Background
Methods
Sheep breeding and tissue collection
Fetal sheep sex genotyping
Total RNA isolation
Reverse Transcription of miRNAs
Real-time PCR expression of conserved miRNAs in fetal gonads
Reverse transcription of mRNAs
Real-time PCR expression of mRNAs in fetal gonads
Histology
In situ hybridization
Results
MiRNA expression in fetal gonads
miRNA | GPR Score | Fold-Change |
---|---|---|
Significantly higher in GD42 ovary | ||
miR-431
| 0.836 | 5.35 |
let-7c
| 0.789 | 3.98 |
miR-328
| 0.688 | 3.85 |
miR-195
| 0.734 | 3.59 |
miR-486
| 0.695 | 3.40 |
let-7d
| 0.727 | 3.13 |
miR-7
| 0.594 | 3.07 |
miR-484
| 0.672 | 2.99 |
miR-423
| 0.656 | 2.81 |
let-7a
| 0.703 | 2.75 |
miR-320
| 0.531 | 2.67 |
let-7e
| 0.586 | 2.23 |
Significantly higher in GD42 testis
| ||
miR-758
| 0.734 | 5.00 |
miR-192
| 0.844 | 4.50 |
miR-223
| 0.797 | 3.74 |
miR-101
| 0.477 | 2.74 |
miR-411
| 0.656 | 2.58 |
miR-369-5p
| 0.523 | 2.55 |
miR-301
| 0.594 | 2.21 |
miR-142-5p
| 0.484 | 2.19 |
miR-27b
| 0.516 | 2.19 |
miR-379
| 0.555 | 2.18 |
miR-142-3p
| 0.484 | 2.08 |
miR-376a
| 0.414 | 2.04 |
miRNA | GPR Score | Fold-Change |
---|---|---|
Significantly higher in GD75 ovary | ||
let-7c
| 0.953 | 100.07 |
miR-146b
| 0.781 | 18.52 |
miR-103
| 0.844 | 17.23 |
miR-125b
| 0.859 | 15.92 |
let-7d
| 0.758 | 15.73 |
miR-484
| 0.859 | 15.55 |
let-7a
| 0.758 | 14.90 |
miR-25
| 0.773 | 10.89 |
miR-125a
| 0.758 | 8.91 |
miR-99a
| 0.805 | 8.78 |
miR-100
| 0.828 | 8.39 |
miR-150
| 0.664 | 6.25 |
miR-130a
| 0.734 | 5.76 |
miR-19b
| 0.664 | 5.33 |
miR-28
| 0.695 | 4.81 |
miR-362
| 0.602 | 4.68 |
miR-16
| 0.484 | 4.35 |
miR-200c
| 0.688 | 4.16 |
miR-200b
| 0.688 | 4.11 |
miR-210
| 0.688 | 3.85 |
miR-19a
| 0.555 | 3.59 |
miR-10a
| 0.648 | 3.12 |
let-7g
| 0.602 | 2.82 |
let-7e
| 0.469 | 2.48 |
miR-92
| 0.430 | 2.42 |
miR-183
| 0.422 | 2.42 |
Significantly higher in GD75 testis
| ||
miR-22
| 0.961 | 4223.97 |
miR-142-3p
| 0.922 | 23.40 |
miR-27a
| 0.734 | 7.77 |
miR-33
| 0.719 | 6.04 |
miR-302d
| 0.727 | 5.24 |
miR-27b
| 0.672 | 4.65 |
miR-410
| 0.508 | 3.81 |
miR-199b
| 0.492 | 3.78 |
miR-455
| 0.570 | 3.27 |
miR-211
| 0.547 | 3.22 |
miR-212
| 0.422 | 3.15 |
miR-379
| 0.523 | 3.02 |
miR-411
| 0.453 | 2.86 |
miR-369-5p
| 0.531 | 2.67 |
miR-301
| 0.453 | 2.63 |
miR-409-5p
| 0.445 | 2.30 |
miR-152
| 0.484 | 2.03 |
miRNA | GPR Score | Fold-Change | miRNA | GPR Score | Fold-Change | miRNA | GPR Score | Fold-Change |
---|---|---|---|---|---|---|---|---|
Significantly higher in GD42 versus GD75 XX gonads | Significantly higher in GD75 versus GD42 XX gonads | Significantly higher in GD42 versus GD75 XY gonads | ||||||
miR-22
| 0.953 | 1935.53 |
let-7c
| 0.883 | 46.01 |
miR-302d
| 0.891 | 16.36 |
miR-302d
| 0.945 | 119.16 |
miR-142-5p
| 0.898 | 35.40 |
miR-200c
| 0.867 | 11.85 |
miR-206
| 0.875 | 14.76 |
miR-19b
| 0.906 | 27.28 |
miR-222
| 0.734 | 5.32 |
miR-222
| 0.875 | 8.43 |
miR-19a
| 0.883 | 16.43 |
miR-200b
| 0.695 | 4.44 |
miR-196a
| 0.844 | 7.40 |
miR-135a
| 0.781 | 12.28 |
miR-362
| 0.609 | 3.95 |
miR-328
| 0.672 | 7.34 |
miR-125b
| 0.883 | 12.18 |
miR-99b
| 0.539 | 3.95 |
miR-433
| 0.781 | 7.23 |
let-7a
| 0.727 | 10.89 |
miR-485-5p
| 0.578 | 3.61 |
miR-486
| 0.813 | 6.81 |
miR-130a
| 0.852 | 9.93 |
miR-382
| 0.586 | 3.22 |
miR-216
| 0.727 | 6.63 |
miR-146b
| 0.648 | 9.92 |
miR-149
| 0.484 | 3.01 |
miR-221
| 0.820 | 5.88 |
miR-100
| 0.883 | 9.48 |
miR-15b
| 0.523 | 2.82 |
miR-196b
| 0.758 | 5.69 |
miR-99a
| 0.859 | 8.91 |
miR-92
| 0.430 | 2.72 |
miR-574
| 0.750 | 4.83 |
let-7d
| 0.453 | 8.76 |
miR-17-5p
| 0.484 | 2.27 |
miR-485-5p
| 0.742 | 4.52 |
miR-101
| 0.688 | 8.52 |
miR-107
| 0.414 | 2.15 |
miR-7
| 0.727 | 4.45 |
miR-484
| 0.750 | 5.00 |
Significantly higher in GD75 versus GD42 XY gonads
| ||
miR-423
| 0.617 | 4.39 |
miR-150
| 0.750 | 4.66 |
miR-142-3p
| 0.938 | 15.89 |
miR-382
| 0.680 | 3.76 |
miR-30b
| 0.758 | 4.41 |
miR-142-5p
| 0.938 | 14.89 |
miR-134
| 0.656 | 3.57 |
miR-103
| 0.664 | 4.32 |
miR-33
| 0.922 | 11.50 |
miR-181b
| 0.703 | 3.54 |
let-7g
| 0.664 | 3.94 |
miR-211
| 0.758 | 6.22 |
miR-200c
| 0.625 | 2.99 |
miR-497
| 0.773 | 3.82 |
miR-193a
| 0.633 | 4.21 |
miR-668
| 0.648 | 2.96 |
miR-25
| 0.688 | 3.71 |
miR-19b
| 0.586 | 3.59 |
miR-149
| 0.586 | 2.90 |
miR-331
| 0.664 | 3.59 |
miR-19a
| 0.602 | 3.53 |
miR-379
| 0.672 | 2.90 |
miR-143
| 0.734 | 3.36 |
miR-199b
| 0.539 | 3.52 |
miR-598
| 0.656 | 2.55 |
miR-199a
| 0.695 | 3.18 |
miR-27a
| 0.406 | 3.35 |
miR-615
| 0.430 | 2.54 |
miR-33
| 0.656 | 2.99 |
miR-199a
| 0.414 | 3.22 |
miR-214
| 0.500 | 2.50 |
miR-335
| 0.680 | 2.89 |
miR-143
| 0.453 | 3.18 |
miR-539
| 0.656 | 2.40 |
miR-21
| 0.680 | 2.84 |
let-7g
| 0.563 | 2.79 |
miR-652
| 0.453 | 2.35 |
miR-15a
| 0.648 | 2.76 |
miR-22
| 0.500 | 2.62 |
miR-17-5p
| 0.578 | 2.31 |
miR-148a
| 0.641 | 2.31 |
miR-30e-5p
| 0.500 | 2.46 |
miR-15b
| 0.641 | 2.27 |
let-7e
| 0.492 | 2.31 |
miR-204
| 0.414 | 2.27 |
miR-296
| 0.602 | 2.21 |
miR-204
| 0.563 | 2.11 |
miR-30b
| 0.492 | 2.25 |
miR-212
| 0.539 | 2.01 |
miR-148b
| 0.633 | 2.08 |
let-7e
| 0.430 | 2.16 |
Predicted Gene Target | miRNA |
---|---|
CYP19
|
Let7 (a, c, d, e, g)
|
ESR1
|
miR-22
|
FOG2 (ZFPM2)
|
miR-200c, miR-142-5p, miR-302d
|
FOXL2
|
miR-302d
|
FST
|
miR-410
|
GATA4
|
miR-200c
|
SOX9
|
miR-101
|
WNT4
|
miR-211
|
Expression of CYP19A1, FST, ESR1, ESR2, SOX9, and WNT4in fetal gonads
MiRNA target and functional enrichment analysis
Pathway | XX | XY | ||
---|---|---|---|---|
GD42 | GD75 | GD42 | GD75 | |
Cell Cycle | 13 | 20 | 15 | 20 |
Chromatin remodelling by hSWI/SNF ATP-dependent complexes | 3 | 1 | 2 | 5 |
Cytokine Network | 6 | 6 | 2 | 3 |
EGF signaling | 4 | 3 | 3 | 2 |
Electron Transport chain | 4 | 3 | 9 | 2 |
G-protein signaling | 8 | 16 | 6 | 15 |
Gap junction proteins connexins | 2 | 1 | 3 | 2 |
Glycolysis and gluconeogenesis | 4 | 3 | 1 | 1 |
Growth hormone signaling | 2 | 2 | 3 | 1 |
Insulin signaling | 3 | 4 | 4 | 2 |
MAPKinase signaling | 10 | 13 | 9 | 12 |
NGF pathway | 3 | 2 | 3 | |
PDGF signaling pathway | 4 | 3 | 3 | |
Phospholipids as signaling intermediates | 6 | 1 | 4 | |
Proteasome degradation | 1 | 7 | 1 | 6 |
Rac 1 cell motility signaling pathway | 1 | 3 | 3 | 2 |
Regulation ER | 5 | 2 | 1 | |
TGFbeta signaling pathway | 2 | 1 | 3 | |
TNFR Signaling | 3 | 3 | ||
Transcription factor CREB and its extracellular signals | 1 | 6 | 3 | 4 |
WNT signaling | 5 | 5 | 7 | 8 |