Supplementary figure SF1. CGC morphology cultivated in different growth media on days 1, 4, 7, 13 and
24. The scale bar corresponds to 100 μm.
Target Forward primer Reverse primer
FSHR TTGGAGGTGATAGAGGCAGA ATGTAGAGCAGGTTGTTGGC AMHR2 CTACAGAGTGCGAGGTGAGC TCCCGGATTACCTGGGAGAA LHR GGATAGAAGCTAATGCCTTTGA ACTTTCTGATGCCTGTGTTAC CYP19A
1 CATGCGAGTCTGGATCTCTG CCAAGTTTGCTGCCGAATC GnRHR TCCGAGTGACGGTTACTTTC CTCCAACAGGTTGGCTAAGG AMH GGTGCTGCTGCTGAAGAT TCCTCCGACAGGCTGATG CYP11A
1 GCTGAAGTGGAGCAGGTACA GCACTCAGAAAGGTCTGGCA
IGF1 ACCACCTCCCTTCATAACCT CTTCTTGACGACTTGCTGCT
IGF2 AGCAATCGGAAGTGAGCAA GGGATGGAACCTGATGGAAAC
IGF1R AAGCAGCATTGGGAGATGTG CTGGCAACAGAGGTTCAGTC
IGF2R CAACGACGGATACAGACCAG AATCTTCCCAATGCCAGAGAC
INSR GGTTTCCACAGTTGCTGCT ACAGGTGCTTTCTTTCCATCT
FGF2 AGCAGAAGAGAGAGGAGTTGT TCCGTAACACATTTAGAAGCCA
FGFR1 CCACCGACAAAGAGATGGAG CGATAGAGTTACCCGCCAAG
FGFR2 TGATGCCACAGAGAAAGACC GCAGGCTCCAAGAAGATTTATG
FGFR3 CTGAAAGACGATGCCACTGA CCCAGCAGGTTGATGATGTT
FGFR4 GCTCAAAGACAACGCCTCT CCGATCAGCTTCATCACCTC
SDHA TGGGAACAAGAGGGCATCTG CCACCACTGCATCAAATTCATG
Supplementary table ST2. Relative gene expression of selected genes normalized to SDHA in CGCs. CGCs were cultivated in either regular, IGF2, FGF2 or IGF2 + FGF2 growth medium, time points were taken on days 4, 13 and 24. The results are shown as mean value, standard deviation (SD), and fold change (FC) relative to day 0 of three independent experiments. Green background represents upregulated genes, red background downregulated genes.
Target Medium
Day 0 Day 4 Day 13 Day 24
Mean SD Mean SD FC Mean SD FC Mean SD FC
FSHR regular
0.076 0.032
0.537 0.287 7.104 0.107 0.063 1.417 0.023 0.013 0.307
IGF2 0.394 0.399 5.211 0.051 0.050 0.670 0.023 0.012 0.301
FGF2 0.243 0.168 3.215 0.043 0.032 0.565 0.002 0.003 0.029
IGF2 + FGF2 0.402 0.203 5.326 0.062 0.039 0.819 0.010 0.015 0.133
AMHR2 regular
0.028 0.007
0.031 0.013 1.117 0.009 0.003 0.336 0.000 0.000 0.000
IGF2 0.022 0.019 0.790 0.006 0.006 0.203 0.000 0.000 0.000
FGF2 0.022 0.013 0.777 0.009 0.004 0.306 0.001 0.001 0.028
IGF2 + FGF2 0.031 0.011 1.122 0.009 0.002 0.330 0.003 0.005 0.106
LHR regular
0.518 0.249
0.300 0.057 0.579 0.180 0.085 0.348 0.020 0.006 0.039
IGF2 0.239 0.217 0.462 0.108 0.109 0.209 0.015 0.011 0.028
FGF2 0.228 0.128 0.440 0.071 0.070 0.138 0.010 0.011 0.019
IGF2 + FGF2 0.369 0.071 0.712 0.126 0.082 0.243 0.009 0.011 0.017
CYP19A1 regular
12.759 3.611
3.104 1.718 0.243 0.049 0.024 0.004 0.020 0.012 0.002
IGF2 2.958 1.077 0.232 0.053 0.029 0.004 0.014 0.015 0.001
FGF2 4.242 3.804 0.333 0.057 0.025 0.005 0.039 0.006 0.003
IGF2 + FGF2 3.666 1.660 0.287 0.059 0.028 0.005 0.028 0.011 0.002
GnRHR regular
0.024 0.002
0.019 0.007 0.786 0.009 0.003 0.395 0.011 0.005 0.450
IGF2 0.019 0.008 0.815 0.007 0.003 0.296 0.012 0.001 0.493
FGF2 0.016 0.007 0.659 0.013 0.004 0.553 0.013 0.007 0.536
IGF2 + FGF2 0.020 0.004 0.825 0.017 0.004 0.732 0.012 0.004 0.502
IGF1 regular
0.000 0.000
0.000 0.000 NA 0.000 0.001 NA 0.005 0.008 NA
IGF2 0.000 0.000 NA 0.000 0.000 NA 0.009 0.013 NA
FGF2 0.000 0.000 NA 0.003 0.005 NA 0.010 0.014 NA
IGF2 + FGF2 0.000 0.000 NA 0.002 0.004 NA 0.003 0.005 NA
IGF2 regular
0.613 0.539
0.247 0.290 0.399 0.168 0.207 0.272 0.118 0.106 0.191
IGF2 0.127 0.119 0.205 0.171 0.207 0.278 0.116 0.024 0.188
FGF2 0.082 0.073 0.132 0.065 0.063 0.105 0.063 0.041 0.101
IGF2 + FGF2 0.254 0.301 0.410 0.082 0.059 0.133 0.080 0.052 0.129
IGF1R regular
4.829 0.445
0.789 0.129 0.164 0.517 0.218 0.107 0.522 0.279 0.108
IGF2 0.770 0.171 0.159 0.586 0.253 0.121 0.605 0.419 0.125
FGF2 0.573 0.106 0.119 0.512 0.267 0.106 0.534 0.300 0.111
IGF2 + FGF2 0.688 0.191 0.142 0.451 0.202 0.093 0.465 0.207 0.096
IGF2R regular
3.105 0.441
1.626 0.506 0.524 3.372 0.844 1.086 3.101 1.586 0.999
IGF2 1.302 0.981 0.419 2.511 1.954 0.809 3.701 2.076 1.192
FGF2 1.293 0.567 0.416 2.071 1.230 0.667 3.206 1.196 1.033
IGF2 + FGF2 1.768 0.338 0.569 2.290 0.654 0.738 2.565 1.705 0.826
INSR regular
0.862 0.088 0.842 0.240 0.977 0.199 0.053 0.230 0.242 0.018 0.281
IGF2 0.552 0.419 0.640 0.132 0.101 0.153 0.256 0.000 0.298
IGF2 + FGF2 0.993 0.223 1.152 0.155 0.033 0.180 0.172 0.137 0.199 FGF2 regular
0.465 0.168
0.161 0.028 0.346 0.861 0.301 1.853 2.691 0.918 5.790
IGF2 0.141 0.063 0.304 0.878 0.574 1.889 2.977 0.718 6.406
FGF2 0.148 0.016 0.317 0.321 0.042 0.690 1.191 0.102 2.563
IGF2 + FGF2 0.151 0.020 0.325 0.308 0.025 0.663 1.017 0.501 2.188
FGFR1 regular
0.223 0.037
0.134 0.021 0.601 0.619 0.085 2.771 0.759 0.171 3.396
IGF2 0.112 0.060 0.501 0.511 0.262 2.286 0.919 0.012 4.114
FGF2 0.143 0.062 0.641 1.950 0.907 8.728 3.465 1.828 15.506
IGF2 + FGF2 0.191 0.040 0.853 2.019 0.235 9.035 2.956 0.926 13.230
FGFR2 regular
0.000 0.000
0.000 0.000 NA 0.000 0.000 NA 0.001 0.002 NA
IGF2 0.000 0.000 NA 0.000 0.000 NA 0.004 0.006 NA
FGF2 0.000 0.000 NA 0.000 0.000 NA 0.001 0.001 NA
IGF2 + FGF2 0.000 0.000 NA 0.000 0.000 NA 0.003 0.005 NA
FGFR3 regular
0.001 0.001
0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
IGF2 0.004 0.007 8.711 0.000 0.000 0.000 0.000 0.000 0.000
FGF2 0.002 0.004 4.478 0.000 0.000 0.000 0.000 0.000 0.000
IGF2 + FGF2 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
FGFR4 regular
0.027 0.009
0.006 0.003 0.210 0.002 0.002 0.089 0.003 0.004 0.117
IGF2 0.009 0.008 0.349 0.003 0.003 0.125 0.002 0.002 0.056
FGF2 0.010 0.008 0.354 0.003 0.003 0.125 0.000 0.000 0.000
IGF2 + FGF2 0.005 0.002 0.199 0.002 0.002 0.085 0.002 0.004 0.084
Supplementary table ST3. Relative gene expression of selected genes normalized to SDHA in CGCs. CGCs were cultivated in either IGF2+FGF2 or IGF2+FGF2+FSH growth medium, time points were taken on days 4, 7, 13 and 24. The results are shown as mean value, standard deviation (SD), and fold change (FC) relative to day 0 of three independent experiments. Green background represents upregulated genes, red background downregulated genes.
Target Medium
Day 0 Day 4 Day 7 Day 13 Day 24
Mean SD Mean SD FC Mean SD FC Mean SD FC Mean SD FC
FSHR IGF2 + FGF2
0.092 0.027
0.239 0.031 2.601 0.180 0.049 1.961 0.032 0.012 0.351 0.000 0.000 0.000 IGF2 + FGF2
+ FSH 0.141 0.012 1.537 0.210 0.026 2.279 0.027 0.013 0.289
AMHR2 IGF2 + FGF2
0.026 0.009
0.045 0.007 1.748 0.016 0.004 0.633 0.009 0.003 0.369 0.001 0.001 0.022 IGF2 + FGF2
+ FSH 0.046 0.006 1.808 0.047 0.006 1.839 0.001 0.001 0.057
LHR IGF2 + FGF2
0.480 0.236
0.768 0.167 1.600 0.277 0.011 0.578 0.049 0.019 0.101 0.015 0.008 0.031 IGF2 + FGF2
+ FSH 2.342 0.214 4.880 1.978 0.276 4.122 0.031 0.019 0.065
CYP19A
1 IGF2 + FGF2
8.380 2.988
2.975 0.679 0.355 0.276 0.064 0.033 0.058 0.016 0.007 0.640 0.381 0.076 IGF2 + FGF2
+ FSH 38.817 4.425 4.632 34.873 13.303 4.162 1.883 1.486 0.225
GnRHR IGF2 + FGF2
0.014 0.024
0.024 0.004 1.780 0.012 0.001 0.892 0.017 0.003 1.276 0.010 0.002 0.716 IGF2 + FGF2
+ FSH 0.025 0.001 1.851 0.030 0.003 2.201 0.014 0.006 1.005
AMH IGF2 + FGF2
0.518 0.305
0.024 0.005 0.046 0.010 0.002 0.020 0.007 0.001 0.013 0.004 0.002 0.008 IGF2 + FGF2
+ FSH 0.035 0.007 0.067 0.033 0.009 0.063 0.009 0.006 0.017
CYP11A
1 IGF2 + FGF2
9.250 2.322
9.553 1.021 1.033 5.482 0.985 0.593 3.615 0.216 0.391 1.388 0.512 0.150 IGF2 + FGF2
+ FSH 0.000 0.000 0.000 32.323 9.801 3.494 0.699 0.275 0.076
IGF1 IGF2 + FGF2
0.003 0.001
0.000 0.000 0.000 0.000 0.000 0.000 0.013 0.010 3.789 0.059 0.024 17.776 IGF2 + FGF2
+ FSH 0.000 0.000 0.000 0.001 0.002 0.398 0.028 0.012 8.524
IGF2 IGF2 + FGF2
0.787 0.137
0.176 0.128 0.224 0.054 0.022 0.068 0.028 0.008 0.036 0.078 0.089 0.099 IGF2 + FGF2
+ FSH 0.958 0.397 1.218 0.374 0.112 0.475 0.113 0.096 0.143
IGF1R IGF2 + FGF2
3.874 0.775
0.692 0.142 0.179 0.349 0.006 0.090 0.328 0.017 0.085 0.438 0.143 0.113 IGF2 + FGF2
+ FSH 0.764 0.129 0.197 0.883 0.131 0.228 0.331 0.077 0.085
IGF2R IGF2 + FGF2
1.988 0.173
1.155 0.168 0.581 1.431 0.062 0.720 1.371 0.043 0.690 1.664 0.293 0.837 IGF2 + FGF2
+ FSH 2.317 0.181 1.166 2.077 0.036 1.045 1.347 0.421 0.678
INSR IGF2 + FGF2
0.744 0.225
0.633 0.184 0.851 0.207 0.015 0.278 0.103 0.035 0.139 0.084 0.014 0.113 IGF2 + FGF2
+ FSH 0.588 0.093 0.790 1.022 0.020 1.373 0.092 0.019 0.123
FGF2 IGF2 + FGF2
0.207 0.021
0.127 0.034 0.612 0.218 0.091 1.051 0.431 0.096 2.076 2.597 1.013 12.522 IGF2 + FGF2
+ FSH 0.068 0.060 0.329 0.189 0.085 0.911 1.589 1.289 7.659
FGFR1 IGF2 + FGF2
0.187 0.026
0.214 0.016 1.143 0.427 0.031 2.281 1.576 0.361 8.420 1.061 0.327 5.666 IGF2 + FGF2
+ FSH 0.181 0.007 0.969 0.316 0.109 1.687 0.707 0.064 3.775
FGFR2 IGF2 + FGF2
0 0
0 0 NA 0 0 NA 0 0 NA 0.017 0.010 NA
IGF2 + FGF2
+ FSH 0 0 NA 0 0 NA 0.007 0.009 NA
0.002 0.002 IGF2 + FGF2
+ FSH 0.000 0.000 0.000 0.000 0.000 0.000 0.001 0.002 0.422
FGFR4 IGF2 + FGF2 0.051 0.031 0.007 0.007 0.133 0.009 0.010 0.169 0.000 0.000 0.000 0.000 0.000 0.000 IGF2 + FGF2
+ FSH 0.009 0.008 0.177 0.003 0.003 0.057 0.000 0.000 0.000
Supplementary table ST4. Relative gene expression of selected genes normalized to SDHA in MGCs. MGCs were cultivated in either IGF2+FGF2 or IGF2+FGF2+FSH growth medium, time points were taken on days 4, 7, 13 and 24. The results are shown as mean value, standard deviation (SD), and fold change (FC) relative to day 0 of three independent experiments. Green background represents upregulated genes, red background downregulated genes.
Target Medium
Day 0 Day 4 Day 7 Day 13 Day 24
Mean SD Mean SD FC Mean SD FC Mean SD FC Mean SD FC
FSHR IGF2 + FGF2
0.114 0.064
0.194 0.033 1.699 0.174 0.028 1.524 0.015 0.006 0.129 0.000 0.000 0.000 IGF2 + FGF2
+ FSH 0.079 0.024 0.693 0.146 0.074 1.273 0.007 0.006 0.060
AMHR2 IGF2 + FGF2
0.024 0.006
0.036 0.009 1.488 0.033 0.005 1.385 0.008 0.001 0.314 0.003 0.003 0.120 IGF2 + FGF2
+ FSH 0.044 0.002 1.819 0.017 0.005 0.697 0.002 0.002 0.092
LHR IGF2 + FGF2
0.412 0.072
0.937 0.162 2.276 0.761 0.353 1.848 0.062 0.021 0.150 0.012 0.004 0.029 IGF2 + FGF2
+ FSH 2.668 0.650 6.481 0.329 0.174 0.800 0.010 0.003 0.025
CYP19A
1 IGF2 + FGF2
8.242 1.815 2.380 0.982 0.289 1.823 0.971 0.221 0.137 0.072 0.017 0.354 0.232 0.043 IGF2 + FGF2
+ FSH 37.703 10.059 4.574 9.764 5.104 1.185 0.700 0.080 0.085
GnRHR
IGF2 + FGF2
0.010 0.018
0.020 0.020 1.869 0.031 0.008 2.952 0.011 0.001 1.019 0.012 0.004 1.109 IGF2 + FGF2
+ FSH 0.032 0.001 3.048 0.013 0.002 1.205 0.010 0.003 0.940
AMH IGF2 + FGF2
0.207 0.110
0.017 0.002 0.084 0.015 0.003 0.071 0.006 0.001 0.028 0.005 0.002 0.024 IGF2 + FGF2
+ FSH 0.031 0.011 0.150 0.019 0.007 0.089 0.006 0.001 0.028
CYP11A
1 IGF2 + FGF2
14.470 1.966
7.450 0.301 0.515 9.959 2.328 0.688 1.854 0.142 0.128 1.087 0.415 0.075 IGF2 + FGF2
+ FSH 36.347 6.833 2.512 6.468 2.723 0.447 0.808 0.125 0.056
IGF1 IGF2 + FGF2
0.060 0.054
0.001 0.002 0.015 0.002 0.004 0.036 0.011 0.001 0.188 0.115 0.023 1.902 IGF2 + FGF2
+ FSH 0.001 0.002 0.016 0.010 0.003 0.159 0.073 0.019 1.212
IGF2 IGF2 + FGF2
1.376 0.261
0.274 0.134 0.199 0.257 0.143 0.187 0.046 0.010 0.033 0.027 0.020 0.020 IGF2 + FGF2
+ FSH 0.353 0.105 0.257 0.372 0.182 0.270 0.081 0.098 0.059
IGF1R
IGF2 + FGF2
3.111 0.569 0.736 0.159 0.237 0.517 0.110 0.166 0.317 0.012 0.102 0.330 0.053 0.106 IGF2 + FGF2
+ FSH 0.639 0.126 0.206 0.406 0.073 0.131 0.353 0.023 0.114
IGF2R
IGF2 + FGF2
1.834 0.184 0.949 0.831 0.517 1.555 0.197 0.848 1.322 0.070 0.721 1.662 0.172 0.906 IGF2 + FGF2
+ FSH 1.882 0.055 1.026 1.711 0.272 0.933 1.617 0.152 0.882
INSR IGF2 + FGF2
0.863 0.081
0.571 0.193 0.662 0.468 0.164 0.542 0.131 0.004 0.152 0.105 0.029 0.121 IGF2 + FGF2
+ FSH 0.656 0.099 0.760 0.253 0.079 0.293 0.102 0.012 0.118
FGF2 IGF2 + FGF2
1.397 1.921
0.180 0.042 0.129 0.149 0.023 0.107 0.909 0.270 0.651 1.723 0.496 1.234 IGF2 + FGF2
+ FSH 0.109 0.023 0.078 0.937 0.276 0.671 2.824 0.526 2.022
FGFR1 IGF2 + FGF2
0.200 0.048
0.220 0.056 1.099 0.351 0.092 1.750 0.659 0.049 3.289 0.705 0.038 3.519 IGF2 + FGF2
+ FSH 0.218 0.054 1.087 0.559 0.126 2.789 0.646 0.062 3.225
FGFR2 IGF2 + FGF2
0 0 0 0 NA 0 0 NA 0.006 0.003 NA 0.044 0.012 NA
IGF2 + FGF2 0.002 0.001 NA 0.006 0.004 NA 0.022 0.004 NA
FGFR3
IGF2 + FGF2
0 0 0.001 0.001 NA 0.000 0.000 NA 0.000 0.001 NA 0.002 0.002 NA IGF2 + FGF2
+ FSH 0.000 0.000 NA 0.000 0.000 NA 0.003 0.002 NA
FGFR4
IGF2 + FGF2
0.014 0.025
0.023 0.013 1.610 0.011 0.013 0.771 0.002 0.001 0.136 0.001 0.001 0.036 IGF2 + FGF2
+ FSH 0.006 0.005 0.398 0.002 0.001 0.141 0.001 0.001 0.075
Supplementary table ST5. p-values after Bonferroni correction of Student’s t-test comparing the effect of FSH on GC markers (a) and other targets (b) between CGCs and MGCs on days 7, 13, and 24. Red background represents p<0.05.
a)
Day FSHR AMHR2 LHR CYP19A1 GnRHR AMH CYP11A1 7 0,3699 0,0702 1,0000 1,0000 0,1521 0,7437 0,0041 13 1,0000 0,0052 0,0028 0,1130 0,0403 0,2926 0,0478 24 0,2151 1,0000 0,9416 1,0000 0,5970 0,6055 1,0000
b)
Day IGF1 IGF2 IGF1R IGF2R INSR FGF2 FGFR1 FGFR2 FGFR3 FGFR4 7 1,0000 0,0874 0,0650 0,0825 0,2890 0,1275 0,0814 0,3875 NA 1,0000 13 0,3623 1,0000 0,0150 0,2208 0,0004 0,1420 0,0409 1,0000 1,0000 0,4033 24 1,0000 1,0000 1,0000 0,4108 1,0000 0,1125 0,5278 0,5590 1,0000 1,0000
