In a first set of experiments we aimed to optimize the MST protocol and to determine whether the manipulation of the spindle-chromosome complex is feasible without impairing the developmental potential of reconstructed oocytes. We performed reciprocal MST among sibling oocytes from the mouse hybrid B6CBAF1 strain (Figure 1a). Enucleation and reconstruction (karyoplast-cytoplast fusion) of oocytes were first assessed with freshly collected oocytes. Enucleation was successful in 98.9% of oocytes (n = 790) and reconstruction was achieved in 96.1% (n = 321), confirmed using a microscope with polarized light that allows visualization of the birefringence of the spindle microtubules (Figure 1b–c and Figure 1—figure supplement 1). Next, MST was carried out with both fresh and cryopreserved B6CBAF1 oocytes that were vitrified and warmed using the open Cryotop system (97.7% survival, n = 600). In this set of experiments, spindles were taken from fresh oocytes and transferred into either fresh (fresh-sp/fresh-cyt) or vitrified-warmed cytoplasts (fresh-sp/vitrified-cyt) and vice-versa, that is spindles from vitrified oocytes transferred to fresh (vitrified-sp/fresh-cyt) or vitrified-warm cytoplasts (vitrified-sp/vitrified-cyt). The resultant oocytes from the different groups were then fixed after reconstruction and processed for evaluation of the spindle apparatus and chromosomes distribution by immunofluorescence microscopy (Figure 1e–f and Figure 1—figure supplement 2). All oocytes analyzed presented a spindle with a normal barrel shape and with the chromosomes aligned at the MII plate (fresh-sp/fresh-cyt n = 20, fresh-sp/vitrified-cyt n = 15, vitrified-sp/fresh-cyt n = 15, vitrified-sp/vitrified-cyt n = 16; Figure 1e–f), regardless of whether fresh or vitrified gametes were used as spindle or cytoplast donors (Figure 1—figure supplement 2). These observations indicated that the conditions used to perform the manipulation of the spindle-chromosome complex were neither damaging to its structure nor altering of the distribution of the chromosomes. Furthermore, there was no evidence that the procedure was inducing premature activation of the oocytes.

Subsequently, in an independent set of samples, we compared the in vitro development of reciprocal MST experiments using fresh and vitrified B6CBAF1 oocytes, after insemination by ICSI (Figure 1j and Figure 1—figure supplement 1). High enucleation (98.7%, n = 399) and fusion (98.2%, n = 394) rates were achieved in all MST groups (see also Table 1) and almost all oocytes that were prepared with fresh (99%, n = 100) or vitrified (100%, n = 90) spindles, and transferred into fresh cytoplasts, developed to the two-cell stage on the next morning (Figure 1h–j and Table 1). Interestingly, a significantly lower proportion of inseminated oocytes composed of vitrified spindles transferred into vitrified cytoplasts (vitrified-sp/vitrified-cyt) developed to the two-cell stage (82.4%, n = 85) compared with non-manipulated fresh (96.8%, n = 94, p=0.001) or vitrified (96.7%, n = 90, p=0.001) controls. Poorer development was also observed for the fresh-sp/vitrified-cyt group (81.1%, n = 90) (Figure 1j and Table 1). On the contrary, when spindles from vitrified oocytes were transferred into fresh cytoplasts (vitrified-sp/fresh-cyt, n = 90), two-cell stage (100%) and blastocyst formation (85.6%) rates were high and equivalent to fresh controls (96.8% and 84.1%, respectively) or to MST oocytes where fresh spindles were transferred into fresh cytoplasts (fresh-st/fresh-cyt, n = 100, 99% and 81%, respectively) (Figure 1j and Table 1). Additionally, the mean number of total cells (mean ± SD, n) in the blastocysts obtained in the fresh-st/fresh-cyt group (177.8 ± 26.7, n = 81) was equivalent to controls (192 ± 29.5; n = 79). No differences were found either in the number of inner cell mass cells that were positive for the Oct4 pluripotency marker between fresh-st/fresh-cyt and control groups (22.4 ± 3.5; n = 14 versus 25.3 ± 5.6; n = 10, see also Figure 1i). Taken together, the experiments performed among sibling B6CBAF1 oocytes, showed that MST is technically feasible in the mouse without impacting the in vitro developmental competence of the oocyte. Experiments indicate that vitrification induces changes that make cryopreserved oocytes unsuitable for use as cytoplasts. However, the spindle apparatus does not appear to be damaged during vitrification or MST procedures. When recipient cytoplasts were derived from fresh oocytes, blastocyst development rates were equivalent to those obtained for non-manipulated controls, regardless of whether the spindle originated from a fresh or vitrified oocyte.

After careful optimization and validation of the different steps of the MST protocol, the effectiveness of the technique as a strategy to overcome embryo developmental arrest was evaluated. Two different oocyte strains were employed: the hybrid B6CBF1 (resultant from the cross between C57BL/6JRj females and CBA/Jrj males), and the New Zealand Black (NZB/OlaHsd) strains. The NZB strain holds two interesting characteristics. Firstly, NZB mice present a poor reproductive performance (Fernandes et al., 1973; Hansen CT and Whitney, 1973) and, secondly, the genetic background of the NZB strain has diverged genetically from most other mouse laboratory strains, including the hybrid B6CBAF1 strain, accompanied by characteristic differences in mtDNA sequences (Bielschowsky and Goodall, 1970). These two features are particularly relevant to the experimental design of this study as, in a clinical context, the NZB strain could be considered analogous to a subfertile patient (especially those with a history of poor in vitro embryo development), and the B6CBAF1 strain, a donor of proven fertility. Additionally, single nucleotide polymorphisms in the divergent mtDNA of the NZB strain provides an opportunity to evaluate the carryover of organelles and resultant heteroplasmy induced by MST procedures (see Materials and methods). Experiments were thus carried out between the two mouse strains, so that meiotic spindles were transferred from fresh NZB oocytes into fresh B6CBAF1 cytoplasts and vice-versa (Figure 2a). Once reconstructed, oocytes were inseminated using ICSI in parallel with non-manipulated oocytes from both strains and cultured in vitro until the blastocyst stage (Figure 2a). Enucleation and fusion rates were identical in both MST groups, and no differences were found in terms of survival to ICSI compared to controls (Figure 2b and Table 2). As expected, NZB control oocytes presented significantly lower fertilization rates than B6CBAF1 control oocytes, measured as two-cell stage development (Figure 2b and Table 2). Additionally, while blastocyst formation rates were close to 80% in the B6CBAF1 control group (77.8%, n = 144), most of the injected oocytes from the NZB control group arrested their development before reaching this stage (5.6% developed into blastocysts, n = 159, Figure 2b and Table 2). Remarkably, when the meiotic spindles from NZB oocytes were transferred into B6CBAF1 cytoplasts (NZB-sp/B6-cyt), the blastocyst formation rates were 10-fold higher (51.4%, n = 212, p<0.0001) compared to the non-manipulated NZB control (Figure 2b and Table 2). In the reciprocal MST group, B6CBAF1 spindles transferred into NZB cytoplasts (B6-sp/NZB-cyt), blastocysts were not obtained (0%, n = 110), indicating that cytoplasmic factors are likely to be responsible for the lower fertilisation and massive developmental arrest observed at preimplantation stages in the NZB strain (Figure 2b,c and Table 2).

At 96 hr post-insemination, embryos produced in the different experimental groups were vitrified and their competence to develop in vivo determined when synchronized pseudo-pregnant females were available for transfer. A total of 65 MST blastocysts from the NZB-sp/B6-cyt MST group were then warmed (100% survival) and transferred non-surgically into six recipients, which resulted in 14 live pups (21.5%) (Figure 2d,e and Table 3). This birth rate is comparable (p>0.05) with results obtained from the B6CBAF1 control group (15 live pups (25.9%) out of 58 blastocysts transferred into five recipients). Consistent with expectations, only six pups developed to term from 44 morulas/blastocysts (13.6%) transferred into five recipients from the control NZB group. All living pups were born healthy and respired normally. Caesarean sections at 18.5 dpc were performed in two recipients of each group to evaluate the size and weight of the placentas and the corresponding pups, with no significant differences found between groups (Table 4). These results suggest that MST procedures do not typically induce an overgrowth phenotype of the type described for certain other techniques, such as somatic cell nuclear transfer (Costa-Borges et al., 2010). Overall, these experiments confirmed that MST, with cytoplast donation from a distantly related mouse strain, is highly effective at overcoming the in vitro developmental arrest phenotype of NZB mice and that the resultant embryos are competent to develop to term with high efficiency.

The extent of mtDNA carryover induced by MST was evaluated in embryos at different preimplantation developmental stages. Spindles from NZB oocytes were transferred into B6CBAF1 cytoplasts and the resultant MST oocytes were fertilized by ICSI and cultured in vitro (Figure 3a). Afterwards, biopsies were performed to remove second polar bodies from embryos at the two-cell stage, single cells (blastomeres) from 6 to 8 cell stage embryos, or to excise a cluster of 4–8 trophectoderm cells from blastocysts (Figure 3—figure supplement 1). The biopsies and their corresponding embryos were then analyzed individually to ascertain whether mtDNA heteroplasmy levels in the biopsied cells are representative of the values found in the complementary embryo (Figure 3a).

To determine mtDNA carryover, a high-throughput sequencing protocol was developed based upon quantification of a single nucleotide polymorphism (SNP) in mtDNA using Ion PGM sequencer (ThermoFisher, see Materials and methods for further details). The SNP utilized for this purpose is located at position m.3932 and exists as a guanine (G) in the B6CBAF1 strain and an adenine (A) in the NZB strain. The presence of different alleles at m.3932 was confirmed by minisequencing analysis using genomic DNA (gDNA) from tail tips of B6CBAF1 and NZB mice (Figure 3—figure supplement 2). This sequencing protocol was carefully validated. Initially, protocol accuracy and sensitivity was assessed by analyzing different ratios of G and A alleles in artificially constructed samples, composed of gDNA from both mouse strains mixed in different ratios. For the purpose of these experiments, the G base (derived from B6CBAF1) was considered the reference allele and the A base (from NZB) the variant allele (Figure 3—figure supplement 2 and Supplementary file 1). To verify validity of mtDNA carryover assessment by analysis of a single SNP and to ensure reliability of the utilized sequencing platform, four additional SNPs (B6CBAF1/NZB: m.2798C/T; m.2814T/C; m.3194T/C; m.3260A/G) were analyzed on a different sequencer (Illumina’s MiSeq). The presence of different alleles was also confirmed by minisequencing (see Materials and methods and Supplementary file 2 for further details; and Figure 3—figure supplement 2).

Analysis of mtDNA carryover after MST in biopsied cells and the complementary embryos (Figure 3a), revealed that the mean variant (NZB) allele frequencies obtained from polar bodies were significantly higher compared to the mean frequencies in the complementary two-cell-stage embryos (6.2 ± 6.2% SD versus 0.5 ± 0.8% SD; p=0.0095) (Figure 3b). By contrast, there was no significant difference in mtDNA allele frequencies between biopsied blastomeres and trophectoderm samples when compared to the corresponding embryos (cleavage-stage: 1.3 ± 1.0% SD versus 1.9 ± 0.6% SD, respectively; blastocyst stage: 1.7 ± 0.9% SD versus 1.9 ± 0.6% SD, respectively). Moreover, the mean heteroplasmy levels were similar between all embryonic samples (except polar bodies) (Figure 3b and Supplementary file 3). These experiments demonstrate that cleavage stage or blastocyst biopsy are preferable over biopsy of second polar bodies as methods for determining the mtDNA carryover levels found in preimplantation embryos. The results also suggest that while some mitochondria remain associated with the meiotic spindle, and are unavoidably transferred to the recipient cytoplast, the vast majority of these organelles do not persist into later developmental stages, with most being expelled into the second polar body at the completion of meiosis II.

To ascertain the long-term health status and fertility of the mice generated by MST, follow up studies were then conducted over five generations. Ten mice (three females and seven males) generated by MST were selected for mating with wild type (WT) mice. At 21 days after birth, the resultant offspring were weaned, and the size and gender ratio of the litters were assessed. All parental MST mice (F1) were fertile and produced a total of 78 pups, with a mean litter size of 7.8 ± 1.4 pups/animal and no significant deviations in the expected male-female ratio (59% and 41% respectively, Supplementary file 4). All pups (F2) were born alive, respired normally and grew to adulthood without manifesting any physiological or behavioral alteration. The fertility of these mice was assessed for a total of 5 generations, by selecting random males and females from litters (n = 9 in F2 and n = 4 between F3 and F5). Similarly, these mice also displayed normal fertility and produced viable offspring, without alterations in the expected gender ratio (Supplementary file 4).

Gross necropsies of the parents and offspring were performed during the five generations, with no pathological findings observed. In the 239 mice analyzed, all organs showed a normal size, texture and morphological appearance. Additionally, F1 mice generated by MST B6-sp/B6-cyt (n = 3), MST NZB-sp/B6-cyt (n = 5) and control B6 (n = 4) groups were also processed for histopathological examinations, which were performed in vital organs including heart, kidney, liver and brain, as well as, in tibial and quadriceps skeletal muscle and urinary bladder smooth muscle. Reproductive systems and accessory glands of both males (testis, epididymis, seminal vesicles, prostate, coagulating glands, ampullary glands and bulbourethral glands) and females (ovaries, oviducts, uterine horns) were also assessed. Except for a pericardium focal inflammation in one animal of the B6 control group, none of the animals showed any lesions or visible abnormalities (Figure 4—figure supplements 1 and 2). Taken together, these results support the notion that MST can efficiently produce viable and fertile offspring.

A source of great concern in MRTs field has been the reversion of mtDNA heteroplasmy observed in embryonic stem cells (ESCs) derived from pronuclear transfer or MST generated embryos (Hyslop et al., 2016; Kang et al., 2016; Paull et al., 2013). To evaluate whether heteroplasmy was transmitted through generations and whether homoplasmy was restored, the ratios of the mtDNA alleles attributable to B6CBAF1 and NZB were assessed through several generations. Multiple organs were assessed, including those with different metabolic demands: brain; heart; liver; kidneys (Jenuth et al., 1997; Sharpley et al., 2012). A total of six mice (four male and two female) from F1 were sacrificed at adult age (12 weeks old). The mean heteroplasmy level in this group of mice was low at 2.3 ± 1.3% (mean ± SD, n = 6) ranging from mean frequencies of undetectable values to 3.5% in individual mice (Figure 4a, Supplementary file 5). Moreover, heteroplasmy levels were similar among different tissue types from the same mouse (Figure 4b) and showed no differences between males and females.

Finally, the fate of the heteroplasmy was examined in adult mice derived from the MST female lineage. Four mice (two males and two females) were selected at random from each litter, through five generations. Mitochondrial DNA heteroplasmy levels were reduced to 0.4 ± 0.6% (mean ± SD, n = 4) on average in F2 mice (Figure 4c and Supplementary file 5) and decreased to undetected levels in subsequent generations (F3 to F5, Supplementary file 5). These quantifications based on a single SNP in an Ion PGM sequencer were corroborated by using an additional sequencing platform (Illumina’s MiSeq) and 5 SNPs, as described above. Artificially constructed samples, composed of gDNA from both mouse strains mixed in different ratios, and gDNA from 5 organs of selected adult mice from F1-3 generations were analyzed (Figure 3—figure supplement 2, Figure 4—figure supplement 3, Supplementary files 2 and 6). These results suggest that low levels of mtDNA heteroplasmy resultant from MST typically result in a homoplasmic state in offspring within a few generations, without reversion (Supplementary files 5 and 6). However, it is acknowledged that different mtDNA haplogroups or mtDNA genomes affected by specific mutations might have differences in the efficiency with which they replicate, influencing the speed at which homoplasmy is attained as well as the risk if reversion.

Animal care and procedures were conducted according to protocols approved by the Ethics Committee on Animal Research (DAMM-7436) of the Parc Cientific of Barcelona (PCB), Spain. Hybrid (B6/CBA) and outbred CD1 females of 5–6 weeks of age (25–30 g), and male mice from the same genetic strains of 8–10 weeks of age (25–30 g) were purchased from Janvier Laboratories (France). New Zealand Black (NZB/OlaHsd) mice were purchased from Envigo (France). Upon arrival, all mice were quarantined and acclimated to the PCB Animals´ facility (PRAL) for approximately 1 week prior to use. Three to four mice were housed per cage in a room with a 12 hr light/dark cycle with ad libitum access to food and water.

For the collection of oocytes, hybrid B6CBAF1 and NZB females were induced to superovulate by intraperitoneal injection of 5 IU of pregnant mare serum gonadotropin (PMSG) followed 48 hr later by 5 IU of human chorionic gonadotropin (hCG). Cumulus–oocyte complexes from the both strains were released from the oviducts by 14–15 hr after hCG administration and treated with hyaluronidase (LifeGlobal) until cumulus cells dispersed. Once denuded, oocytes with good morphology were washed several times and kept in culture medium (Global total, LifeGlobal) under oil (Lifeguard, LifeGlobal) at 37.3°C, in an atmosphere with 7%CO2% and 7%O2 in air, until use. Sperms were collected from cauda epididymis and then diluted and incubated in medium supplemented with glucose (Global total for fertilization, LifeGlobal) at 37.3°C, in an atmosphere with 7%CO2% and 7%O2 in air, until use.

Oocytes from B6CBAF1 or NZB strains were used as spindle chromosome-complex and cytoplasts donors. Procedures were performed using a piezo-driven (PiezoXpert, Eppendorf) micromanipulator. Oocytes first were exposed to small drops of hepes-buffered medium (Global total w/hepes, LifeGlobal) containing 5 µg/mL cytochalasin B (Sigma) covered with mineral oil for 3–5 min at 37°C. Afterwards, the meiotic spindle was aspirated into an enucleation pipette (Humagen) trying to remove the minimum amount of surrounding cytoplasm possible, and enucleation confirmed using a microtubule birefringence system (PolarAide, Vitrolife) to visualize the spindle apparatus (Figure 1—figure supplement 1). If the karyoplast removed contained a larger amount of cytoplasm, the extra cytoplasm was eliminated by pressing the cytoplasm against the zona pellucida. Karyoplasts were inserted below the zona pellucida of another enucleated oocyte (cytoplast) and fused using inactivated Sendai virus HVJ-E (GenomeOne, Cosmo Bio). All manipulations were performed on a 37°C heated stage (Okolab) of an Olympus IX73 inverted microscope, using Eppendorf micromanipulators. Non-manipulated control oocytes and those generated by MST were inseminated using a modified piezo-actuated ICSI technique, known as the ‘hole removal technique’. Briefly, this procedure is based on withdrawing the ICSI pipette and applying rapid suction simultaneously just after the sperm head has been injected to seal the oocyte membrane, which increases survival chances. The injected oocytes were then cultured in Global total medium (LifeGlobal) under oil at 37.3°C, in K-Minc incubators (Cook Medical), in an atmosphere with 7% CO2 and O2 in air.

Embryos generated by MST were biopsied at different developmental stages, including: two-cell, morula or blastocyst stage. Regardless of the developmental stage, biopsies were performed in individual 5 µL droplets of Global total w/hepes medium covered with oil using a biopsy pipette with 19 µm of internal diameter (Eppendorf) with the assistance of laser shots to open a hole in the zona pellucida or to weaken the trophectoderm cells in the case of the blastocyst biopsy. After biopsy, both the biopsied cells and the complementary embryo were transferred individually to empty PCR tubes and stored at −80°C until processed for mtDNA allele frequencies determination.

For analysis of the spindle structure and chromosomes distribution, control and MST oocytes were fixed and extracted for 30 min at 37°C in a microtubule stabilizing buffer (MTSB-XF). A triple-labeling protocol was then used for the detection of microtubules, microfilaments and chromatin by fluorescence microscopy, as described previously (Messinger and Albertini, 1991). Briefly, fixed oocytes were first incubated in a mixture of mouse monoclonal anti α/β-tubulin antibodies, and then in a mixture of secondary antibody (chicken anti-mouse IgG) conjugated to Alexa Fluor 488 and of Alexa Fluor 594 phalloidin. Finally, all oocytes were washed in PBS blocking solution, incubated in Hoechst 33258, and put on a mounting solution droplet on a glass slide. Blastocysts processed for total cell counts were fixed in 4% PFA and permeabilized in 2.5% Triton-X100 for 25 min at room temperature. Afterwards, blastocysts were incubated overnight in blocking solution and then in rabbit monoclonal anti-Oct-4, washed 3 times in PBS blocking solution for 10 min at 37°C. After, they were incubated in secondary antibody (goat anti rabbit IgG) conjugated with Alexa Fluor 594, washed and incubated in Hoechst (10 µg/ml) for 10 min at room temperature and finally mounting solution droplet on a glass slide. Stained oocytes or blastocysts were examined using an epifluorescence microscope (Nikon E1000) fitted with specific filters for Hoechst, Fluorescein and Texas Red and a 50W mercury lamp. Digital images were acquired with E1000 Nikon software.

Oocytes and blastocysts were vitrified following the instructions provided by the manufacturer (Kitazato BioPharma Japan). Briefly, samples were exposed to equilibration solution (ES) for 15 min, transferred to VS1 for 30 s and then to VS2 for additional 30 s. Afterwards, they were loaded onto the surface strip of a classic Cryotop (Kitazato BioPharma Japan) and directly plunged into liquid N2. For warming, the Cryotop strip was transferred from the liquid nitrogen into a TS solution for 1 min at 37°C and then gradually moved to dilution solution (DS) for 3 min, to washing solution (WS) 1 for 5 min and, finally, to WS2 for an additional 1 min. Exposures to DS and WS solutions were performed at room temperature. After warming, samples were extensively washed and kept in culture medium under oil at 37.3°C, in an atmosphere with 7% CO2 and O2 in air.

Embryo transfers were performed non-surgically using a commercial non-surgical embryo transfer protocol (NSET, Paratechs). Briefly, an NSET device was coupled to a P2 pipette with volume adjusted to 1.8 μl. Between 8 and 12 blastocysts were loaded in each device within a culture medium droplet under a stereomicroscope. After loading the blastocysts, the volume in the P2 pipette was re-adjusted to 2 μl to create an air bubble and to avoid the loss of the embryos by capillarity. The recipient female assigned for transfer was then immobilized, and a NSET small speculum was carefully introduced in the vagina. With the animal still immobilized, the NSET device loaded with the embryos was introduced by the speculum through the cervix. When the base of the device got in contact with the speculum, the blastocysts were transferred by pressing the plunger of the pipette. Having the plunger of the pipette still pressed, NSET device was removed and checked under the stereomicroscope to confirm that all embryos had been correctly transferred. Finally, the speculum was removed and the female returned to its corresponding cage.

In the majority of transferred females natural delivery was controlled at the day 20 of pregnancy (P20), while in a few cases, cesarean sections were performed on embryonic day 18.5 to collect information on the weight and size of the placentas and pups. Pups (F1) resultant from the embryo transfer procedures were checked for health status and grown up until sexual maturity age was reached. Having reached the adult age, F1 males and females from each experimental group were randomly selected for crossing with wild-type (WT) B6CBAF1 mice, so that their health status and fertility competency could be assessed. At day 21 after birth, the offspring of the F1xWT = F2 mice were weaned and the F2 animals were checked and sexed. The same strategy was repeated for a total of 5 generations, by selecting random males and females from litters (n = 9 in F2 and n = 4 between F3 and F5).

For histological evaluation, tissue samples from 4 ICSI-B6 control, 3 B6-sp/B6-cyt MST and 5 NZB-sp/B6-cyt MST mice were collected at 6 weeks of age. Mice were perfused with PBS and 5% formaldehyde solution. Subsequently, tissues were fixed overnight at 4°C in 5% formaldehyde and embedded in paraffin wax, sliced in 4 μm sections and stained with hematoxylin and eosin staining (H and E). The atrium, valves and myocardium of heart, kidney, liver and gall bladder, forebrain, midbrain and hindbrain, tibial and quadriceps muscle, urinary bladder and reproductive organs (testis, epididymis, accessory glands, ovary and uterus) were evaluated. Histological analysis was carried out blindly using mouse identification codes for group assignment that were unknown to the evaluator.

Analysis of mitochondrial DNA carryover mtDNA carryover in embryo specimens and adult mouse tissues was determined by SNP quantification using a high-throughput sequencing protocol. Prior to sequencing, polymerase chain reaction (PCR) was performed to amplify the SNP located at m.3932 in the mtDNA (B6CBAF1: A; NZB: G). DNA from embryo specimens was obtained by alkaline lysis. After the addition of 0.75 μl nuclease-free water, 1.25 μl 0.1M DL-Dithiothreitol and 0.5 μl 1.0M Sodium hydroxide solution (per sample), cells were lysed at 65°C for 10 min. Genomic DNA (gDNA) from organs (tail tips, hearts, brains, livers and kidneys) was extracted using the DNeasy Blood and Tissue Kit from Qiagen. A single PCR mixture consisted of 1.5 μl HotMaster Taq DNA Buffer with Magnesium (5 Prime), 0.6 μl of 100 μm primer pool (5’-CCATACCCCGAAAACGTTGG-3’ and 5’-GGTTGGTGCTGGATATTGTGA-3’), 0.3 μl 10 nM dNTP Mix and 0.09 μl HotMaster Taq DNA Polymerase (5 Prime). The PCR mix was added to lysed embryo specimens along with 7.99 μl nuclease-free water and 2.5 μl 0.4M Tricine (per sample) and to 0.5 μl of gDNA along with 12.49 μl nuclease-free water (per sample). PCRs were performed using the following conditions: 96.0°C for one minute; 35 cycles of 94.0°C for 15 s, 58°C for 15 s and 65.0°C for 45 s; 65.0°C for two minutes. Successful amplification was verified by gel electrophoresis. Sequencing libraries were prepared from PCR amplicons using the Ion Plus Fragment Library Kit from ThermoFisher. Libraries were sequenced on the Ion Personal Genome Machine (PGM; ThermoFisher). The Torrent Variant Caller plugin (ThermoFisher) was used for SNP allele quantification. In order to increase variant calling accuracy the settings for ‘Somatic’ variants were set to ‘High Stringency’, to enable low frequency variant detection at a minimal false-positive call rate. The read depth was downsampled to 20,000 to increase accuracy of variant calls. A ‘HotSpot Region’ BED file, defining the exact genomic coordinate of the assessed nucleotide, in addition to a ‘Target Region’ BED file, was used.

Prior to the analysis of embryo specimens and tissue samples, validation experiments were performed. Minisequencing was used to confirm SNP alleles A and G at position m.3932 in B6CBAF1 and NZB mouse strains, respectively. PCR amplicons (1 μl) from gDNA (extracted from tail tips) of the B6CBAF1 and NZB mouse strains were treated with 0.5 μl EXOSAP-it (Affymetrix) and incubated at 37°C for 15 min and 80°C for 15 min. PCR amplicons (1.5 μl) were combined with 0.5 μl water, 2.5 μl SNaPshot Multiplex Ready Reaction Mix (ThermoFisher) and 0.5 μl primer (2 μM; 5’-AATAAATCCTATCACCCTT-3’; 5’-ATTGTGAAGTAGATGATGG-3’). Mixtures were incubated using the following conditions: 25 cycles of 96.0°C for ten seconds, 50°C for 5 s and 60°C for 30 s. Products were analyzed by capillary electrophoresis on a genetic analyser (ThermoFisher). The resulting data were analyzed using GeneMapper v4.0 software (Applied Biosystems). DNA mixing experiments were performed to ensure accuracy and sensitivity of the SNP quantification protocol. Sample mixtures were created by combining gDNA (extracted from tail tips) from both mouse strains at different ratios (B6CBAF1/NZB: 100/0; 98/2; 96/4; 94/6; 92/8; 90/10; 75/25; 50/50; 25/75; 0/100). Samples were sequenced and obtained ratios compared to those expected.

To ensure both the validity of assessment of a single SNP for mtDNA carryover analysis and the accuracy of the utilized sequencing platform (Ion PGM); a second set of experiments was performed, which included analysis of four additional SNPs (B6CBAF1 > NZB: m.2798C > T; m.2814T > C; m.3194T > C; m.3260A > G) utilizing Illumina’s MiSeq System sequencing platform. Minisequencing was used to confirm presence/absence of SNPs in B6CBAF1 and NZB mouse strains. PCR and minisequencing procedures were performed as described above. In brief, PCR was performed to amplify additional SNPs in gDNA from B6CBAF1 and NZB tail tips (m.2798 and m.2814: 5’-AACACTCCTCGTCCCCATTC-3; and 5’-TGGACCAACAATGTTAGGGC-3’; m.3194 and m.3260: 5’-GCCGTAGCCCAAACAATTTC-3’ and 5’-GGTCAGGCTGGCAGAAGTAA-3’). Amplicons were subjected to minisequencing with following primers: 5’-TCGTCCCCATTCTAATCGC-3’ and 5’-TGTTAGGAAGGCTAT-3’ (m.2798T); 5’-ATAGCCTTCCTAACA-3’ and 5’-AAGATTTTGCGTTCTACTA-3’ (m.2814); 5’-ATGAAGTAACCATAGCTAT-3’ and 5’-ATAGAACTGATAAAAGGAT-3’ (m.3194); 5’-CACTTATTACAACCCAAGA-3’ and 5’-GCAGAAGTAATCATATGTG-3’ (m.3260). Sequencing libraries were prepared from PCR amplicons using the TruSeq DNA Nano LT kit from Illumina and sequenced on the MiSeq System. Analysis was performed with Miseq Reporter and Illumina’s Somatic Variant Caller. Again, gDNA mixtures (B6CBAF1/NZB: 100/0; 99/1; 97/3; 90/10; 75/25; 50/50; 0/100) were sequenced and obtained ratios compared to those expected. Furthermore, gDNA samples from organs of three individual mice were sequenced and results compared to those obtained by PGM sequencing.

Experiments involving micromanipulation procedures were usually repeated between 6 to 9 times. Results obtained in the different replicates were pooled and analyzed together. Oocytes used for manipulation were always taken randomly from a common pool of oocytes collected from the 4–6 female mice used on each experimental day. In all experiments that involved embryo culture, control groups with non-manipulated oocytes were always processed and cultured in parallel together with the manipulated groups. All statistical analyses were performed using Prism 6.0 program (GraphPad). For comparisons of mean cell numbers, placentas and mice weights, mtDNA carryover values in embryo species and adult mouse tissues, a t-test or one-way ANOVA was performed, where the significance was set at p<0.05. For the analysis of oocyte/embryo proportions, chi-square test was performed and a p-value<0.05 was considered significant.

- 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.