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From the * Institute of Reproductive Medicine of
the University, Münster, 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.
| Correspondence to: Eberhard Nieschlag, Institute of Reproductive Medicine of the University, Domagkstrasse 11, 48129 Münster, Germany (e-mail: Eberhard.Nieschlag{at}ukmuenster.de). |
| Received for publication March 16, 2007; accepted for publication November 19, 2007. |
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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-1 as a specific surface marker and
magnetic-activated cell sorting as a separation approach. 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
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, 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 physical contacts 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 from the 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 environmental features allowing recognition and settlement of SSCs. They also might provide crucial factors needed for 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 conventional 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 established to 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 methodology for 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 with the
STAPUT technique (Dirami et al,
1999), fluorescence-activated cell sorting
(Shinohara et al, 2000;
Fujita et al, 2005;
Guan et al, 2006), or
magnetic-activated cell sorting (MACS; von
Schönfeldt 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 SSCs is 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 Schönfeldt et al,
1999; Buageaw et al,
2005). To isolate the population of undifferentiated spermatogonia
in mice, marker proteins such as GDNF family receptor-alpha-1 (Gfr-1;
Meng et al, 2000;
von Schönfeldt et al,
2004), Cd-9 (tetraspanin transmembrane protein;
Kanatsu-Shinohara et al,
2004b), and Thy-1 (glycosyl phosphatidylinositol–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 (Kubota et 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 this study, we aimed to
characterize a novel three-dimensional culture system and determine the
survival, expansion, and differentiation of germ cells.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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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, Gaithersburg, 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 37°C
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 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).
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-1, Cd-9, and Thy-1, and secondary or
tertiary anti-rabbit or anti-biotin antibodies carrying ferromagnetic
particles (von Schönfeldt et al,
1999). In brief, the cells were incubated with a polyclonal rabbit
anti–Gfr-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 6°C–10°C. 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 µL degassed buffer. The Gfr-1–labeled
cell suspension (Table 1) was
resuspended in degassed buffer and poured into the column reservoir.
Gfr-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 µL 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 µL 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 microscopically evaluated using Trypan blue staining.
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Flow Cytometry
The efficiency of spermatogonial enrichment was quantitatively 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 6°C–8°C. 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 determined by comparison with the
control sample. A minimum of 105 cells was analyzed in each run
(according to von Schönfeldt 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 embedded in paraffin using an
automated processor. Cultured cells (within the agar phase) were fixed in 4%
paraformalde-hyde (PFA) for 24 hours at 6°C–8°C 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 µm
and were immunohistochemically stained for Cd9, Gfr-1 (spermatogonial
markers), cAMP reponse element modulator (Crem; postmeitotic spermatids), and
5-bromodesoxyuridine (BrdU; proliferation marker;
Figure 1C). Polyclonal primary
antibodies against the peptide ETQEDAQKILQEAEKLNYKDKKLN (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 80°C. 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–Gfr-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 x 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
x 5 minutes). Afterwards, the sections were incubated with biotinylated
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 µg/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 horseradishperoxidase 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 Gfr-1 expression in unsorted and enriched MAC-sorted
fractions, single-cell solutions were stained for Gfr-1 with an
FITC-labeled secondary antibody (Sigma) in combination 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 Gfr-1 on the cell surface of undifferentiated
spermatogonia was evaluated by confocal microscopy (TCS SL; Leica, Wetzlar,
Germany).
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, Loughborough, 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 37°C, avoiding heat-induced cellular stress and
premature coagulation of the agar. Culture conditions were 35°C 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 µm.
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 Reverse Transcription–Polymerase Chain Reaction of Fresh 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 µg total RNA with 0.5 µg
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 µL Tris-HCl-MgCl reaction buffer,
supplemented with dithiothreitol, dinucleotriphosphates (0.5 mM; Roche
Molecular Biochemicals), and RNase inhibitor (40 units; Roche Molecular
Biochemicals). The reverse transcriptase (RT) reaction was performed for 1
hour at 37°C and stopped for 10 minutes at 75°C. The volume of 20
µL was subsequently filled up to 60 µL with water. Negative controls for
the reverse transcriptase reaction (RT–) 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 performed with negative controls of the RT reaction (RT–) and without cDNA (cDNA–). The PCR reactions were carried out on a Cycler II System Thermal Cycler (Ericomp, San Diego, California). A total of 20 µL 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 µMACS oneStep cDNA Kit
(Miltenyi), following the manufacturer'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
x g for 3 minutes to separate the mRNA. After separation, the
lysate was mixed with 50 µL 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 µL
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 42°C for 1 hour. Subsequently, cDNA
was washed 2 times with equilibration/wash buffer within the column. To
release the cDNA from the magnetic beads, 20 µL cDNA-Release solution
(Miltenyi) was applied for 10 minutes at 42°C on the top of the column.
Synthesized cDNA was eluted with 50 µL cDNA Elution buffer (Miltenyi). The
efficiency of cDNA synthesis was verified by PCR amplification of the
β-actin gene. Specific expression of different spermatogenic
stage-specific markers (Table
2) was investigated by PCR amplification. PCR conditions were 2
minutes at 94°C, 35 x 50 seconds at 94°C, 50 seconds at
58°C, and 1 minute at 72°C (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 ± SD is given in the
figures as described in the legends.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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-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
Gfr-1 and Cd-9. In the juvenile testis, Gfr-1 and Cd-9 were
expressed in single spermatogonial cells
(Figure 2B, C, H, and I). In
the adult testis, the predominant Gfr-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 the absence
of any specific positive staining in the juvenile and adult tissue sections
(Figure 2A, D, G, and J).
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-1 (Figure 3A through
C), anti–Cd-9 (Figure 3D
through F) and, as a third marker of 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
Gfr-1 showed the highest percentage of events in the cell population of
the enriched fraction. Therefore, the staining pattern and the flow cytometric
analysis indicated that Gfr-1 is the most suitable of the 3 markers
analyzed for isolation of undifferentiated spermatogonia.
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 Gfr-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 Gfr-1 (Figure
4G). RNA analysis revealed an expression profile typical for
spermatogonial cells in the enriched fraction after MACS only. The expression
of Gfr-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%.
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-1–positive cells. The use of magnetically labeled secondary
antibodies resulted in a twofold to threefold (up to 42% positive cells)
enrichment rate for Gfr-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.
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Evaluation of Germ Cell Development With and Without Supporter Cells Using the SACS
Three experimental approaches were selected to compare 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.
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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).
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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 ± 39.1 [SD]; with supporter cells: 357.6 ± 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 µMACS kit. The small
amount of cell numbers resulted in very low levels of mRNA. Therefore, we
could only analyze expression profiles on 1 day of culture
(Figure 6E and F). In the 2
approaches using Gfr-1–isolated spermatogonia
(Figure 6E and F), a strong
expression of Cd-9 (undifferentiated spermatogonia;
Figure 6E and F, lane Q),
-smooth muscle (peritubular cells;
Figure 6E and F, lane N), and
β-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 the
approach containing all testicular cells, mRNA of different spermatogenic
stages was observed (Figure
3G). In this approach, the mRNA expression was detectable for
spermatogonial and meiotic genes.
Characterization of the cultured cells in the gel phase by
immunohistochemistry revealed the presence of Gfr-1–positive
cells in histologic sections and on filters after vacuum filtration at
different time points (Figure 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 performed immunohistochemical
localization of Boule (Figure 7E through
J) and Crem (Figure 7K and
L). Boule is 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).
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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). Immunohistochemistry 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).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Successful enrichment and separation of isolated testicular cells from
mouse tissue using MACS has previously been described
(von Schönfeldt 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).
Therefore, Gfr-1, Cd-9, and Thy-1 were analyzed as putative mouse SSC
markers (Meng et al, 2000;
Kanatsu-Shinohara et al,
2004b; von Schönfeldt 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 spermatogonia indicated that Cd-9 is a marker for
Asingle and Aaligned spermatogonia. In contrast,
Gfr-1 appeared to be expressed exclusively in single spermatogonia,
rendering out our favorite marker for SSC separation. Previous studies using
MACS confirmed that Gfr-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 Gfr-1 in type A spermatogonia in 6
dpp murine testes. Taken together, these and our results indicate an
expression of Gfr-1 in SSCs before the initial differentiation and
expansion into pairs and chains starts, which is 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 of isolation depends on the enhancement of a rather weak cellular
labeling. This indicates that even low expression of Gfr-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 Gfr-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% Gfr-1–positive cells in
immature preparations prior to sorting. In addition, isolated spermatogonia
from immature mice showed better viability
(Creemers et al, 2002) and
differentiation potential (Nagano et al,
2003). Therefore, the use of juvenile male germ 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 containing the enriched SSCs.
Colony morphology was different when the cultured spermatogonia were grown in different SACS approaches; once established, it did not change during continued culture. During the first 24 hours of SACS spermatogonial 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 first wave of apoptosis occurs at day 16 pp (Zheng et al, 2006). We also observed an apoptotic wave in 16-dayold 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 wave was 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 be initiated 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 all testicular 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 all meiotic 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 later differentiation. Hence, in the other experiments, we did not find this expression pattern, and maybe for this 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.
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Acknowledgments |
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