coculture of spermatogonia with somatic cells in a novel three-dimensional soft-agar-culture-system

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.


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.


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.


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.


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.


Table 3. Quantitative and flow cytometric evaluation of MACS by the use of standard MicroBeads compared with anti-biotin MicroBeadsa


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.


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.


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.


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


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.


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).


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


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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coculture of spermatogonia with somatic cells in a novel three-dimensional soft-agar-culture-system

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