Maternal spindle transfer overcomes embryo developmental arrest caused by ooplasmic defects in mice
- Embryotools, Parc Cientific de Barcelona, Spain
- Nuffield Department of Women's and Reproductive Health, University of Oxford, United Kingdom
- Juno Genetics, Winchester House, Oxford Science Park, United Kingdom
- Genomics and Bioinformatics, Centre for Genomic Regulation, Spain
- PCB Animal Facility, Parc Cientific de Barcelona, Spain
Figures
Figure 1 with 2 supplements
Maternal spindle transfer (MST) between sibling fresh B6CBAF1 mouse oocytes does not impair embryo development.
(a) Schematic representation of the experimental design used to validate the different steps of the technique. (b) Detail of the enucleation procedure with confirmation of the spindle isolation …
Figure 1—figure supplement 1
Representative images of the MST protocol.
Mouse oocyte arrested at MII stage before (a), during (b) and after (c) spindle removal. (d) Spindle being exposed to inactivated Sendai virus HVJ-E. (e-g) Reconstruction of enucleated oocytes and …
Figure 1—figure supplement 2
Representative immunofluorescence images of B6CBAF1 strain fresh (left) or vitrified (right) oocytes that have been non-manipulated (control) or used as spindle (sp) donors for MST procedures.
Microfilaments of actin are labeled in red, tubulin in green and DNA in blue.
Figure 2
Meiotic spindle transfer between NZB/OlaHsd and B6CBAF1 oocytes.
(a) Schematic representation of the experimental design. (b) Comparison between in vitro developmental rates in MST embryos and controls. (c) Representative blastocyst images from NZB oocytes …
Figure 3 with 2 supplements
Analysis of mtDNA carryover in biopsied cells and complementary embryos from MST between NZB/OlaHsd and B6CBAF1 strain oocytes.
(a) Schematic representation of the experimental design. (b) Variant allele frequencies detected in embryo specimens. Dots represent allele frequencies of individual samples. Unpaired t-test was …
Figure 3—figure supplement 1
Representative pictures of biopsy procedure in MST embryos for mtDNA heteroplasmy quantification.
(a-c) Second polar body biopsy of a 2 cell stage MST embryo. (d-f) Blastomere biopsy of a 8 cell stage MST embryo. (g-i) Trophoectoderm biopsy of a MST blastocyst embryo.
Figure 3—figure supplement 2
Validation of established high-throughput sequencing protocol for mtDNA carryover analysis.
(a) Results of SNP minisequencing analysis. The presence of guanine (G) or adenine (A); and cytosine (C) or thymine (T) at position m.3932 on the forward and reverse mtDNA strands of B6CBAF1 or NZB …
Figure 4 with 3 supplements
Analysis of mitochondrial heteroplasmy levels in adult mice born by MST.
(a) Mitochondrial heteroplasmy levels in several organs from 4 male and two female adult MST mice (F1) are maintained below 6%. (b) Mitochodrial heteroplasmy levels are not significantly different …
Figure 4—figure supplement 1
Hematoxylin and eosin (H and E) stained sections of adult mice generated from B6 ICSI control embryos, B6 reciprocal MST and NZB-sp/B6-cyt MST embryos.
Representative images from liver (a-c), hippocampus (d-f), kidney cortex (g-i), heart (j-m), skeletal muscle from the tibia (n-p) and smooth muscle from the bladder (q-s). All analyzed sections …
Figure 4—figure supplement 2
Hematoxylin and eosin (H and E) stained sections of the reproductive organs of adult mice generated from B6 ICSI control embryos, B6 reciprocal MST and NZB-sp/B6-cyt MST embryos.
Representative images from uterus (a-c), ovary (d-f), testis (g-i), and male urethra (j-m). All analyzed sections showed the typical morphology of a healthy tissue.
Figure 4—figure supplement 3
Validation of established sequencing protocol for mtDNA carryover analysis using two sequencing platforms.
Allele frequencies at positions m.2798, m.2814, m.3194, m.3260 and m.3932 in five adult mouse tissues from NZB-sp/B6-cyt MST mice measured with MiSeq (orange) and SNP m.3932 in Ion PGM sequencer …
Tables
Table 1
Efficiency and in vitro developmental rates of B6CBAF1 mouse oocytes processed by MST using fresh and vitrified oocytes.
| n oocytes processed by MST | In vitro development for up 96 hr post-ICSI | ||||||||
|---|---|---|---|---|---|---|---|---|---|
| Group | n initial | Enucleated (%) | Fused (%) | ICSI survival (%) | n cultured | Two-cells (%) | Blastocysts (%) | Total cell counts (± SD) | Oct4+ |
| Control fresh | 98 | N/A | N/A | 94 (95.9) | 94 | 91 (96.8)* | 79 (84.1)* | 192.1 (29.5) | 25.3 (5.6) |
| Control vitrified | 102 | N/A | N/A | 90 (88.2) | 90 | 87 (96.7)* | 71 (78.9)*,† | N/A | N/A |
| MST FreshSp/FreshCyt | 107 | 107 (100) | 103 (96.2) | 100 (97.1) | 100 | 99 (99)* | 81 (81)* | 177.8 (26.7) | 22.4 (3.5) |
| MST FreshSp/VitriCyt | 96 | 96 (100) | 95 (98.9) | 90 (94.7) | 90 | 73 (81.1)† | 65 (72.2)*,‡ | N/A | N/A |
| MST VitriSp/FreshCyt | 98 | 96 (97.9) | 96 (100) | 90 (93.8) | 90 | 90 (100)* | 77 (85.6)* | N/A | N/A |
| MST VitriSp/VitriCyt | 98 | 95 (96.9) | 93 (97.9) | 85 (91.4) | 85 | 70 (82.4)† | 56 (65.9)†,‡ | N/A | N/A |
-
*, †, ‡ Values with different superscripts differ significantly within the same column (p<0.05; Chi-square test or Fisher's test).
Table 2
Efficiency and in vitro developmental rates of non-manipulated control and MST oocytes.
| n oocytes processed by MST | In vitro development for up 96 hr post-ICSI | |||||||
|---|---|---|---|---|---|---|---|---|
| Group | n initial | Enucleated (%) | Fused (%) | ICSI survival (%) | Cultured | Two-cell (%) | Morula (%) | Blastocysts (%) |
| Control B6CBAF1 | 155 | N/A | N/A | 149 (96.1) | 144 | 144 (100.0)* | 121 (84.1)* | 112 (77.8)* |
| Control NZB | 193 | N/A | N/A | 181 (93.7) | 159 | 129 (81.1)† | 36 (22.6)† | 9 (5.6)† |
| MST B6-St/NZB-Cyt | 156 | 149 (95.5) | 144 (96.6) | 132 (91.7) | 110 | 93 (70.5)† | 11 (8.3)† | 0 (0.0)‡ |
| MST NZB-St/B6-Cyt | 270 | 238 (88.1) | 228 (95.7) | 221 (96.9) | 212 | 208 (98.1)* | 169 (79.7)* | 109 (51.4) § |
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*,†,‡,§ Values with different superscripts differ significantly within the same column (p<0.05; Chi-square test or Fisher's test).
Table 3
In vivo developmental rates of non-manipulated control and MST oocytes.
| In vivo development | |||
|---|---|---|---|
| Group | n transferred | n implantation sites (%) | n full-term (%) |
| Control B6CBAF1 | 58 | 23 (39.7)* | 15 (25.9) |
| Control NZB | 44 | 7 (15.9)† | 6 (13.6) |
| MST B6-St/NZB-Cyt | N/A | N/A | N/A |
| MST NZB-St/B6-Cyt | 65 | 30 (46.1)* | 14 (21.5) |
-
*, † Values with different superscripts differ significantly within the same column (p<0.05; Chi-square test or Fisher's test).
Table 4
Average weights of placentas and pups generated from control and MST oocytes.
| Average weight | |||
|---|---|---|---|
| Group | n | Placentas (± SD) | Pups (± SD) |
| Control B6CBAF1 | 3 | 134.1 (23.3) | 802.1 (153.2) |
| Control NZB | 3 | 171.1 (27.9) | 747.9 (76.9) |
| MST B6-St/NZB-Cyt | N/A | N/A | N/A |
| MST NZB-St/B6-Cyt | 4 | 168.3 (14.1) | 923.5 (146.5) |
Additional files
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Source data 1
Variant site coverages referring to Supplementary file 1.
- https://cdn.elifesciences.org/articles/48591/elife-48591-data1-v2.xlsx
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Source data 2
Variant site coverages referring to Supplementary file 2.
- https://cdn.elifesciences.org/articles/48591/elife-48591-data2-v2.xlsx
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Source data 3
Variant site coverages referring to Supplementary file 3.
- https://cdn.elifesciences.org/articles/48591/elife-48591-data3-v2.xlsx
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Source data 4
Variant site coverages referring to Supplementary file 4.
- https://cdn.elifesciences.org/articles/48591/elife-48591-data4-v2.xlsx
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Source data 5
Variant site coverages referring to Supplementary file 6.
- https://cdn.elifesciences.org/articles/48591/elife-48591-data5-v2.xlsx
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Supplementary file 1
Validation of established sequencing protocol for mtDNA carryover analysis measured by Ion PGM sequencer.
Allele frequencies at position m.3932 in homoplasmic samples and artificially constructed heteroplasmic sample mixtures. See also Source data 1.
- https://cdn.elifesciences.org/articles/48591/elife-48591-supp1-v2.xlsx
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Supplementary file 2
Validation of established sequencing protocol for mtDNA carryover analysis using MiSeq sequencer.
Allele frequencies at positions m.2798, m.2814, m.3194, m.3260 and m.3932 in homoplasmic samples and artificially constructed heteroplasmic sample mixtures. See also Source data 2.
- https://cdn.elifesciences.org/articles/48591/elife-48591-supp2-v2.xlsx
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Supplementary file 3
mtDNA heteroplasmy analysis of biopsies and complementary embryos using Ion PGM platform.
Allele frequencies at position m.3932 are shown. See also Source data 3.
- https://cdn.elifesciences.org/articles/48591/elife-48591-supp3-v2.xlsx
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Supplementary file 4
Analysis of fertility and developmental potential of MST litters through five generations.
- https://cdn.elifesciences.org/articles/48591/elife-48591-supp4-v2.xlsx
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Supplementary file 5
Allele frequencies at position m.3932 in adult MST mouse tissues through five generations using Ion PGM platform.
See also Source data 4.
- https://cdn.elifesciences.org/articles/48591/elife-48591-supp5-v2.xlsx
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Supplementary file 6
Validation of established sequencing protocol for mtDNA carryover analysis using MiSeq platform.
Allele frequencies at positions m.2798, m.2814, m.3194, m.3260 and m.3932 in adult MST mouse tissues. See alsoSource data 5.
- https://cdn.elifesciences.org/articles/48591/elife-48591-supp6-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/48591/elife-48591-transrepform-v2.docx
