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Coculture of Spermatogonia With Somatic Cells in a NovelThree-Dimensional Soft-Agar-Culture-System
JAN-BERND STUKENBORG,* JOACHIM WISTUBA,* C. MARC LUETJENS,*
MAHMOUD ABU ELHIJA,{ MAHMOUD HULEIHEL,{5 EITAN LUNENFELD,{ JORG GROMOLL,*
EBERHARD NIESCHLAG,* AND STEFAN SCHLATT§5
From the *Institute of Reproductive Medicine of the University, Munster, Germany; the �Shraga Segal Department of
Microbiology and Immunology and the `Department of Obstetrics and Gynecology, Faculty of Health Sciences and
Soroka University Medical Center, Ben-Gurion University of the Negev, Beer-Sheva, Israel; and the §Department of
Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.
ABSTRACT: Isolation and culture of spermatogonial stem cells
(SSCs) has become an approach to study the milieu and the factors
controlling their expansion and differentiation. Traditional conven-
tional cell culture does not mimic the complex situation in the
seminiferous epithelium providing a basal, intraepithelial, and
adluminal compartment to the developing male germ cells. SSCs
are located in specific stem cell niches whose features and functional
parameters are thus far poorly understood. It was the aim of this
study to isolate SSCs and to explore their expansion and
differentiation potential in a novel three-dimensional Soft-Agar-
Culture-System (SACS). This system provides three-dimensional
structural support and multiple options for manipulations through the
addition of factors, cells, or other changes. The system has
revolutionized research on blood stem cells by providing a tool for
clonal analysis of expanding and differentiating blood cell lineages. In
our studies, SSCs are enriched using Gfra-1 as a specific surface
marker and magnetic-activated cell sorting as a separation ap-
proach. At termination of the culture, we determined the type and
number of germ cells obtained after the first 24 hours of culture. We
also determined cell types and numbers in expanding cell clones of
differentiating germ cells during the subsequent 15 days of culture.
We analyzed a supportive effect of somatic cell lineages added to the
solid part of the culture system. We conclude that our enrichment
and culture approach is highly useful for exploration of SSC
expansion and have found indications that the system supports
differentiation up to the level of postmeiotic germ cells.
Key words: Spermatogonial stem cells, spermatogenesis, early
culture effects, in vitro meiosis.
J Androl 2008;29:312–329
Analysis of male germ cell proliferation and
differentiation under various in vitro conditions
has increasingly come into focus. Thereby, substances
influencing maturation processes have been intensively
investigated. These studies aimed to specifically analyze
spermatogonial stem cell (SSC) physiology in different
approaches using feeder layer (Nagano et al, 2003),
serum-free (Creemers et al, 2002; Kubota et al, 2004b;
Kanatsu-Shinohara et al, 2005a), or feeder-free culture
conditions (Kanatsu-Shinohara et al, 2005a).
The physiologic conditions needed to maintain and
differentiate cultured SSCs were previously analyzed in
conventional culture systems by addition of testicular
cells (Lee et al, 1997; Nagano et al, 2003) and/or certain
factors (eg, leukemia inhibitory factor [LIF], de Miguel
et al, 1996; Kanatsu-Shinohara et al, 2003; glia cell line–
derived neurotrophic factor [GDNF], Meng et al, 2000;
Kanatsu-Shinohara et al, 2003; Kubota et al, 2004b;
basic fibroblast growth factor, Kanatsu-Shinohara et al,
2003; Kubota et al, 2004b; or stem cell factor [SCF],
Allard et al, 1996; Blanchard et al, 1998; de Rooij et al,
1998) that had been identified for spermatogonial
propagation. All of these factors have been proposed
to be crucial for premeiotic germ cell development.
The conditions allowing male germ cells to enter
meiosis are unknown. However, entry into meiosis relies
on the integrity of the testicular microenvironment, as it
is easily achieved in organ culture (Schlatt et al, 1999)
but rarely observed in cell culture. Therefore, we and
others assume that testicular somatic cells create unique
physical and paracrine support for the developing germ
cells, allowing them to enter meiosis (Hofmann et al,
Supported by the German-Israeli Foundation (grant 1: 760-205.2/
2002) and a grant from the medical faculty of the University of
Munster (IZKF Project No. WI 2/023/07). J.W. was supported by the
Deutsche Forschungsgemeinschaft WI 2723/1-1, and S.S. by a U54
grant from the National Institutes of Health.
5 These authors contributed equally to this article and share
coauthorship.
Correspondence to: Eberhard Nieschlag, Institute of Reproductive
Medicine of the University, Domagkstrasse 11, 48129 Munster,
Germany (e-mail: [email protected]).
Received for publication March 16, 2007; accepted for publication
November 19, 2007.
DOI: 10.2164/jandrol.107.002857
Journal of Andrology, Vol. 29, No. 3, May/June 2008Copyright E American Society of Andrology
312
1992; Lee et al, 1997; Nagano et al, 2003). In vivo, the
seminiferous tubule offers three compartments for germ
cells. 1) The basal compartment, offering physicalcontacts with the basement membrane, peritubular cells,
Sertoli cells, and other premeiotic germ cells. Here, the
germ cell receives paracrine and endocrine signals from
the interstitium. 2) The intraepithelial compartment,
offering only contact with the Sertoli cells and other
meiotic and postmeiotic germ cells. 3) The adluminal
compartment, allowing contact with Sertoli cells and
postmeiotic germ cells, as well as signal molecules fromthe luminal fluid. The stem cell niches are part of the
basal compartment, which offers the most versatile
compartment within the seminiferous tubules. Stem cell
niches could be established through specific extracellular
matrix–specific contacts or specific signaling cascades
and will provide specific physical support and environ-
mental features allowing recognition and settlement of
SSCs. They also might provide crucial factors neededfor maintenance of pluripotent abilities of SSCs
(Spradling et al, 2001).
In general, mammalian SSC culture experiments have
been performed in conventional ‘‘two-dimensional’’ cell
culture approaches using culture dishes or flasks (eg,
Dirami et al, 1999; Feng et al, 2002; Hasthorpe, 2003;
Nagano et al, 2003; Kanatsu-Shinohara et al, 2004a,
2005a,b). The physical support for SSCs in a conven-tional culture is different from the natural niche
environment zof the seminiferous epithelium, and it
remains conjectural whether stem cell niches can be
reestablished in a monolayer culture of Sertoli cells.
Hence, a three-dimensional culture approach might
offer more appropriate opportunities for cell growth.
The Soft-Agar-Culture-System (SACS), a three-
dimensional cell culture approach, was first establishedto characterize clonal expansion of bone marrow cells
and to identify factors involved in the regulation of their
proliferation and differentiation (Lin et al, 1975; Quaroni
et al, 1979; Huleihel et al, 1993; Horowitz et al, 2000).
Applied to testicular stem cells, it might also provide an
improved structural environment for clonal expansion of
germ cells. Here, we are testing this hypothesis to explore
whether SACS can be used as an innovative methodologyfor analysis of germ cell development. Previously
published studies demonstrated the importance of a
three-dimensional structure for the differentiation of
mouse and human testicular cells and the support of in
vitro spermatogenesis (Lee et al, 2006, 2007).
To isolate spermatogonia from testicular tissues,
particularly from immature animals, several approaches
are available, such as gravity sedimentation to separate
cells of different size in percoll (Koh et al, 2004) or withthe STAPUT technique (Dirami et al, 1999), fluores-
cence-activated cell sorting (Shinohara et al, 2000; Fujita
et al, 2005; Guan et al, 2006), or magnetic-activated cell
sorting (MACS; von Schonfeldt et al, 1999; Buageaw et
al, 2005). The MACS system is fast and causes minimal
stress to the spermatogonial cells during isolation and
enrichment. One of the most crucial steps to enrich SSCsis the availability of highly specific markers. Signaling
pathway proteins or receptors exclusively expressed on
the surface of spermatogonia can be specifically utilized
for cell separation by MACS (von Schonfeldt et al, 1999;
Buageaw et al, 2005). To isolate the population of
undifferentiated spermatogonia in mice, marker pro-
teins such as GDNF family receptor-alpha-1 (Gfra-1;
Meng et al, 2000; von Schonfeldt et al, 2004), Cd-9(tetraspanin transmembrane protein; Kanatsu-Shino-
hara et al, 2004b), and Thy-1 (glycosyl phosphatidyli-
nositol–anchored surface antigen; Kubota et al, 2004a;
Oatley et al, 2007) have been suggested to show
prevalence for this cell type. MAC-sorted cells have
previously been cultured using standard procedures.
These studies showed the possibility of maintaining
proliferating SSCs in vitro for up to 6 months (Kubotaet al, 2004b). However, the in vitro production of
meiotic and postmeiotic germ cells, which would
indicate an optimal culture condition not only for SSCs,
but also for survival and differentiation of their
progeny, turned out to be extremely difficult. Thus far,
no culture system was able to maintain the viability of
differentiating spermatogonia and to support the
meiotic and postmeiotic spermatogenic progress. In thisstudy, we aimed to characterize a novel three-dimen-
sional culture system and determine the survival,
expansion, and differentiation of germ cells.
Materials and Methods
Animals
Testes were obtained from juvenile (10 days post partum [dpp])
and mature (30 dpp) CD-1 mice from our institutional colony.
All animal experimental procedures were performed in
accordance with the German federal law on the handling of
experimental animals (animal license no. A87/05).
Testicular Cell Isolation
Testicular cells were isolated on day 10 pp from CD-1 mice
($20 animals per isolation). Testes were removed from the
scrotum and decapsulated. The tissue was minced with fine
scissors and transferred into culture medium (Dulbecco
modified Eagle medium DMEM/HAM F12; Gibco, Gaithers-
burg, Maryland) containing collagenase type 1A (1 mg/mL;
Sigma Chemical Co, St Louis, Missouri) and DNase (0.5 mg/
mL; Sigma). Digestion was performed at 37uC for 10 minutes
in a shaking water bath operated at 110 cycles per minute.
After this step, we obtained a fraction of tubular fragments
and single cells, which were separated by sedimentation at unit
Stukenborg et al N Soft-Agar-Culture of Spermatogonia 313
gravity. To obtain an enriched fraction of interstitial cells, no
DNase was added to the collagenase, the supernatant was
removed after 10 minutes, and the cell fraction was washed
and stored in ice-cold DMEM/HAM F12.
To obtain a fraction of tubular cells (designated unsorted
fraction) consisting mainly of Sertoli cells and germ cells, the
fragments of seminiferous tubules obtained after the first
digestion step were washed once in DMEM and further digested
in a mixture of collagenase type I (1 mg/mL; Sigma), DNase
(0.5 mg/mL; Sigma), and hyaluronidase (0.5 mg/mL; Sigma;
Wistuba et al, 2002). The single-cell suspension (Table 1) was
washed successively with medium and phosphate-buffered
saline (PBS) containing 2 mM EDTA (Sigma) and 0.5% fetal
calf serum (Gibco; Figure 1A). Efficiency of the digestion, cell
number, and concentration were established microscopically
using a Thoma chamber (Hecht, Sondheim, Germany).
Magnetic Labeling and Separation of Cells
Aliquots of cell suspensions from the unsorted fraction with a
concentration of around 7.5 6 107 cells/mL (Table 2) were
subjected to indirect labeling using a previously described
protocol based on primary polyclonal antibodies against Gfra-
1, Cd-9, and Thy-1, and secondary or tertiary anti-rabbit or
anti-biotin antibodies carrying ferromagnetic particles (von
Schonfeldt et al, 1999). In brief, the cells were incubated with a
polyclonal rabbit anti–Gfra-1 immunoglobulin G (IgG)
antibody (H-70, diluted 1:20; Santa Cruz Biotechnology,
Santa Cruz, California), polyclonal rabbit anti–Cd-9 IgG
antibody (H-110, diluted 1:20; Santa Cruz Biotechnology), or
monoclonal mouse anti–Thy-1 IgG antibody (HIS51, diluted
1:20; Santa Cruz Biotechnology) for 15 minutes at 6uC–10uC.
Afterwards, cells were washed with PBS (supplemented with
EDTA and fetal calf serum as described above), labeled with
goat anti-rabbit IgG MicroBeads (dilution 1:5; Miltenyi,
Bergisch Gladbach, Germany) or anti-biotin MicroBeads
(dilution 1:5; Miltenyi), and washed again. Before the use of
anti-biotin MicroBeads, the cells were incubated with anti-
rabbit IgG biotin conjugate (B-8895; Sigma) or anti-mouse
IgG biotin conjugate (B-0529; Sigma) for 15 minutes. A
separation column (MS separation column; Miltenyi) was
placed in a strong magnetic field and flushed with 500 mL
degassed buffer. The Gfra-1–labeled cell suspension (Table 1)
was resuspended in degassed buffer and poured into the
column reservoir. Gfra-1–positive cells were retained in the
magnetic field within the matrix of the column, whereas
nonlabeled cells passed through and were collected and
designated as a depleted cell fraction (Table 1; Figure 1A).
This depleted fraction was added in coculture experiments as
somatic cell fraction to support spermatogonia in the SACS.
To deplete unlabeled cells from the magnetic fraction, the
column was rinsed 3 times with 500 mL degassed buffer. Cells
eluting in these washing steps were added to the depleted
fraction. In order to retrieve the enriched fraction (Table 1),
the column was removed from the magnet, and 500 mL
degassed buffer was added to the reservoir. The cells were
flushed out of the column using a plunger. Cell size and
nuclear size and shape were evaluated and documented under
phase-contrast microscopy and were used as criteria to
determine the homogeneity/heterogeneity of the unfixed fresh
cell suspensions. Viability of MAC-sorted cells was micro-
scopically evaluated using Trypan blue staining.
Flow Cytometry
The efficiency of spermatogonial enrichment was quantita-
tively assessed by flow cytometry. Forward and sideward
scatter were used to gate cell populations. This gating by size
and granularity allowed the exclusion of FITC-positive cell
debris. Fractions from the unsorted, enriched, and depleted
cell suspensions (Table 1) were stained with FITC-conjugated
anti-rabbit antibody (F-0382; Sigma) for 30 minutes at 6uC–
8uC. To determine the proportion of FITC-positive cells, the
cells were analyzed on a Beckman Coulter flow cytometer
FC500 (Krefeld, Germany) equipped with a 15-mW argon-ion
laser at an excitation wavelength of 488 nm. The green signals
of FITC plotted on a log scale of each cell fraction were
collected using a 520-band pass filter (505–545 nm). A marker
was set in the FITC histogram as the cutoff between
background signals and positive staining, which was deter-
mined by comparison with the control sample. A minimum of
105 cells was analyzed in each run (according to von
Schonfeldt et al, 1999).
Immunohistochemistry
Tissue samples were fixed in Bouin solution for up to 12 hours
before being transferred into 70% ethanol, and were routinely
Table 1. List of cell types of different cell suspensions used for SACSa
Cell Fractions Cell Types
Unsorted fraction (MACS) Tubular cells (undifferentiated spermatogonia up to leptotene spermatocyes and Sertoli
cells)
Enriched fraction (MACS) Tubular cells (undifferentiated spermatogonia [enriched: 42%–54%] up to leptotene
spermatocytes and Sertoli cells)
Depleted fraction (MACS) Tubular cells (undifferentiated spermatogonia [depleted: 11%–19%] up to leptotene
spermatocytes and Sertoli cells)
Testicular cell fraction Tubular and interstitial cells (undifferentiated spermatogonia up to leptotene
spermatocytes, peritubular cells, macrophages, Sertoli cells, and Leydig cells)
a The separation of the unsorted cell fraction with MACS resulted in 2 different tubular cell suspensions, the depleted and the enriched
fraction. The percentage of Gfra-1–positive spermatogonia is decreased in the depleted fraction, whereas the enriched fraction contained
more Gfra-1–positive spermatogonial cells. The testicular cell fraction is a mixture of tubular and interstitial cells. The different cell types of
10 dpp murine testis are also shown in Figure 2A through C (Cd-9 expression) and Figure 2G through I (Gfra-1 expression).
314 Journal of Andrology N May �June 2008
embedded in paraffin using an automated processor. Cultured
cells (within the agar phase) were fixed in 4% paraformalde-
hyde (PFA) for 24 hours at 6uC–8uC before transfer into 30%
(24 hours) and 50% (24 hours) ethanol and embedding as
described before. Tissue and cultured cells were cut into
sections of 5–7 mm and were immunohistochemically stained
for Cd9, Gfra-1 (spermatogonial markers), cAMP reponse
element modulator (Crem; postmeitotic spermatids), and 5-
bromodesoxyuridine (BrdU; proliferation marker; Figure 1C).
Polyclonal primary antibodies against the peptide ETQE-
DAQKILQEAEKLNYKDKKLN (common to all 3 human
BOULE isoforms) were used for detection of murine Boule
protein (provided by R.A. Reijo Pera, San Francisco,
California). Briefly, sections were deparaffinized in paraclear
and rehydrated in a graded series of ethanol. For antigen
retrieval, sections were heated in a microwave oven in Glycin/
HCl buffer (50 mM, pH 3.5) for 12 minutes at 80uC.
Endogenous peroxidase activity was quenched by treatment
with hydrogen peroxide (3% for 5 minutes), followed by
blocking of nonspecific antibodybinding with 5% normal horse
serum supplemented with bovine serum albumin (BSA; 0.1%)
for 20 minutes at room temperature. All antibodies were diluted
in Tris buffered saline (TBS)/BSA (0.1%). The slides were
incubated with a primary antibody (rabbit anti-Cd9 antibody
[H-110; 1:50], rabbit anti–Gfra-1 [H-70; 1:50], rabbit anti–
Crem-1 [X-12; 1:50]; Santa Cruz Biotechnology; rabbit anti-
BrdU [Bu20a; 1:30]; Sigma-Aldrich, Taufkirchen, Germany)
and a polyclonal primary antibody against the peptide
ETQEDAQKILQEAEKLNYKDKKLN (Boule detection:
1:300) at room temperature in a humidified chamber for 1 hour
and rinsed in TBS (10 mM TBS, 150 mM NaCl, pH 7.6) for 3
6 5 minutes between each of the following incubations.
Sections incubated in TBS/BSA without primary antibody
served as negative control. Juvenile and adult testicular tissues
were immunostained using an LSAB2 kit (DAKO Cytomation,
Hamburg, Germany). Washing steps followed incubation with
primary antibody and were carried out in TBS (2 6 5 minutes).
Afterwards, the sections were incubated with biotinylated
Figure 1. Scheme of the Soft-Agar-Culture-System (SACS) in combination with vacuum filtration, mRNA/total RNA analysis, and fixation forimmunohistochemistry. The scheme contains 3 parts with the time course of the experiment. The cell separation and the magnetic-activatedcell sorting (MACS) is described in A, the Soft-Agar-Culture in B, and the analysis by vacuum filtration, mMACS/RNA isolation and staining ofparaffin embedded SACS sections in C.
Stukenborg et al N Soft-Agar-Culture of Spermatogonia 315
swine–anti-rabbit IgGs (15 minutes), washed, and covered with
streptavidin-horseradish-peroxidase (HRP) solution (15
minutes), and staining was finally visualized using 3,3-
diaminobenzidine tetrahydrochloride (DAB) in urea buffer for
5 to 20 minutes (Sigma-Aldrich). Positive staining appeared as a
brown precipitate in the cells. Vacuum-filtrated cell colonies
were stained with LSAB2 kit under identical conditions, as
described before.
Staining for apoptosis was performed by the DeadEnd
Colorimetric TUNEL System (Promega, Madison, Wisconsin).
Sections were deparaffinized in paraclear and rehydrated in a
graded series of ethanol. After washing with 0.9% NaCl and
PBS for 5 minutes, all sections were fixed before and after
incubation with proteinase K (20 mg/mL) for 15 minutes in 4%
PFA. The recombinant terminal deoxynucleodityl transferase
(rTdT) reaction was maintained for 1 hour in a humidified
chamber, followed by 15 minutes of twofold SSC incubation.
All sections were immunostained with streptavidin horseradish-
peroxidase solution after washing with PBS for 30 minutes. To
visualize positive TUNEL-stained cells, DAB in urea buffer was
added for 8 minutes. All sections were counterstained in
hematoxylin, mounted, and analyzed by light microscopy.
Additionally, the analysis of anti-BrdU was counterstained with
Hoechst 33528 (Sigma) for 15 minutes. These sections were
analyzed by light and fluorescence microscopy.
To analyze Gfra-1 expression in unsorted and enriched MAC-
sorted fractions, single-cell solutions were stained for Gfra-1
with an FITC-labeled secondary antibody (Sigma) in combina-
tion with Hoechst 33528 for 30 minutes. Immunohistochemical
results were documented by digital imaging using a fluorescence
microscope (Axiovert 200; Zeiss, Oberkochen, Germany).
The expression of Gfra-1 on the cell surface of undifferen-
tiated spermatogonia was evaluated by confocal microscopy
(TCS SL; Leica, Wetzlar, Germany).
Table 2. Primer sequences and sizes for different murine spermatogenic stages used for analysis of total and mRNAexpressionsa
Spermatogenic Cell Stage and Marker Primer Product Size
Spermatogonia (undifferentiated and
differentiated)
Oct3/4 Forward 59-AGAAGGAGCTAGAACAGTTTGC-39 416 bp
Reverse 59-CGGTTACAGAACCATACTCG-39
Kit Forward 59-GCATCACCATCAAAAACGTG-39 331 bp
Reverse 59-GATAGTCAGCGTCTCCTGGC-39
Cd-9 Forward 59-ATGGCTTTGAGTGTTTCCCGCT-39 374 bp
Reverse 59-ATCTTCTGGCTCGCTGGCATT-39
Gfra-1 Forward 59-GGCCTACTCGGGACTGATTGG-39 462 bp
Reverse 59-GGGAGGAGCAGCCATTGATTT-39
a-6-integrin Forward 59-AGGAGTCGCGGGATATCTTT-39 502 bp
Reverse 59-CAGGCCTTCTCCGTCAAATA-39
Dazl Forward 59-TTCAGGCATATCCTCCTTATC-39 262 bp
Reverse 59-ATGCTTCGGTCCACAGACTTC-39
Spermatocytes
Prohibitin Forward 59-GTGGCGTACAGGACATTGTG-39 306 bp
Reverse 59-AGCTCTCGCTGGGTAATCAA-39
Scp-3 Forward 59-ACAACAAGAGGAAATACAGAA-39 618 bp
Reverse 59-GAGAGAACAACTATTAAAACA-39
Srf-1 Forward 59-TCCATTCAGCACCTTCAACA-39 303 bp
Reverse 59-TCATCCAAATGGAAAGAGCC-39
Spermatids (postmeiotic)
Ldh Forward 59-GCACGGCAGTCTTTTCCTTAGC-59 585 bp
Reverse 59-TCGCGCCAGATCAGTCACAG-39
Protamine-2 Forward 59-GGCCACCACCACCACAGACACAGGCG-39 188 bp
Reverse 59-TTAGTGATGGTGCCTCCTACATTTCC-39
Sp-10 Forward 59-GGAGCACCACCAGGTCAG-39 734 bp
Reverse 59-GACCTTGTTGCAGAGAGG-39
Stertoli cells
Abp Forward 59-GGAGAAGAGAGACTCTGTGG-39 900 bp
Reverse 59-GCTCAAGACCACTTTGACTC-39
Peritubular cells
a-Smooth muscle Forward 59-CGATAGAACACGGCATCATC-39 524 bp
Reverse 59-CATCAGGCAGTTCGTAGCTC-59
Positive control
b-actin Forward 59-AGAGGGAAATCGTGCGTGAC-39 463 bp
Reverse 59-GCCGGACTCATCGTACTCCT-39
a Spermatogonial markers (undifferentiated and differentiated): Oct3/4, C-kit, Gfra-1, Cd-9, and a-6-integrin; meiotic germ cell stages:
Prohibitin, Scp-3, and Srf-1; postmeiotic germ cell stages: Ldh, Protamine-2, and Sp-10; Sertoli cells: Abp; peritubular cells: a-smooth
muscle; positive control: b-actin.
316 Journal of Andrology N May �June 2008
SACS
The enriched fraction (Table 1) was used for culture in the gel
phase of SACS. The cells were added to the gel-agar medium
(0.35% [w/v]) settled on a solid-agar base (0.5% [w/v];
Figure 1B; Lin et al, 1975; Kimball et al, 1978; Hofmann et
al, 1992; Huleihel et al, 1993). Depending on the experimental
setup, the solid base was either empty or supplemented with
cells from the depleted fraction (Table 1). To establish the final
concentrations of agar and cells, 0.7% (w/v) agar and 1.0%
(w/v) agar were dissolved in distilled water to prepare the gel
and solid phases, respectively (Fisher Scientific, Loughbor-
ough, United Kingdom). This solution was mixed with the
same volume of DMEM high glucose (Gibco, pH 7.4) to
achieve a final concentration of 0.35% and 0.5% (Figure 1B).
Cell suspensions were added to the DMEM prior to mixing
with the agar. The agar and the cells in DMEM were mixed at
37uC, avoiding heat-induced cellular stress and premature
coagulation of the agar. Culture conditions were 35uC in 5%
CO2. For standard cell culture experiments, regular 24-well
plates (Nunc, Wiesbaden, Germany) and 24-well plates with
standard Transwell inserts (Corning, New York) were used. To
investigate cell proliferation, BrdU (B-5002; Sigma) was added
in a final concentration of 100 M to the cell/DMEM
suspension before mixing with the agar solution.
Vacuum Filtration
The gel-agar phase containing cultured cells was separated
from the solid-agar phase by pipetting and subsequent vacuum
filtration using Whatman 47 filters (Whatman, Maidstone,
United Kingdom) with a pore size of 0.2 mm. After filtration,
cells were fixed on the filter material in 4% PFA and washed
twice with PBS. After washing, cell nuclei were stained with
Hoechst 33258 for 30 minutes before they were washed again.
For evaluation of the cultured cells, 10 micrograph images at a
fivefold magnification (Axiovert microscope, CCD camera;
Zeiss) of each experiment were scored for cell numbers
(Figure 1C), and the total cell number per filter was calculated.
Three filters per time point were evaluated in the 24-hour
approach, and 12 filters per time point in the 1- to 16-day
approach.
Total RNA Extraction, cDNA Synthesis, and ReverseTranscription–Polymerase Chain Reaction ofFresh Tissue
Total RNA was extracted from immature (10 dpp) mouse
testes using the EZ-RNA Reagent protocol (Biological
Industries, Beit Haemek, Israel). First-strand cDNAs were
synthesized from 2.5 mg total RNA with 0.5 mg random
oligonucleotide primers (Roche Molecular Biochemicals,
Mannheim, Germany) and 200 units of Moloney-Murine
Leukemia Virus–Reverse Transcriptase (M-MLV-RT; Life
Technologies Inc, Paisley, Scotland, United Kingdom) in a
total volume of 20 mL Tris-HCl-MgCl reaction buffer,
supplemented with dithiothreitol, dinucleotriphosphates
(0.5 mM; Roche Molecular Biochemicals), and RNase inhib-
itor (40 units; Roche Molecular Biochemicals). The reverse
transcriptase (RT) reaction was performed for 1 hour at 37uCand stopped for 10 minutes at 75uC. The volume of 20 mL was
subsequently filled up to 60 mL with water. Negative controls
for the reverse transcriptase reaction (RT2) were prepared in
parallel using the same reaction preparations with the same
samples and without M-MLV-RT. The polymerase chain
reaction (PCR), performed subsequently, contained cDNA
samples in final dilution of 1:15 with 2 pairs of oligonucleotide
primers (Sigma) which were exon spanning (Table 2).
To assess the absence of genomic DNA contamination in
RNA preparations and RT-PCR reactions, PCR was per-
formed with negative controls of the RT reaction (RT2) and
without cDNA (cDNA2). The PCR reactions were carried out
on a Cycler II System Thermal Cycler (Ericomp, San Diego,
California). A total of 20 mL of each PCR product was run on
2% agarose gel containing ethidium bromide and was
photographed under ultraviolet light (Figure 1C).
Messenger RNA Isolation from SACS-Cultured Cells
Messenger RNA from 10 dpp murine testicular cells and from
testicular cells cultured with SACS were isolated using the
mMACS oneStep cDNA Kit (Miltenyi), following the manu-
facturer’s protocol. In brief, the cells were prepared fresh or
were snap frozen before mRNA isolation. The samples were
thawed in Lysis/Binding buffer (Miltenyi) on ice and were
lysed by mixing and additional vortexing for 5 minutes.
Afterwards, the sheared lysate samples were placed in a
LysateClear column (Miltenyi) and centrifuged at 26 450 6 g
for 3 minutes to separate the mRNA. After separation, the
lysate was mixed with 50 mL Oligo(dt) MicroBeads (Miltenyi).
Magnetic separation was preceded using a prepared column
within a magnetic field of the thermoMACS Separator
(Miltenyi). Magnetically labeled mRNA was retained in the
column during washing steps to remove rRNA and DNA
according to the manufacturer’s protocol. To proceed with
cDNA synthesis, 100 mL equilibration/wash buffer was added
2 times to the column, followed by incubation with the enzyme
mix (Miltenyi) for 1 hour. During cDNA synthesis the
thermoMACS separator was set to 42uC for 1 hour. Subse-
quently, cDNA was washed 2 times with equilibration/wash
buffer within the column. To release the cDNA from the
magnetic beads, 20 mL cDNA-Release solution (Miltenyi) was
applied for 10 minutes at 42uC on the top of the column.
Synthesized cDNA was eluted with 50 mL cDNA Elution
buffer (Miltenyi). The efficiency of cDNA synthesis was
verified by PCR amplification of the ß-actin gene. Specific
expression of different spermatogenic stage-specific markers
(Table 2) was investigated by PCR amplification. PCR
conditions were 2 minutes at 94uC, 35 6 50 seconds at
94uC, 50 seconds at 58uC, and 1 minute at 72uC (annealing
and extension). Messenger RNA isolation was performed
using the same cell numbers (Figure 1C).
Statistics
Statistic evaluation was performed by Student’s t test
(SigmaStat3; Statcon, Witzenhausen, Germany). Mean 6 SD
is given in the figures as described in the legends.
Stukenborg et al N Soft-Agar-Culture of Spermatogonia 317
Results
Immunohistochemical and Flow Cytometric Evaluation ofSpermatogonial Markers
Immunoreactivity of anti–Gfra-1 and anti–Cd-9 for
subpopulations of SSCs was confirmed by staining of
representative histologic sections in day 10 pp and day 30
pp mice (Figure 2A through L). Positive spermatogonial
cells were observed at the basal membrane of the
seminiferous tubules. Differentiating germ cells and
Sertoli cells were negative for Gfra-1 and Cd-9. In thejuvenile testis, Gfra-1 and Cd-9 were expressed in single
spermatogonial cells (Figure 2B, C, H, and I). In the
adult testis, the predominant Gfra-1–positive cells were
isolated single spermatogonia (Figure 2K and L). In
contrast, Cd-9 labeling was often detected in groups and
chains of spermatogonia (Figure 2E and F). Omission of
the primary antibodies or the use of rabbit IgGs (data not
shown) as negative control showed identical results in theabsence of any specific positive staining in the juvenile
and adult tissue sections (Figure 2A, D, G, and J).
Figure 2. Expression of Cd-9 (A–F) and Gfra-1 (G–L) in the juvenile (A–C, G–I) and the adult (D–F, J–L) mouse testis. In the adult tissue, Cd-9is expressed in aligned (E, F) and Gfra-1 in single spermatogonia (K, L). The staining is confined to spermatogonia (arrowheads). Sertoli andLeydig cells remain unstained. A representative control of the method specificity shows no specific staining within the tubular compartment.Scale bars 5 50 mm.
318 Journal of Andrology N May �June 2008
To confirm these results and to demonstrate both
markers to be sufficient to isolate undifferentiated
spermatogonia, flow cytometric analysis was performed
on unsorted, depleted, and enriched fractions (Table 1)
after MACS with anti–Gfra-1 (Figure 3A through C),
anti–Cd-9 (Figure 3D through F) and, as a third markerof undifferentiated cells, anti–Thy-1 (Figure 3G through
I) antibodies. All markers showed separation of cell
populations of the same size and granularity pattern in
the depleted and the enriched fractions (Figure 3),
whereas Gfra-1 showed the highest percentage of events
in the cell population of the enriched fraction. There-
fore, the staining pattern and the flow cytometric
analysis indicated that Gfra-1 is the most suitable ofthe 3 markers analyzed for isolation of undifferentiated
spermatogonia.
Figure 3. Images of flow cytometric analysis of Gfra-1, Cd-9, and Thy-1 unsorted and MAC-sorted cell fraction. Three different cell fractions(unsorted [A, D, G], depleted [B, E, H], and enriched [C, F, I]) are shown for MACS using anti–Gfra-1 (A–C), anti–Cd-9 (D–F), and anti–Thy-1(G–I) to separated undifferentiated spermatogonia. All three antibodies detect the same cell population defined by size (forward scatter [FS log:x-axis]) and granularity (side scatter [SS log: y-axis]). The dominant fractions in the depleted, and enriched fractions are surrounded with ablack line.
Stukenborg et al N Soft-Agar-Culture of Spermatogonia 319
Table 3. Quantitative and flow cytometric evaluation of MACS by the use of standard MicroBeads compared with anti-biotin MicroBeadsa
320 Journal of Andrology N May �June 2008
Cell Separation
The 2-step enzymatic digestion resulted in a single-cell
suspension (Table 1), which was used for cell separation
by MACS. The eluted depleted fraction contained a
heterogeneous suspension of living cells similar to the
unsorted cell suspension (Figure 4A and B). After
separation, the enriched fraction of fresh cells was
homogenous, containing clusters of cells with similar
sizes and shapes and comparable nuclear-cytoplasm
ratio and nuclear morphology (Figure 4C). Microscopic
imaging of immunohistochemical staining of isolated
cells confirmed that the enriched fraction contained a
higher number of Gfra-1–positive cells compared with
the unsorted and depleted fraction (Figure 4D through
F). Total RNA isolation and analysis were performed to
analyze the enriched fraction after MACS with Gfra-1
(Figure 4G). RNA analysis revealed an expression
profile typical for spermatogonial cells in the enriched
fraction after MACS only. The expression of Gfra-1 on
the cell surface of undifferentiated spermatogonia is
proven by confocal microscopy (Figure 4H through K).
Cell survival was verified by trypan blue exclusion test at
around 90%.
Flow Cytometric Analysis of the Separation Technique
Flow cytometric analysis allowed a quantification of
enrichment efficiency. Table 3 presents a comparison of
the two methods used to establish the unsorted
(Table 3A and D), depleted (Table 3B and E), and
enriched fractions (Table 3C and F). Recovery rates
were threefold higher when MACS was performed with
biotin-labeled instead of magnetically labeled secondary
antibodies. The unsorted fraction from the juvenile
testes contained 21%–24% Gfra-1–positive cells. The
use of magnetically labeled secondary antibodies result-
ed in a twofold to threefold (up to 42% positive cells)
enrichment rate for Gfra-1–positive cells derived from
10 dpp murine cells. These enriched and depleted
fractions from the day 10 pp testes were used for the
SACS experiments.
Evaluation of Germ Cell Development With and WithoutSupporter Cells Using the SACS
Three experimental approaches were selected to com-
pare the outgrowth of colonies in the gel phase of the
three-dimensional culture system: 1) cells from the
enriched fraction growing in the gel phase (Figure 5A,
D, and G) without additional supporting cells; 2) cells
from the enriched fraction growing in the gel phase(Figure 5B, E, and H) and cells from the depleted
fraction added to the solid phase; and 3) cells from the
enriched fraction growing in the gel phase (Figure 5C,
F, and I) together with cells of the interstitial and
depleted fractions.
Morphologic analysis of colonies in the gel phase
revealed a different growth pattern over time in response
to the 3 different conditions (Figure 5). Colonies in the
absence of supporter cells were heavily compacted and
exhibited a round shape with sharp edges (Figure 5A,
D, and G). The colonies growing in the gel phase in the
presence of supporter cells in the solid phase were lessdense and showed single cells and small groups of cells
in loose contact with the colony (Figure 5B, E, and H).
Those colonies that also contained interstitial cells
(Table 1) in the gel phase show similar colony structures
(Figure 5C, F, and I).
Evaluation of cell numbers on filters demonstrated a
positive effect of supporter cells in the solid phase on the
number of cells in the gel phase as consistently
throughout all time points (days 1–16); a higher number
of cells was determined in these groups (Figure 6A).
A high loss of total cells but a positive effect of
supporter cells in the solid phase were also determined
during the first 24 hours of SACS (Figure 6B). To
determine the cause for the 40%–70% loss of cells from
the gel phase during the initial 24 hours of culture, we
evaluated the number of apoptotic cells after 24 hours ofculture. In all groups, we determined rates of 37.2%–
47.7% (+10% SD) TUNEL-positive cells independent of
the presence of supporter cells. The proliferation analysis
showed a significant increase of BrdU-positive cells
between the 12th and 20th hour of culture in the
r
a Absolute cell numbers/mL of all 3 MACS fractions (unsorted, enriched, and depleted) are shown in white columns. The enrichment and
depletion of Gfra-1–positive cells in the obtained fractions after isolation are shown for both MicroBeads (standard MicroBeads: unsorted
[A], depleted [B], and enriched [C]; anti-biotin MicroBeads: unsorted [D], depleted [E], and enriched [F]; y-axis: events; x-axis: FL1 log for
positive FITC signals). Both MicroBeads show a depletion in FITC-positive signals in the depleted fraction (B, E) and an enrichment of
FITC-positive signals in the enriched fraction (C, F) compared with the unsorted ones (A, D). The absolute cell numbers/mL of Gfra-1–
positive cells of the same fractions are shown in gray columns. In the anti-biotin MicroBeads-enriched fraction, more Gfra-1–positive cells
were found compared with cell numbers enriched by standard MicroBeads. The unsorted fraction showed a similar content of cells in both
MACS variations. The use of anti-biotin MicroBeads resulted in fewer Gfra-1–positive cells in the depleted fraction compared with the use of
standard MicroBeads. Results are given as mean (%) 6 SD of 3 and 7 experiments.
Stukenborg et al N Soft-Agar-Culture of Spermatogonia 321
experiment with isolated spermatogonia in the gel phase
supported by cells from the depleted fraction in the solid
phase (Figure 6C), whereas no significant increase could
be observed in the approach without supporting cells in
the solid phase. Analysis of apoptotic events during the
culture period of the third approach, when all testicular
cells were used, showed a decrease of apoptosis (to around
20%) between day 6 and day 11 of culture (Figure 6D).
To exclude a potential migration of cells from the
solid phase into the gel phase, we separated the 2 phases
by a cell-impermeable membrane. The cell numbers were
evaluated by counting cells in 40 paraffin sections per
culture approach after 24 hours of culture. In this
approach we obtained no difference in cell number
proportion (without supporter cells: 101.2 6 39.1 [SD];
with supporter cells: 357.6 6 79.0 [SD]) compared with
the approach without using a membrane, and therefore
no indices for cell migration between the 2 phases.
To investigate spermatogenic development during
culture in all 3 experimental settings, mRNA was
isolated with the mMACS kit. The small amount of cell
numbers resulted in very low levels of mRNA.
Figure 4. Images of fluorescence-labeled cells showing freshly and fixed isolated cells from mouse testes (10-day-old mice). A heterogeneouscell suspension containing single cells is observed in the unsorted cell fraction after digestion of cells before the separation procedure (A) and inthe depleted fraction after MAC sorting with Gfra-1 (B). A homogeneous cell suspension is observed in the enriched fraction (C). These cellstend to form clusters of variable size. Images of fluorescent cells depicting fixed spermatogonia before and after MAC sorting. The mentionedfractions are shown after immunofluorescent labeling with anti–Gfra-1 (unsorted fraction [D], depleted fraction [E], and enriched fraction [F];arrowheads: Gfra-1–positive cells [FITC]). DNA staining by Hoechst: A–F. Expression analysis of different spermatogenic marker genes(murine spermatogonial stages: Oct3/4, C-kit, Gfra-1, Cd-9, and a-6-integrin; murine meiotic stages: Prohibitin and Srf-1; murine postmeioticstages: Ldh, Protamine-2, and Sp-10; positive control: b-actin before (G; unsorted [us] lane) and after sorting with anti–GFRa-1 (G; enriched [+]lane). Confocal microscopy images of fluorescent-labeled cells showed expression of Gfra-1 (arrows) on the cell surface of spermatogonialcells (H–J; overlay K). Scale bar 5 20 mm.
322 Journal of Andrology N May �June 2008
Therefore, we could only analyze expression profiles on
1 day of culture (Figure 6E and F). In the 2 approachesusing Gfra-1–isolated spermatogonia (Figure 6E and
F), a strong expression of Cd-9 (undifferentiated
spermatogonia; Figure 6E and F, lane Q),
a-smooth muscle (peritubular cells; Figure 6E and F,
lane N), and b-actin (positive control; Figure 6E and F,
lane P), and weak expression of prohibitin and Srf-1
(meiotic spermatocytes; Figure 6E and F, lanes G and
H) could be observed after 1 day of culture. In theapproach containing all testicular cells, mRNA of
different spermatogenic stages was observed (Fig-
ure 3G). In this approach, the mRNA expression wasdetectable for spermatogonial and meiotic genes.
Characterization of the cultured cells in the gel phase
by immunohistochemistry revealed the presence of
Gfra-1–positive cells in histologic sections and on filters
after vacuum filtration at different time points (Fig-
ure 7A through D). To determine the degree of
differentiation of germ cells in the gel phase when
exposed to different culture conditions and to confirm
these data obtained by mRNA analysis, we performedimmunohistochemical localization of Boule (Figure 7E
through J) and Crem (Figure 7K and L). Boule is
Figure 5. Images of clonal expansion of SACS-cultured spermatogonia after different culture periods: without supporter cells (A, day 6; D, day11; G, day 16) and with cell support: depleted fraction (tubular cells): (B, day 6; E, day 11; H, day 16); testicular cells (tubular and interstitialcells): (C, day 6; F, day 11; I, day 16) found in the gel phase. Colonies found in somatic cell–supported SACS showed fewer connected cells atthe edge of the colonies (arrowheads) or even highly condensed structures in the inner region (arrows). Scale bar 5 50 mm.
Stukenborg et al N Soft-Agar-Culture of Spermatogonia 323
Figure 6. Evaluation of testicular cells cultured in a three-dimensional SACS for 16 days and 24 hours with (dark bars, A–C) or without (lightbars, A–C) intratubular supporter cells (depleted fraction) in the solid phase or supplementation with all somatic testicular cells (D). Analysis ofvacuum-filtrated cells showed different cell numbers in the approach with or without supporter cells after 16 days of culture (A) and 24 hours(B). Proliferation rate analysis shows a significant increase of proliferating cell numbers during the 12th and 20th hours in the approach withsupport of intratubular cells in the solid phase (C). Evaluation of TUNEL-positive cells in the approach using all testicular cells in the gel phasedepicts a decrease of apoptosis at day 6 and day 11 of culture (D). Results of mRNA isolation and expression after 1 day of
324 Journal of Andrology N May �June 2008
considered a reliable marker for meiotic germ cells (Xu
et al, 2001), and its expression in murine testis was
observed earliest in the stage of late pachytene
spermatocytes. Immunohistochemistry of tissue sections
confirms that Boule is present after day 15 pp in the late
spermatocyte stage (Figure 7N), but is not expressed at
an earlier time point of development (Figure 7M).
No Boule-positive cells were observed in cell fractions
after SACS without supporter cells or with cells from the
depleted fraction in the solid phase (Figure 7E through
H). However, when interstitial cells and cells from the
depleted fraction were added to the gel phase, Boule-
positive cells were detected consistently when the cultures
were maintained for at least 13 days (Figure 7I).
Crem is considered a marker for postmeiotic cells at
the stage of round spermatids (Delmas et al, 1993;
Wistuba et al, 2002). In the third approach containing
all testicular cells, positive signals for this postmeiotic
spermatogenic stage of round spermatids were observed
for at least 21 days of culture (Figure 7K). Immunohis-
tochemistry of tissue sections confirms that Crem is
present 3 weeks after birth in the spermatogenic stage of
round spermatids (Figure 7Q) but not in cells at the time
point when cell isolation was performed (Figure 7P).
Discussion
Many studies have analyzed in vitro culture effects on
gonocytes or SSCs (Hasthorpe et al, 2000; Hasthorpe
2003; Izadyar et al, 2002, 2003; Kanatsu-Shinohara et
al, 2003, 2004a, 2005a; Nagano et al, 2003; Kubota et al,
2004b). In general, conventional culture approaches
were employed. These did not provide structural
conditions that would closely resemble the natural
testicular environment. Therefore, our study aimed to
establish and validate a novel method for spermatogo-
nial cell culture to improve propagation and differenti-
ation of these undifferentiated germline cells. In contrast
to conventional culture performed in dishes or flasks,
the three-dimensional agar structure of SACS offers
conditions mimicking some structural features of the in
vivo situation. SACS was used to examine supporting
and limiting effects of somatic testicular cells cocultured
in the solid phase of the system. As previously
published, the use of different supporter cell types
revealed aspects of SSC culture (single spermatogonia;
Asingle; de Rooij et al, 2000) in terms of SSC line
establishment (Shinohara et al, 2000; Nagano et al,
2003; Kubota et al, 2004b; Kanatsu-Shinohara et al,
2005a) and differentiation into more developed sper-
matogenic stages (Tres et al, 1983; Gerton et al, 1984;
Hue et al, 1998; Tesarik et al, 1998a,b; Feng et al, 2002;
Sousa et al, 2002). However, a culture system that allows
complete spermatogenesis to occur is still far from
routine methodology.
Successful enrichment and separation of isolated
testicular cells from mouse tissue using MACS has
previously been described (von Schonfeldt et al, 1999;
Kubota et al, 2004b; Buageaw et al, 2005; Oatley et al,
2007). Supplementation with somatic cells resulted in a
stabilized and more differentiated in vitro population of
germ cells. The experimental setting combining MACS
separation of the testicular cell fraction and SACS
allowed the use of the various fractions achieved by the
MAC sorting. A successful MACS separation depends
on the use of cell surface markers expressed exclusively
on undifferentiated SSCs (Sofikitis et al, 2005). There-
fore, Gfra-1, Cd-9, and Thy-1 were analyzed as putative
mouse SSC markers (Meng et al, 2000; Kanatsu-
Shinohara et al, 2004b; von Schonfeldt et al, 2004;
Oatley et al, 2007; He et al, 2007). The fact that all 3
markers detect the same cell population (same size and
granularity in flow cytometric analysis of the enriched
fractions) and that we localized exclusively single
spermatogonia and small chains/groups of spermatogo-
nia indicated that Cd-9 is a marker for Asingle and
Aaligned spermatogonia. In contrast, Gfra-1 appeared to
be expressed exclusively in single spermatogonia,
rendering out our favorite marker for SSC separation.
Previous studies using MACS confirmed that Gfra-1 is
an excellent marker for SSCs as a co-enrichment of Oct-
3/4, which is considered a specific marker for pluripotent
cells and germline stem cells (primordial germ cell,
embryonic germ cells, and embryonic stem cells), was
observed in the enriched fraction (Ohbo et al, 2003;
Buageaw et al, 2005). Furthermore, our results are
strongly supported by a recently published study by He
et al (2007), who showed the double expression of Oct-3/
4 and Gfra-1 in type A spermatogonia in 6 dpp murine
r
culture of cells in the gel phase (E–G). In both approaches with isolated spermatogonia (without intratubular supporter cells in the solid phase[E]; with intratubular supporter cells in the solid phase [F]), strong expression of Cd-9 and a-smooth muscle is shown, whereas the approachcontaining all testicular cells shows a variety of markers different spermatogenic stages (G). Primer used for analysis: A/O standard marker(500-bp band is shown on both sites), B: Oct3/4; C: Gfra-1; D: C-kit; E: a-6-integrin; F: Dazl; G: Prohibitin; H: Srf-1; I: Ldh; J: Protamine-2; K:Scp-3; L: Sp-10; M: Abp; N: a-smooth muscle; P: ß-actin; Q: Cd-9. Pooled results are shown as mean 6 SD of 12 (A) and 3 (B–D) experiments.*P , .05; **P , .01; ***P , .001.
Stukenborg et al N Soft-Agar-Culture of Spermatogonia 325
Figure 7. Expression Gfra-1 (A, B, D) in SACS-cultured cells. The staining is confined to spermatogonia (DAB-positive, A, B [brown precipitate;arrowheads] and FITC-positive, D) directly in the gel phase in associated cell groups (B, D) and single cells (A) after 24 hours (A, B) and16 days (D). An image of a control labeling omitting a primary antibody is shown in C. Expression of the Boule protein (E–I) and Crem (K) inSACS-cultured cells and in the murine testis at different developmental stages (M–R). Culture approaches without (E, 11 days of culture; andG, 16 days of culture) and with tubular cell (depleted fraction) support (F, 11 days of culture; and H, 16 days of culture) showed no positiveexpression of Boule after vacuum filtration, whereas stained paraffin sections of the approach with all testicular cells (tubular and interstitialcells) as supporter resulted in associated cell groups confined to meiotic cells (DAB-positive, arrowheads [I]) directly in the gel phase after13 days of culture. Crem-positive cells are shown in this approach after at least 21 days of culture. Images of a control labeling omitting aprimary antibody are shown for Boule in J and for Crem in L. In the 10 dpp old tissue (used in our experiments as starting material for SACS),Boule and Crem are not expressed in any tubular cell type (for Boule: M; for Crem: P). We observed positive Boule expression in the testisstarting with day 16 pp (day 16: N). The staining is confined to meiotic cells in the stage of pachytene spermatocytes (arrows, N). An image of acontrol labeling omitting a primary antibody is shown (O). We observed positive Crem expression in the testis starting with day 21 pp (day 21:Q). The staining is confined to postmeiotic cells in the stage of round spermatids (open arrowheads [Q]). An image of a control labeling omittinga primary antibody is shown (R). Scale bars: 10 mm (A–L) and 20 mm (M–R).
326 Journal of Andrology N May �June 2008
testes. Taken together, these and our results indicate an
expression of Gfra-1 in SSCs before the initial differen-
tiation and expansion into pairs and chains starts, whichis also indicated by expression of Cd-9.
To explore the effect of more sensitive separation
approaches, we compared indirect approaches with
magnetically labeled secondary antibodies to strategies
using biotin-labeled secondary antibodies and antibiotin
magnetic MicroBeads. The better result in cell numbers
but similar outcome in the degree of enrichment using
the latter approach let us conclude that the efficiency ofisolation depends on the enhancement of a rather weak
cellular labeling. This indicates that even low expression
of Gfra-1 on the cell surface could be detected. This
finding also confirms previous observations that sub-
populations of SSCs exist which are characterized by
different levels of Gfra-1 expression (Buagaew et al,
2005).
On day 10 pp in the juvenile immature mouse testis,the SSC proportion is up to 100-fold higher compared
with adult tissue (de Rooij et al, 2000; McLean et al,
2003; Aponte et al, 2005). We determined a proportion
of 21%–24% Gfra-1–positive cells in immature prepa-
rations prior to sorting. In addition, isolated spermato-
gonia from immature mice showed better viability
(Creemers et al, 2002) and differentiation potential
(Nagano et al, 2003). Therefore, the use of juvenile malegerm cells seems to be beneficial for spermatogonial in
vitro development.
The SACS we used consists of 2 phases of different
agar concentrations forming a gel and a solid phase
according to Huleihel et al, 1993. This arrangement
allows addition of different supplemental factors or
supporter cell lines (eg, Sertoli cells) to the solid agar
phase without contaminating the gel phase containingthe enriched SSCs.
Colony morphology was different when the cultured
spermatogonia were grown in different SACS approach-
es; once established, it did not change during continued
culture. During the first 24 hours of SACS spermato-
gonial cell number decreased independently of the
presence of tubular cells and was shown to occur via
apoptosis due to abundant TUNEL-positive cells.However, cell survival was enhanced when germ cells
are cocultured with cells of the tubular and interstitial
fraction, and this early effect was sustained throughout
the culture period up to 16 days. Better survival of
spermatogonia in the presence of somatic cells confirms
findings from conventional in vitro experiments (Dirami
et al, 1999; Izadyar et al, 2003).
During murine male germ cell development, a firstwave of apoptosis occurs at day 16 pp (Zheng et al,
2006). We also observed an apoptotic wave in 16-day-
old germ cells (isolated at day 10 pp and maintained for
6 days in vitro). This response could reflect the first
wave of apoptosis found in vivo. However, when a
somatic cell supported the germ cell, this apoptotic wavewas not seen. It can be speculated that factors produced
by Sertoli cells and/or Leydig cells have a positive effect
on the cultured spermatogonia.
During the development of the immature testis,
Sertoli cells differentiate terminally (eg, Tarulli et al,
2006). Sertoli cells produce 2 isoforms of SCF, a
paracrine growth factor, which has inhibiting effects
on apoptosis in early spermatogenesis (Print et al, 2000;Huleihel et al, 2004). The soluble form is predominantly
expressed and important in the juvenile testis; the
membrane-bound isoform is crucial for adult spermato-
genesis (Blanchard et al, 1998; de Rooij et al, 1998).
Considering the antiapoptotic effect of SCF, our data
obtained from SACS suggest an effect on apoptotic
inhibition during spermatogonial differentiation.
If optimal culture conditions exist, meiosis should beinitiated and completed in vitro. Boule is a meiosis
marker highly expressed in mice in late pachytene or
diplotene stage spermatocytes (Xu et al, 2001). We
confirmed here that Boule protein in immature mouse
testis is not detected before day 16 pp. In SACS-cultured
germ cells, we found Boule expression at day 13 of
culture when the spermatogonia were cocultured with all
other somatic testicular cells in the gel phase of the agar,but not when spermatogonia were cultured alone or
with the tubular somatic fraction. As an additional
marker to determine meiotic processes in vitro, we
analyzed Crem expression indicating for postmeiotic/
round spermatid stages. Crem is known to be expressed
in round spermatids (Delmas et al, 1993; Wistuba et al,
2002), and is therefore the optimal marker to analyze
meiosis completion. In the SACS approach using alltesticular cells (intratubular and interstitial; Table 1), we
show that Crem-positive cells appear at least at day 21
of culture. This might be an effect of testosterone as a
product of Leydig cells located in the interstitium, which
is considered to be a crucial factor inhibiting apoptotic
events during meiosis (Print et al. 2000).
The observed mRNA expression profile supports the
results obtained by immunohistochemistry. In the
approach containing all testicular cells, almost allmeiotic genes were expressed at the mRNA level already
after 1 day of culture. This can be explained by the well-
known shift between transcription and translation of
genes during spermatogenesis (Kleene et al, 1984;
Kleene, 1996; Iguchi et al, 2006). Although only the
germ cells that were supported by all other testicular
cells progress up to meiosis, the early expression of these
mRNAs might be a necessary step preparing the laterdifferentiation. Hence, in the other experiments, we did
not find this expression pattern, and maybe for this
Stukenborg et al N Soft-Agar-Culture of Spermatogonia 327
reason we also failed to detect meiosis. Therefore, these
results indicate that our coculture approach allowed
germ cells to enter meiosis in vitro without any addition
of growth factors.
The hypothesis that in vitro meiosis is even possible
without a direct cell-cell contact has to be investigated
further. Therefore, additional experiments combining
the supporting factors (eg, LIF, GDNF, SCF, and/or
hormones like testosterone) with a three-dimensional
environment might result in completed spermatogenesis
in vitro.
AcknowledgementsThe authors thank Prof Dr R. Reijo at the University of California,
San Francisco, CA, for providing us with BOULE antibodies. J. Salzig
and H. Kersebom are thanked for technical assistance, and M.
Heuermann, G. Stelke, and O. Damm for animal caretaking. Finally,
we are grateful to Prof M. Simoni for comments on the manuscript and
Susan Nieschlag, MA, for language editing.
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