Primates (2016) 57:129–135
DOI 10.1007/s10329-015-0500-4

ORIGINAL ARTICLE

Sphere-formation culture of testicular germ cells in the common
marmoset, a small New World monkey
Zachary Yu-Ching Lin1 • Orie Hikabe1 • Sadafumi Suzuki1 • Takamasa Hirano2 •Haruhiko Siomi2 • Erika Sasaki1,3,4 • Masanori Imamura1,5 • Hideyuki Okano1

Received: 20 August 2015 / Accepted: 21 October 2015 / Published online: 3 November 2015
Ó Japan Monkey Centre and Springer Japan 2015

Abstract Spermatogonia are specialized cells responsible
for continuous spermatogenesis and the production ofoffspring. Because of this biological property, in vitro
culture of spermatogonia provides a powerful methodology
to advance reproductive biology and engineering. However, methods for culturing primate spermatogonia arepoorly established. We have designed a novel method for
culturing spermatogonia in the common marmoset (Callithrix jacchus), a small primate. By using our method witha suite of growth factors, adult marmoset testis-derived
germ cells could be cultured in the form of a floating spherefor several weeks. Notably, this method could be applied
not only to freshly isolated cells but also to cryopreserved
cell stocks. The spheres enriched spermatogonia and earlyspermatocytes, and could be assembled from a C-KIT?
spermatogonial population. Techniques for culturing& Masanori Imamura
imamura.masanori.2m@kyoto-u.ac.jp& Hideyuki Okano
hidokano@a2.keio.jp
1

Department of Physiology, School of Medicine, Keio
University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582,
Japan

2

Department of Molecular Biology, School of Medicine, Keio
University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582,
Japan

3

Department of Applied Developmental Biology, Central
Institute for Experimental Animals, 3-25-12 Tonomachi,
Kawasaki 210-0821, Japan

4

PRESTO Japan Science and Technology Agency, Tokyo,
Japan

5

Molecular Biology Section, Department of Cellular andMolecular Biology, Primate Research Institute, Kyoto
University, Inuyama, Aichi 484-8506, Japan

spermatogonia could facilitate increased understanding ofprimate reproduction as well as the preservation of valuable biomaterials from nonhuman primates.
Keywords
cultureCommon marmoset  Testis  Germ cell

Introduction
Spermatogonia are specialized cells responsible for producing sperm throughout a male’s life. To guarantee continuous spermatogenesis, spermatogonial stem cells renew
themselves while giving rise to a large number of differentiated cells. In the context of advancing our understanding of spermatogonial physiology, in vitro culture
systems provide a powerful method of investigating their
biological characteristics and the underlying molecularmechanisms. In rodents, this has led to successful longterm culture of spermatogonia, denoted germline stem cells(GSCs; Kanatsu-Shinohara et al. 2003). GSCs proliferate
stably in culture and resume spermatogenesis upon transplantation into seminiferous tubules. Notably, GSCs areavailable as a biological vector to preserve or create animals with a particular genetic mutation (Takehashi et al.
2010). Thus, techniques for culturing spermatogonia could
potentially allow the conservation of valuable bioresources
and reproductive engineering.
In contrast, the methodology for culturing primate
spermatogonia has not been established, due mainly to alack of accessibility to primate samples, although theimportance of primate research has increased in recent
years. Among nonhuman primates, the common marmoset(Callithrix jacchus), a small New World monkey, hasmany advantages as an experimental model for life

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sciences: it produces many offspring during its lifetime anddisplays physiological and developmental similarities tohumans (Li et al. 2005; Okano et al. 2012). Furthermore,
artificial reproductive technologies have been developed to
produce offspring in marmosets (Sasaki et al. 2009;
Tomioka et al. 2012; Takahashi et al. 2014). A recent study
also attempted to culture marmoset spermatogonia (Langenstroth et al. 2014); however, long-term culture has notbeen achieved.
In this study, we aimed to develop a long-term culture ofmarmoset testicular germ cells, especially spermatogonia.
Using our method, we were able to maintain marmoset
testicular germ cells in culture for at least 2 months. Cell
culture could be initiated not only from freshly isolated
testis cells but also from cryopreserved cells. This technique may therefore represent a conventional method ofpreserving and culturing testicular germ cells from valuable nonhuman primate species for reproductive biology.

Methods
Animals and tissue collection
The marmosets used in this study were housed in cages
measuring 82 9 61 9 160 cm with wire mesh floors,which were maintained at 25–26 °C with 45–55 %humidity and illuminated for 12 h per day at the Central
Institute for Experimental Animals (CIEA). Animal care
was conducted in accordance with the recommendations of
the Guide for the Care and Use of Laboratory Animals(Institute of Laboratory Animal Resources, 1996). All
animal experiments were approved by the Institutional
Animal Care and Use Committee of CIEA (license numbers: 12025, 13071, 14030) and performed in accordancewith CIEA and Keio University guidelines concordant with
the Guidelines for Proper Conduct of Animal Experiments
by the Science Council of Japan (2006). For tissue collection, testes were obtained from more than 10 adultmarmosets (2, 3, 4, 7, 8, and 10 years of age) that wereeuthanized because of disease, accidents, or otherexperiments.
Cell culture
For cell culture, seminiferous tubules were physicallyexcised and digested in Dulbecco’s modified Eagle’s
medium (DMEM) containing 10 mM Hepes, 20 ng/ml
collagenase, and 4 mg/ml dispase II at 37 °C for 15 minwith shaking. The cell suspension was consecutively passed through 70- and 40-lm cell drainers, and then seeded ata density of 1.5 9 105 cells/well in 6-well low-attachment
plates with knockout DMEM supplemented with 20 %123

Primates (2016) 57:129–135

knockout serum replacement, 100 lM non-essential aminoacids, 1 mM L-glutamine, 1 mM 2-mercaptoethanol,
20 ng/ml human GDNF, 200 ng/ml human GFR-a1, 10 ng/
ml human bFGF, 10 ng/ml mouse SCF, 12.5 ng/ml human
FGF9, and 103 U/ml LIF. The cells were cultured at 37 °C
in a humidified atmosphere of 5 % CO2 and 95 % air. Halfof the medium was exchanged every 2 days. For storage,
the cell suspension was cryopreserved with CELLBANKER 2 (Takara) in liquid nitrogen.
Cell sorting
For cell sorting, cells were suspended in ice-cold Hank’sbalanced salt solution at 1 9 107 cells/ml and incubatedwith a mouse anti-C-KIT antibody (Mar117-22; Oriental
Yeast) for 30 min on ice. After two washes with ice-cold
phosphate-buffered saline (PBS), 1 9 106 cells were gently
resuspended in ice-cold 70 % ethanol and incubated at
4 °C for 2 h. The fixed cells were washed again with PBS
and then treated with 25 lg/ml RNase A. After incubation
at 37 °C for 1 h, propidium iodide (PI) was added at a final
concentration of 50 lg/ml, and the cell suspension was
incubated at 4 °C in the dark for 1 h. Flow-cytometricanalysis and cell sorting were performed using a FACS
Aria III (BD Biosciences).
Immunofluorescence microscopy
Alkaline phosphatase (AP) staining was performed according
to the manufacturer’s instructions (Sigma). For whole-mountimmunofluorescence microscopy, spheres were adhered tofibronectin-coated coverslips on the day before experiments.
The spheres were then fixed with 10 % formaldehyde, permeabilized with 0.5 % Triton-X, blocked with 5 % skim milk,
and incubated with primary antibodies. For microtomy,
spheres were solidified in iPGell (Genostaff), fixed in 4 %PFA, embedded in paraffin, and sliced at a thickness of 5 lm,
as previously described (Lin et al. 2015). The primary antibodies used were rabbit anti-DDX4 antibody (ab13840;
Abcam), rabbit anti-DAZL antibody (ab34139; Abcam),mouse anti-MARWI antibody (2D9-1H6 clone), mouse antiKi67 antibody (NCL-Ki67 MM1; Leica Biosystems), andrabbit anti-SALL4 antibody (ab29112; Abcam). Nuclei were
stained with 10 ng/ml Hoechst 33342.
RT-PCR analysis
RNA extraction and RT-PCR were performed as described
previously (Lin et al. 2012). Briefly, total RNA preparedwith TRIzol reagent was isolated using an RNeasy Mini
Kit (Qiagen). First-strand cDNA was synthesized with
SuperScript III reverse transcriptase (Invitrogen) and an
oligo-dT18 primer. PCR was semiquantitatively performed

Primates (2016) 57:129–135

131

with Ex Taq Hot Start Version (Takara) using three different escalatory cycles, and only representative images areshown in the results.

Results
Surgically isolated adult marmoset testes were dissociatedfor cell culture. The testicular cells were cultivated insuspension, in which they formed cell clumps around day
3. Small spheres formed by week 1 and grew during(a) Week 1

4 weeks of culture (Fig. 1a). The spheres could be formed
not only from freshly isolated testicular cells but also fromcryopreserved cells (Fig. 1b). Compared with freshly isolated cells, cryopreserved cells formed morphologicallygood spheres with less debris, possibly due to the elimination of inappropriate somatic cells via the freeze-thawing
process (Langenstroth et al. 2014). Sphere formation was
induced in a growth-factor-dependent manner; no spheres
were formed in the absence of growth factor cocktail(Fig. 1c). These spheres, named testicular spheres (TeS),
enclosed AP-positive germ cells (Fig. 1d). TeS could be

Week 3

Week 4100 m(b)

Week 1
Fresh cells

200 m

Week 2

Week 3200 m

Week 4Cryopreserved
cells

(c)

Growth factor

Week 2

Week 4

(d)

+
(e)

-

(d)

Fig. 1 Sphere formation culture of marmoset testicular cells. a Morphology of TeS during 4 weeks of culture. b TeS formation culture
from freshly isolated or cryopreserved testicular cells. Scale bar

500 lm. c Growth-factor-dependent formation of TeS. d Alkaline
phosphatase staining. Scale bar 100 lm. e Morphology of 60-day TeS

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Primates (2016) 57:129–135

cultured for at least 2 months without passaging while
retaining AP activity, although there were fewer spheres inculture that had been performed for over a month (Fig. 1e).
Immunofluorescence microscopy showed that DAZL?
(spermatogonia-spermatocyte) and VASA? (spermatocytespermatid) cells were incorporated in the 1-week TeS(Fig. 2a). In the 2-week TeS, quiescent (SALL4) and active(Ki67) spermatogonia markers as well as a spermatocytespermatid marker (MARWI) were detected (Fig. 2b).

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DAZL

Merge

VASA

Merge

(b) Ki67

SALL4

Merge

Week 2

(a)

MARWI

SALL4

Merge

MARWI

SALL4

Merge

Week 4

Fig. 2 Immunofluorescenceanalyses of spermatogonia andspermatocyte markers in TeS.
a Whole-mountimmunofluorescence analyses of
DAZL and VASA proteins.
Nuclei were counterstained with
Hoechst 33342. Scale bar
200 lm. b Immunofluorescenceanalyses of Ki67, SALL4, and
MARWI in the TeS sections.
Scale bar 50 lm

However, MARWI protein disappeared by week 4, while
SALL4 protein was still detectable. Time-course RT-PCR
analyses of TeS revealed that genes associated with laterstages of spermatogenesis disappeared earlier during culture. The 1-week TeS expressed all spermatogenesis-related genes examined except for STRA8 (Fig. 3). However,in prolonged culture, the expression of post-meiotic
markers (TEKT1, HAPRIN. etc.) decreased or stopped, in
contrast to that of spermatogonia and early spermatocyte

Primates (2016) 57:129–135

133

TeS culture (week)
0

1

2

3

4 H2O

SALL4
PLZF
LIN28
C-KIT
PRDM1

Indeed, the transcripts of spermatogonia markers (SALL4,
PLZF etc.) were enriched in C-KIT? cell populations(Fig. 4b), while meiotic, post-meiotic, and Leydig cellmarkers were predominant in C-KIT- cell populations.
Furthermore, when both cell populations were supplied for
TeS culture, only C-KIT? cell populations formed spheres(Fig. 4c), suggesting that TeS formation in culture could be
attributed to spermatogonial activity.

NANOS2GFRα1STELLADAZLDNMT3L
GCNF
VASA
MARWI
NANOS3
SYCP1
SYCP3
PRM1
GASZ
MAEL
PIWIL2
TEKT1
BOULE
PRDM9
HAPRIN
STRA8
MIS
SOX9
LHR
SF1
3βHSD
β-ACTIN
Fig. 3 Enrichment of early spermatogenic cells during TeS culture.
RT-PCR analyses of spermatogenesis-related genes during 4 weeks
of TeS culture. b-actin was analyzed as an internal control and water
was used as a negative control

markers, which remained even later during culture. As forsomatic cells, the expression of Sertoli cell markers (MIS,
SOX9) was regressed upon cultivation, while the expression of Leydig cell markers gradually decreased (LHR) or
remained constant (SF1, 3bHSD).
To confirm whether the genes that were highly enriched in
the later TeS reflected the character of early spermatogenic
cells, especially spermatogonia, we sorted and characterized
C-KIT? dividing/differentiating spermatogonia (Fig. 4a).

Discussion
Sophisticated methods have enabled us to culture rodent
spermatogonia (Kanatsu-Shinohara et al. 2003), while thesituation in primates is quite different; no methods for germ
cell culture have been established yet (Imamura et al.
2013). Previous studies have addressed primate testicularcell culture, but they achieved only short-term culture of
spermatogonia and/or considerable expansion of somatic
cells (Eildermann et al. 2012; Albert et al. 2012; Kossack
et al. 2013; Langenstroth et al. 2014; Zheng et al. 2014).
Our recent work has revealed some of the characteristics ofmarmoset spermatogenesis (Lin et al. 2012; Hirano et al.
2014; Lin et al. 2015), so methods of cultivating marmoset
spermatogonia are now required. In the present work, we
succeeded in cultivating testicular germ cells of adultmarmosets in the form of floating spheres named TeS.
Gene expression and cell sorting analyses suggested thatTeS were preferentially constituted by spermatogonia andearly spermatocytes. This is reasonable because, among
spermatogenic cells, only spermatogonia undertake mitotic
division, and therefore proliferate continuously. Notably,
the TeS formation culture was applicable not only to
freshly isolated cells but also to cryopreserved cell stocks.
Thus, cryopreservation and culture of nonhuman primate
spermatogonia, together with other stem cell technologies
including induced pluripotent stem cells (Imamura et al.
2012, 2014), appears be a promising method of preserving
unique genetic material of endangered or mutant nonhuman primates (Oishi et al. 2014; Suzuki et al. 2010; Onishi
et al. 1999) as well as a useful research tool for basic andapplied reproductive biology.
Two major concerns remain in relation to further
improving marmoset spermatogonia culture. The first is theclonal propagation of marmoset spermatogonia in culture.
Marmoset TeS comprise heterogeneous cell populations
while rodent GSC can be clonally derived from single
spermatogonia (Kanatsu-Shinohara et al. 2005). We andothers have tried to establish clonal culture of marmoset
spermatogonia but have not yet succeeded (Langenstrothet al. 2014). The second concern is reconstructing the entire
process of spermatogenesis in vitro. In prolonged culture,
TeS lost the ability to express post-meiotic genes,

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lls
O

C-KIT

H2

FL3-H: PI

10

ce

3

(+) (-)

le

10

(b)

PI

ho

4

W

(a) 102

SALL4

Viable cells

PLZF

101

LIN28
10

26.2

0

0
10

200

400
600
FSC-H: FSC

800

1000

4

C-KIT
10

FL1-H: FITC

Fig. 4 Isolation and TeS
formation culture of C-KIT?
testicular cells. a Sorting of
C-KIT? cells fromcryopreserved testicular cells.
After live cell selection by PI
staining, the C-KIT? cell
fraction enclosed in a box withpurple borders were collected.
b Gene expression of C-KIT?
and C-KIT- cell populations
separated from whole testicular
cells. b-actin was analyzed as aninternal control and water wasused as a negative control.
c TeS formation from a C-KIT?
cell population. AP staining was
performed. Scale bar 200 lm

Primates (2016) 57:129–135

10

2

MARWI
GFR 1

C-KIT (+)
cells

13.7

DAZL

0

100

(c)

GPR125PRDM1NANOS2

3

101

10

C-KIT

101

102
FL2-H: PE

103

104

STELLAVASA

C-KIT+ TeS

SYCP1
HAPRINLHR
3 HSD-ACTINsuggesting that (1) meiotic germ cells left over from testis
samples gradually become diminished, and (2) de novo
spermatogenesis does not occur in TeS culture. To resume
spermatogenesis, especially meiosis, in vitro, we stimulated TeS with retinoic acid and/or testosterone; however,
the meiotic/post-meiotic genes were not induced (data notshown). Since spermatogenesis can be reconstructed byculturing testis fragments in mice (Sato et al. 2011), similar
methods should be developed for primates as well.
Advances in these techniques, combined with germ celltransplantation (Langenstroth et al. 2014), will provide thefundamental steps needed to produce cultured spermatogonia-derived sperm.
In conclusion, we have demonstrated a method of cultivating germ cells derived from freshly prepared or cryopreserved marmoset testes. This should make it easy toaddress primate reproductive biology and should also
facilitate the preservation of valuable biomaterials in
nonhuman primates.
Acknowledgments This work was supported by grants from the
Ministry of Education, Culture, Sports, Science, and Technology of123

Japan (MEXT); the Ministry of Health, Labour, and Welfare; theJapan Society for the Promotion of Science (JSPS); the National
Institute of Biomedical Innovation and the Strategic Research Program for Brain Sciences from MEXT; the Funding Program forWorld-Leading Innovative R&D in Science and Technology (FIRST);a Keio University Grant-in-Aid for the Encouragement of Young
Medical Scientists; Leave a Nest Grant Life Technologies Japan
Award; Interuniversity Bio-Backup Project for Basic Biology; theBrain Sciences Project of the Center for Novel Science Initiatives(CNTI), National Institutes of Natural Sciences (NINS); and Otsuka
Toshimi Foundation.
Compliance with ethical standards
Conflict of interest H.O. is a Scientific Advisory Board Member of
San Bio Co. Ltd. The other authors have no competing interests.

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