Background
Methods
Animal care and ethics statement
Collection of mouse oocytes and zygotes
Parthenogenetic activation of oocytes
Embryo culture in vitro
Embryo hypotonicity
Zona pellucida drilling by Peizo micromanipulation
Live image with embryo
Immunofluorescence (IF) staining
Detection of apoptosis by TUNEL assay
Real-time PCR relative quantitation of gene expression
Statistical analysis
Results
Both the oxygen concentration and genetic material affect the blastocyst hatch process in vitro
Groups | Oxygen concentration % | Replicates No. | Total embryos No. | 96 h | 120 h |
---|---|---|---|---|---|
No. (%) Blastocysts+ | No. (%) Hatched Blastocysts | ||||
FE | 20 | 7 | 324 | 312 (96.2 ± 1.4) | 179 (58.2 ± 4.2)a |
5 | 7 | 350 | 337 (97.3 ± 1.1) | 83 (23.8 ± 2.6)b | |
PA | 20 | 9 | 346 | 336 (97.6 ± 0.8) | 278 (81.2 ± 2.7)c |
5 | 9 | 346 | 335 (96.8 ± 1.0) | 306 (90.8 ± 1.8)d |
The allocation of ICM cells is affected diversely in fertilized and parthenogenetic blastocysts in vitro
Groups | No. embryos | 120 h Blastocysts+ | No. embryos | 120 h Blastocysts | ||
---|---|---|---|---|---|---|
No. total cells | No. Oct4 positive cells | Oct4 positive cells/Total cells (%) | No. apoptosis cells | |||
HFE | 37 | 116.0 ± 3.2a | 25.2 ± 1.2 | 21.7 ± 0.8e | 14 | 1.9 ± 0.6 |
LFE | 39 | 129.4 ± 4.4b | 24.5 ± 1.2 | 18.8 ± 0.6f | 13 | 1.2 ± 0.6 |
HPA | 49 | 105.7 ± 2.6c | 23.2 ± 1.0 | 21.9 ± 0.7e | 11 | 1.4 ± 0.4 |
LPA | 60 | 112.6 ± 2.7acd | 27.6 ± 0.9 | 24.7 ± 0.6g | 12 | 1.3 ± 0.4 |
Oxygen concentration affects the gene expression in fertilized and parthenogenetic blastocysts before hatching
Sperm-drilling breach is not the main reason for different hatch dynamics between fertilized and parthenogenetic embryos
Embryo types | Diameter of hole on ZP | No. embryos | 96 h Blastocysts | 120 h Hatched Blastocysts (%) | Total Hatched time (repeats) |
---|---|---|---|---|---|
FE | Sperm-size | 23 | 23 | 10 (43)a | 41.7 ± 5.1c (9) |
PA | None | 24 | 24 | 17 (71)b | 6.0 ± 1.8d (10) |
PA-ZPI | ~ 1 μm | 10 | 10 | 8 (80)b | |
PA-ZPI | ~ 5 μm | 20 | 20 | 15 (75)b |