| | | |
Analysis level 1
|
Level 2
|
Level 3
|
Level 4
|
|---|
| |
Sample A
|
Sample B
|
Sample A to B
|
Hits
|
Common in G1R and G2R
|
Hits
|
Common in MGCs and sperm
|
Hits
|
TGI
|
Hits
|
|---|
|
Group 1: CpG_G1R_MGC
|
Oil
|
BPA
|
Test 1
|
112
|
Test A (test 1 v 7)
|
1 (1)
| | | | |
| |
BPA
|
Oil
|
Test 2
|
72
|
Test B (2 v 8)
|
3 (3)
| | | | |
| |
Oil
|
VZ
|
Test 3
|
129
|
Test C (3 v 9)
|
0 (0)
|
Test Q (3 v 13)
|
1 (1)
|
Test C v G
|
0 (0)
|
| |
VZ
|
Oil
|
Test 4
|
81
|
Test D (4 v 10)
|
0 (0)
|
Test R (4 v 14)
|
2 (1)
|
Test D v H
|
0 (0)
|
| |
Oil
|
DEHP
|
Test 5
|
130
|
Test E (5 v 11)
|
4 (1)
| | | | |
| |
DEHP
|
Oil
|
Test 6
|
120
|
Test F (6 v 12)
|
6 (3)
| | | | |
|
Group 2: CpG_G2R_MGC
|
Oil
|
BPA
|
Test 7
|
61
| | | | | | |
| |
BPA
|
Oil
|
Test 8
|
98
| | | | | | |
| |
Oil
|
VZ
|
Test 9
|
59
| | |
Test S (9 v 15)
|
4 (2)
| | |
| |
VZ
|
Oil
|
Test 10
|
135
| | |
Test T (10 v 16)
|
2 (1)
| | |
| |
Oil
|
DEHP
|
Test 11
|
89
| | | | | | |
| |
DEHP
|
Oil
|
Test 12
|
167
| | | | | | |
|
Group 3: CpG_G1R_SPERM
|
Oil
|
VZ
|
Test 13
|
76
|
Test G (13 v 15)
|
4 (4)
| | | | |
| |
VZ
|
Oil
|
Test 14
|
76
|
Test H (14 v 16)
|
4 (3)
| | | | |
|
Group 4:
| | | | | | | | | | |
|
CpG_G2R_SPERM
|
Oil
|
VZ
|
Test 15
|
196
| | | | | | |
| |
VZ
|
Oil
|
Test 16
|
99
| | | | | | |
|
Group 5: Imp_G1R_MGC
|
Oil
|
BPA
|
Test 17
|
46
|
Test I (17 v 23)
|
0 (0)
| | | | |
| |
BPA
|
Oil
|
Test 18
|
35
|
Test J (18 v 24)
|
0 (0)
| | | | |
| |
Oil
|
VZ
|
Test 19
|
93
|
Test K (19 v 25)
|
1 (1)
|
Test U (19 v 29)
|
1 (1)
|
Test K v O
|
0 (0)
|
| |
VZ
|
Oil
|
Test 20
|
51
|
Test L (20 v 26)
|
1 (1)
|
Test V (20 v 30)
|
0 (0)
|
Test L v P
|
0 (0)
|
| |
Oil
|
DEHP
|
Test 21
|
83
|
Test M (21 v 27)
|
0 (0)
| | | | |
| |
DEHP
|
Oil
|
Test 22
|
59
|
Test N (22 v 28)
|
1 (0)
| | | | |
|
Group 6: Impr_G2R_MGC
|
Oil
|
BPA
|
Test 23
|
30
| | | | | | |
| |
BPA
|
Oil
|
Test 24
|
45
| | | | | | |
| |
Oil
|
VZ
|
Test 25
|
29
| | |
Test X (25 v 31)
|
0 (0)
| | |
| |
VZ
|
Oil
|
Test 26
|
120
| | |
Test Y (26 v 32)
|
4 (4)
| | |
| |
Oil
|
DEHP
|
Test 27
|
27
| | | | | | |
| |
DEHP
|
Oil
|
Test 28
|
111
| | | | | | |
|
Group 7: Impr_G1R_SPERM
|
Oil
|
VZ
|
Test 29
|
56
|
Test O (29 v 31)
|
3 (2)
| | | | |
| |
VZ
|
Oil
|
Test 30
|
58
|
Test P (30 v 32)
|
1 (0)
| | | | |
|
Group 8: Impr_G2R_SPERM
|
Oil
|
VZ
|
Test 31
|
114
| | | | | | |
| |
VZ
|
Oil
|
Test 32
|
93
| | | | | | |
- After in utero exposure of G1 MGC to vehicle control (‘oil’) or one of the EDs, the level of DNA methylation was measured in reprogrammed G1R and G2R fetal MGCs and adult spermatozoa by MIRA-chip and Nimblegen microarrays. CpG-promoter arrays were used in groups 1 to 4 and custom imprinting arrays were used in groups 5 to 7. At the first level of analysis, we performed 32 tests comparing the log2 ratio MIRA versus input values. To detect a change between sample A and B, peaks were identified in the average value (n = 3 for MGC and n = 2 for sperm) of A samples first and were compared with the MIRA intensity of the average of sample B at the same locations. The number of hits where a change occurred with greater than ±5% and Fisher’s exact test P value (P <0.05) were tabulated. At level two, we compared the results of two level-1 tests (in parentheses) to find common changes between G1R and G2R in MGCs (Tests A-F and I-N) and in sperm (Tests G-H and O-P); the number of common hits is provided and those that changed in the same direction are given in parentheses. At level three, we compared the results of two level-1 tests to find common changes between MGC and sperm in the same generation, G1R (Test Q-R and U-V) or G2R (S-T and X-Y). At level four, we compared the results of four level-1 tests in search for true transgenerational epigenetic inheritance: immediate effect in G1R MGCs, maintained into G1R sperm, G2R prospermatogonia, and G2R sperm, thereby having the potential to affect G3 soma. Note the low number of hits in levels 1 to 3 and lack of hits at level 4, even at the low cutoff values applied.
