneux LA, Richardson TE, Chapman KM, Hamra FK (2009) Spermatogonial culture medium: an effective and efficient nutrient mixture for culturing rat spermatogonial stem cells. Biol Reprod 81(1):77–86 Yoshinaga K, Nishikawa S, Ogawa M, Hayashi S, Kunisada T, Fujimoto T, Nishikawa S (1991) Role of c-kit in mouse spermatogenesis: identification of spermatogonia as a specific site of c-kit expression and function. Development 113(2):689– 699 Zhao Y, Yin X, Qin H, Zhu F, Liu H, Yang W, Zhang Q, Xiang C, Hou P, Song Z, Liu Y, Yong J, Zhang P, Cai J, Liu M, Li H, Li Y, Qu X, Cui K, Zhang W, Xiang T, Wu Y, Zhao Y, Liu C, Yu C, Yuan K, Lou J, Ding M, Deng H (2008) Two supporting factors greatly improve the efficiency of human iPSC generation. Cell Stem Cell 3(5):475–479
