Spermatogonial Stem Cell Self-Renewal by Suppressing WNT5A Ex- pression in Sertoli ... protein kinase, AMP-activated, alpha 2 catalytic subunit. 2.71. Plbd1.
Stem Cell Reports, Volume 7
Supplemental Information
The Luteinizing Hormone-Testosterone Pathway Regulates Mouse Spermatogonial Stem Cell Self-Renewal by Suppressing WNT5A Expression in Sertoli Cells Takashi Tanaka, Mito Kanatsu-Shinohara, Zhenmin Lei, C.V. Rao, and Takashi Shinohara
Table S1. Microarray analyses. Top 50 upregulated genes in Lhcgr KO testes. Fold Gene symbol change
Gene description
8.03
Ppp2r2c
protein phosphatase 2, regulatory subunit B, gamma
5.24
Acsl6
acyl-CoA synthetase long-chain family member 6
4.49
Cbln3
cerebellin 3 precursor protein
4.38
Gm10879
4.30
Sfrp4
secreted frizzled-related protein 4
4.25
Bcan
brevican
4.10
Ahsg
alpha-2-HS-glycoprotein
4.02
Dlk1
delta-like 1 homolog (Drosophila)
3.96
4930422I07Rik
RIKEN cDNA 4930422I07 gene
3.92
Negr1
neuronal growth regulator 1
3.85
Wnt5a
wingless-type MMTV integration site family, member 5A
3.73
Bdh1
3-hydroxybutyrate dehydrogenase, type 1
3.58
Shisa6
shisa family member 6
3.12
Greb1
gene regulated by estrogen in breast cancer protein
3.07
Mapk13
3.07
Gjc3
3.02
Necab1
3.00
Ins2
2.98
Prss12
protease, serine 12 neurotrypsin (motopsin)
2.93
Thbs2
thrombospondin 2
2.91
Sfrp1
secreted frizzled-related protein 1
2.88
E130309F12Rik
RIKEN cDNA E130309F12 gene
2.87
Utf1
2.85
Hs6st2
heparan sulfate 6-O-sulfotransferase 2
2.82
Pde3b
phosphodiesterase 3B, cGMP-inhibited
2.78
Spp1
secreted phosphoprotein 1
2.77
Pde7b
phosphodiesterase 7B
2.74
Myh6|Myh7
2.73
Prkaa2
protein kinase, AMP-activated, alpha 2 catalytic subunit
2.71
Plbd1
phospholipase B domain containing 1
2.68
Stc1
2.66
5430435G22Rik
2.60
Hepacam
2.58
Phospho1|Abi3|Zfp652
2.55
Filip1
filamin A interacting protein 1
2.54
Wfdc9
WAP four-disulfide core domain 9
2.53
Slc38a1
solute carrier family 38, member 1
2.53
Duxbl|Gm10394
predicted gene 10879
mitogen-activated protein kinase 13 gap junction protein, gamma 3 N-terminal EF-hand calcium binding protein 1 insulin II
undifferentiated embryonic cell transcription factor 1
myosin, heavy polypeptide 6, cardiac muscle, alpha|myosin, heavy polypeptide 7, cardiac muscle, beta
stanniocalcin 1 RIKEN cDNA 5430435G22Rik gene hepatocyte cell adhesion molecule phosphatase, orphan 1|ABI gene family, member 3|zinc finger protein 652
double homeobox B-like 1|predicted gene 10394
2.51
Terc
telomerase RNA component
2.50
Adcyap1r1
2.48
Arhgef4
Rho guanine nucleotide exchange factor (GEF) 4
2.47
Slc24a5
solute carrier family 24, member 5
2.43
Antxr1
anthrax toxin receptor 1
2.42
Cxadr
coxsackie virus and adenovirus receptor
2.39
Clstn2
calsyntenin 2
2.39
Tbc1d30
2.38
Thbd
thrombomodulin
2.34
Cdh12
cadherin 12
2.34
Gm10044
2.33
Vtn
adenylate cyclase activating polypeptide 1 receptor 1
TBC1 domain family, member 30
predicted gene 10044 vitronectin
Top 50 downregulated genes in Lhcgr KO testes. Fold Gene symbol Gene description change -109.8
Hsd3b6
hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 6
-83.2
Spint4
serine protease inhibitor, Kunitz type 4
-73.5
Crisp1
cysteine-rich secretory protein 1
-43.5
Sult1e1
sulfotransferase family 1E, member 1
-28.4
Ptgds
-26.7
Serpina1f
-26.2
Lcn2
-23.9
Gm15070
-19.8
Spink11
serine peptidase inhibitor, Kazal type 11
-19.3
Adam7
a disintegrin and metallopeptidase domain 7
-16.6
Bhmt
-16.3
Defb23
defensin beta 23
-15.7
Ces1d
carboxylesterase 1D
-15.6
Bhmt|Bhmt2
-15.5
Serpina3n
-15.0
Klk1b27
-13.5
Aqp8
aquaporin 8
-12.0
Expi
Extracellular peptidase inhibitor
-11.6
Cd36
CD36 antigen
-11.6
Rnf128
-10.4
BC018473
cDNA sequence BC018473
-9.6
Npy6r
neuropeptide Y receptor Y6
-9.4
Actn3
actinin alpha 3
-9.3
Ust
-9.2
Defb1
defensin beta 1
-9.0
Lrg1
leucine-rich alpha-2-glycoprotein 1
-8.7
Hsd3b3
prostaglandin D2 synthase (brain) serine (or cysteine) peptidase inhibitor, clade A, member 1F lipocalin 2 predicted gene 15070
betaine-homocysteine methyltransferase
betaine-homocysteine methyltransferase|betaine-homocysteine methyltransferase 2 serine (or cysteine) peptidase inhibitor, clade A, member 3N kallikrein 1-related peptidase b27
ring finger protein 128
uronyl-2-sulfotransferase
hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 3
-8.7
Ly6f
lymphocyte antigen 6 complex, locus F
-8.6
Gm10267
predicted gene 10267
-8.4
Rnase12
ribonuclease, RNase A family, 12 (non-active)
-8.4
Prnd
-8.2
Olfr872
olfactory receptor 872
-8.0
Cwh43
cell wall biogenesis 43 C-terminal homolog
-7.6
Abcb5
ATP-binding cassette, sub-family B (MDR/TAP), member 5
-7.4
Myoz2
myozenin 2
-7.4
Spink8
serine peptidase inhibitor, Kazal type 8
-7.4
Serpina3a
-7.3
Spink10
serine peptidase inhibitor, Kazal type 10
-7.3
Igk-V1
immunoglobulin kappa chain variable 1 (V1)
-7.1
D17H6S56E-5
-7.1
Spon1
-7.1
9230104L09Rik
-7.0
Slc7a11
solute carrier family 7 (cationic amino acid transporter, y+ system), member 11
-6.9
Klk1b24
kallikrein 1-related peptidase b24
-6.7
Tspan8
tetraspanin 8
-6.6
Cuzd1
CUB and zona pellucida-like domains 1
-6.5
Car7
carbonic anhydrase 7
-6.2
Cldn8
claudin 8
-6.2
Gpt2
glutamic pyruvate transaminase (alanine aminotransferase) 2
-6.2
Exo1
exonuclease 1
prion protein dublet
serine (or cysteine) peptidase inhibitor, clade A, member 3A
DNA segment, Chr 17, human D6S56E 5 spondin 1, (f-spondin) extracellular matrix protein RIKEN cDNA 9230104L09 gene
Table S2. PCR primers Genotyping
Fshb
Mutant: GCCAGAGGCCACTTGTGTAG Common: CTTGATCTCCCTGTCCGTCT WT: CAGCGGCAGAAAACTCCTAT WT, 261 bp; Mutant, 168 bp.
Lhcgr
WT forward: TGACCTGTTCCTGGGGCTGCTG WT reverse: AAATGGCCTCAACGGGTGTGCA Mutant forward: ATGGGATCGGCCATTGAACAAG Mutant reverse: TCAGAAGAACTCGTCAAGAAGGC WT, 294 bp; Mutant, 804 bp.
Real-time PCR
Forward
Reverse
Hprt
GCTGGTGAAAAGGACCTCT
CACAGGACTAGAACACCTGC
Gdnf
GCCACTTGGAGTTAATGTCC
CTTCGAGAAGCCTCTTACCG
Fgf2
CTCTACTGCAAGAACGGCG
TTCCTCTCCTGGGAGTCCAATG
Csf1
GGGGTGGAAGACATTCTTGA
TGTCAGTCTCTGCCTGGATG
Nrg1
TGAGGAATACGAAACGACCC
GAACTTGGGTTGCTGTCCAT
Cxcl12
GCTCTGCATCAGTGACG
CCAGGTACTCTTGGATCC
Igf1
TGGATGCTCTTCAGTTCGTG
GCAACACTCATCCACAATGC
Wnt5a
TCCTATGAGAGCGCACGCAT
CAGCTTGCCCCGGCTGTTGA
Dlk1
CGTGATCAATGGTTCTCCCT
CAGAAGTTGCCTGAGAAGCC
Sfrp1
AGCGAGTTTGCACTGAGGAT
CGTTCTTCAGGAACAGCACA
Sfrp4
CATGCCAAAATAAAAGCCGT
GGAATTGGTGATGAGAGGGA
Amh
GAACACAAGCAGAGCTTCCC
AGGGTAGCCTGGGAGTCAGT
Krt18
AAGGTGAAGCTTGAGGCAGA
CTGCACAGTTTGCATGGAGT
Pdpn
TGACCCCAGGTACAGGAGAC
AGAGGTGCCTTGCCAGTAGA
Gata1
AGCAACGGCTACTCCACTGT
TGCTGACAATCATTCGCTTC
Table S3. Antibodies Immunostaining Antigen
Name
Company
SYCP3
Rabbit anti-human SYCP3
abcam (ab15093)
GATA4
Rabbit anti-human GATA4
abcam (ab84593)
GATA4
Goat anti-mouse GATA4
Santa Cruz Biotechnology (sc-1237)
GFRA1
Goat anti-rat GFRA1
R&D Systems (AF560)
CDH1
Rat anti-mouse CDH1
Takara Bio Inc. (M108)
KIT
Rat anti-mouse KIT
eBioscience (14-1171)
WNT5A
Goat anti-mouse WNT5A
R&D Systems (AF645)
CLDN11
Rabbit anti-human CLDN11
abcam (ab53041)
Secondary reagents Alexa Fluor 647-conjugated goat anti-rabbit IgG
Invitrogen (A21245)
Alexa Fluor 647-conjugated donkey anti-rabbit IgG
Invitrogen (A31573)
Alexa Fluor 555-conjugated donkey anti-goat IgG
Invitrogen (A21432)
Rhodamine Red-X-conjugated donkey anti-rat IgG
Jackson ImmunoResearch (712-295-150)
Alexa Fluor 568-conjugated donkey anti-rabbit IgG
Invitrogen (A11011)
Rhodamine labeled Peanut Agglutinin (PNA)
Vector Laboratory (RL-1072)
Western blot Antigen
Name
Company
GDNF
Rabbit anti-human GDNF
Santa Cruz Biotechnology (sc-328)
FGF2
Goat anti-human FGF2
Santa Cruz Biotechnology (sc-1390)
NRG1
Rabbit anti-human NRG1
abcam (ab53104)
ACTB
Mouse anti-ACTB
Sigma (A5441)
WNT5A
Goat anti-mouse WNT5A
R&D Systems (AF645)
AMH
Goat anti-human AMH
Santa Cruz Biotechnology (sc-6886)
KRT18
Mouse anti-human KRT18
abcam (ab668)
PDPN
Mouse anti-mouse PDPN
R&D Systems (AF3244)
GATA1
Rat anti-mouse GATA1
Santa Cruz Biotechnology (sc-266)
AR
Rabbit anti-human AR
Santa Cruz Biotechnology (sc-816)
CLDN11
Rabbit anti-human CLDN11
abcam (ab53041)
Secondary reagents HRP (horseradish peroxidase)-conjugated horse anti-rabbit IgG
Cell Signaling (7074)
HRP-conjugated horse anti-mouse IgG
Cell Signaling (7076)
HRP-conjugated donkey anti-goat IgG
Jackson ImmunoResearch (705-035-147)
HRP-conjugated rabbit anti-rat IgG
DakoCytomation (P0162)
Supplemental Experimental Procedures Transplantation procedure Testis cells were dissociated by two-step enzymatic digestion procedure using type IV collagenase and trypsin (both from Sigma, St Lois, MO), as described previously (Ogawa et al., 1997). Approximately 4- or 10 µl of cell suspension were injected through the efferent duct into W or busulfan-treated mice, because W mice have smaller testes. Each injection filled 75-85% of all seminiferous tubules. For serial transplantation experiments, whole testis sample from a primary recipient were dissociated into single cells by enzymatic digestion, and a portion of the cells were transplanted into a single secondary recipient mouse. For in vivo Wnt5a OE, approximately 10 µl of Wnt5a or control lentivirus particles (8 × 105 infectious unit/testis) was microinjected into busulfan-treated BDF1 mice or into untreated green mice. The Institutional Animal Care and Use Committee of Kyoto University approved all of our animal experimentation protocols.
Analysis of recipient testes For counting the colony numbers, recipient animals were killed two months after transplantation. Their testes were examined under UV light, and donor cell clusters were defined as colonies when the entire basal surface of the tubule was occupied and were at least 0.1 mm in length. In some experiments, recipient testes were bisected and fixed in 4% paraformaldehyde. Half of the bisected testis sample was embedded in Tissue-Tek OCT compound (Sakura Finetek, Tokyo, Japan) and processed for cryosectioning, while the other piece was embedded in paraffin for staining in hematoxylin and eosin. For immunohistochemistry, cryosection samples were treated with 0.1% Triton-X in phosphate-buffered saline (PBS). After immersing them in blocking buffer (0.1%Tween 20, 3% bovine serum albumin and 10% goat serum in PBS) for > 1 h, samples were incubated with primary antibodies at 4°C
overnight. Secondary antibodies were incubated for 1 h at room temperature. Samples were counterstained with Hoechst 33342 (Sigma). The images were collected using a confocal microscope (Fluoview FV1000D; Olympus, Tokyo, Japan). The antibodies used are listed in Table S3. To evaluate the donor cell colonization by histological analysis, two sections were made from each testis at 12 mm intervals. Each slide was viewed at 400 × magnification to confirm spermatogenesis in the testis. All sections were stained with hematoxylin and eosin.
Reverse transcription (RT)-PCR Total RNA was isolated using TRIzol Reagent (Invitrogen, Carlsbad, CA). First-strand cDNA was produced using a Verso cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA) for RT-PCR. For real-time PCR, StepOnePlusTM Real-Time PCR System (Applied Biosystems, Warrington, UK) and FastStart Universal SYBR Green Master Mix (Roche Applied Science, Mannheim, Germany) were used according to the manufacturers’ protocols. Transcript levels were normalized relative to those of Hprt. The real-time PCR conditions were 95°C for 10 min, 40 cycles at 95°C for 15 sec, and then 60°C for 1 min. Each PCR was performed at least in triplicate and gene expression levels were determined with at least three biological repeats. The primers used for PCR are listed in Supplementary Table S2.
Western blotting Samples were separated by SDS-PAGE and transferred onto Hybond-P membranes (GE Healthcare, Piscataway, NJ). The membrane was incubated with primary antibodies. Antibodies used in the experiments are listed in Supplementary Table S3.
Microarray To examine genome-wide gene expression profiles, total RNA samples were extracted with TRIzol reagent (Invitrogen) and purified using the RNeasy cleanup system (QIAGEN, Valencia, CA). Two biological replicates were prepared for each sample. To generate a labeled whole-transcript sense target, purified total RNA was converted to sense strand cDNA using the Ambion Whole Transcript Expression Kit for Affymetrix GeneChip Whole Transcript Expression Arrays (Applied Biosystems). The resultant cDNA samples were labeled using a GeneChip Whole Transcript Terminal Labeling Kit (Affymetrix, Santa Clara, CA). Labeled
cDNAs were then hybridized to Mouse Gene 1.0 ST Arrays (Affymetrix) and scanning was performed with a GeneChip Scanner 3000 7G (Affymetrix). Data analyses were performed using GeneSpring 12.0 software (Agilent Technologies, Santa Clara, CA). The probeset signals were calculated with the PLIER algorithm. The annotation is based on na32 (mm9).
Measurement of intratesticular testosterone Total testicular teststerone was measured by enzyme immunoassay with Testosterone EIA Kit (Cayman, Ann Arbor, MI). The testosterone from the testicular lysate was extracted with diethyl ether and was assayed as manufacturer’s protocol.
Cell culture GS cells expressing Egfp transgene were described previously (Kanatsu-Shinohara et al., 2003). Cells were maintained on mouse embryonic fibroblasts that had been treated with mitomycin C (Sigma). GS cells were maintained in Iscove’s modified MEM-based medium (Sigma), which was supplemented with 10 ng/ml human FGF2, 15 ng/ml recombinant rat GDNF (both from Peprotech, London, UK) and 1% fetal bovine serum (FBS), as described previously (Kanatsu-Shinohara et al., 2014). For co-culture experiments, NIH3T3 cells (ATCC, Manassas, VA) were infected with CSII-EF-IRES2-Venus vector that contains mouse Wnt5a, Sfrp1, and Sfrp4 (Open Biosystems, Huntsville, AL), or Dlk1 (gift from Dr. A. Fersuson-Smith, University of Cambridge, UK) 7 days before initiation of co-culture. The multiplicity of infection was for NIH3T3 cells 10.0, which was determined by infection of 293T cells using flow cytometry. GS cells (1 × 105) were plated on NIH3T3 cells (1.5 × 105/3.8 cm2) and cell recovery was determined 7 days after co-culture.
Tracer experiment Biotin tracer experiment was carried out as described previously (Takashima et al., 2011). In brief, approximately 15 ml of PBS containing Sulfo-NHS-LC-biotin (7.5 mg/ml; Thermo Fisher Scientific) was microinjected into the interstitium of 4-6-week-old Lhcgr KO mouse testes. After 30 min, the animals were killed, and their testes were fixed in 4% paraformaldehyde at 4°C overnight. The samples were embedded in
Tissue-Tek OCT compound and processed for cryosectioning. Sections were incubated with phycoerythrin (PE)-conjugated streptavidin (BD Biosciences) before counterstaining with 5 mg/ml Hoechst 33342.
Supplemental References Kanatsu-Shinohara, M., Ogonuki, N., Inoue, K., Miki, H., Ogura, A., Toyokuni, S., and Shinohara, T. (2003). Long-term proliferation in culture and germline transmission of mouse male germline stem cells. Biol. Reprod. 69, 612-616.
Kanatsu-Shinohara, M., Ogonuki, N., Matoba, S., Morimoto, H., Ogura, A., and Shinohara, T. (2014). Improved serum- and feeder-free culture of mouse germline stem cells. Biol. Reprod. 91, 88.
Ogawa, T., Aréchaga, J.M., Avarbock, M.R., and Brinster, R.L. (1997). Transplantation of testis germinal cells into mouse seminiferous tubules. Int. J. Dev. Biol. 41, 111-122.
Takashima, S., Kanatsu-Shinohara, M., Tanaka, T., Takehashi, M., Morimoto, H., and Shinohara, T. (2011). Rac mediates spermatogonial stem cell homing to germline niches by regulating transmission through the blood-testis barrier. Cell Stem Cell 9, 463-475.
