Reproductive BioMedicine Online (2014) 29, 708–716
w w w. s c i e n c e d i r e c t . c o m w w w. r b m o n l i n e . c o m
ARTICLE
Effects of in-vitro or in-vivo matured ooplasm and spindle-chromosome complex on the development of spindle-transferred oocytes Chenhui Ding1, Tao Li1, Yanhong Zeng, Pingping Hong, Yanwen Xu, Canquan Zhou * Reproductive Medicine Center, First Affiliated Hospital of Sun Yat-sen University, 58 Zhongshan Road II, Guangzhou, Guangdong 510080, China * Corresponding author. E-mail address: [email protected] (C Zhou).
1
Tao Li and Chenhui Ding contributed equally to this paper.
Chenhui Ding has been working in the IVF lab at the Reproductive Medicine Center, First Affiliated Hospital of Sun Yat-sen University since 2009. He received his PhD in Zoology from the Kunming institute of Zoology, Chinese Academy of Sciences in 2006 and completed his post-doctoral studies in reproductive biology at the Institute of Zoology, Chinese Academy of Sciences in 2010. Current interests include medical laboratory science and most aspects of reproductive sciences, including sperm biology, early embryos development and genome reprogramming.
To study the effects of in-vitro matured ooplasm and spindle-chromosome complex (SCC) on the development of spindletransferred oocytes, reciprocal spindle transfer was conducted between in-vivo and in-vitro matured oocytes. The reconstructed oocytes were divided into four groups according to their different ooplasm sources and SCC, artificially activated and cultured to the blastocyst stage. Oocyte survival, activation and embryo development after spindle transfer manipulation were compared between groups. Survival, activation, and cleavage rates of reconstructed oocytes after spindle transfer manipulation did not differ significantly among the four groups. The eight-cell stage embryo formation rates on day 3 and the blastocyst formation rate on day 6 were not significantly different between the in-vitro and in-vivo matured SCC groups when they were transplanted into in-vivo matured ooplasm. The rate of eight-cell stage embryo formation with in-vitro matured ooplasm was significantly lower (P < 0.05) than that of embryos with in-vivo matured ooplasm, and none of the embryos developed to the blastocyst stage. Therefore, SCC matured in vitro effectively supported the in-vitro development of reconstructed oocytes. Ooplasm matured in vitro, however, could not support the development of reconstructed oocytes, and may not be an appropriate source of ooplasm donation for spindle transfer.
Abstract
© 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. KEYWORDS: in-vitro maturation, mtDNA, oocytes, parthenogenetic activation, spindle transfer, spindle-chromosome complex
http://dx.doi.org/10.1016/j.rbmo.2014.08.012 1472-6483/© 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.
Spindle transfer of human oocytes
Introduction Mature human oocyte contains between 19,000 and 25,000 mitochondria based on electron microscopic morphometry, and the estimated mtDNA copies per oocyte range from 90,000 and about 150,000, according to measurements of the mitochondrial gene ATPase 6 (Van Blerkom, 2004, 2008). In contrast, sperm cells only contain between 10 and 700 copies of mtDNA (Hecht et al., 1984; Shitara et al., 2000), and sperm mtDNA is eliminated quickly after fertilization by ubiquitination followed by selective digestion by endonucleases (Sutovsky et al., 1999, 2003). Therefore, mtDNA is transmitted entirely through the maternal line via the mitochondria contained in the ooplasm. Since the first report of diseases caused by mtDNA mutations in 1988 (Wallace et al., 1988), more than 150 mtDNA mutations associated with serious human disorders have been identified. These disorders include myopathies, neurodegenerative diseases, diabetes, cancer, and infertility (Solano et al., 2001). The transfer of the nuclear genome into an enucleated oocyte containing normal mitochondria is probably the first choice to effectively prevent the transmission of mtDNA disorders (Tachibana et al., 2009). Although prenatal genetic diagnosis can select embryos with a reduced mutation load, variation between blastomeres in single embryos limits the effectiveness of such screening. Spindle transfer, essentially a modified cloning technique, transfers the meiotic spindle and attached chromosomes (spindle-chromosome complex, SCC) from one mature oocyte to another to select for a cytoplasm or mtDNA background. This technology has been used to generate both cattle and mice after subsequent fertilization (Bai et al., 2006; Bao et al., 2003; Wakayama et al., 2004; Wang et al., 2001), and has generated live monkeys (Macaca mulatta) after sperm injection (Tachibana et al., 2009). Similar techniques, such as germinal vesicle transfer and pronuclear transfer, have also been used to prevent the transmission of mtDNA from one generation to the next (Craven et al., 2010; Cummins, 1998; Fulka, 2004; Takeuchi et al., 2001; Trounson, 2001). Potential problems could arise, however, as the transferred germinal vesicle or pronuclear transfer is still obviously surrounded by mitochondria, which will also be carried over into the donor ooplasm. As pronuclear transfer also involves the destruction of a zygote, usage may be restricted because of ethical and moral considerations. Recent studies using donated human in-vivo matured oocytes for spindle transfer after ovarian hyperstimulation treatment are promising. Tachibana et al. (2009, 2013) demonstrated the feasibility and outcomes of spindle transfer with human oocytes donated by healthy volunteers on the basis of their prior studies in a monkey model. They recruited seven volunteers who underwent ovarian stimulation. A total of 106 in-vivo matured metaphase II (MII) oocytes were retrieved in their study (Tachibana et al., 2013). Although these pioneering works are encouraging, more studies with human oocytes are needed to further optimize spindle transfer protocols and ensure that these procedures are safe. Donated human oocytes matured in vivo are difficult to acquire, hindering the further development of spindle transfer techniques and its possible use in preventing inherited mtDNA diseases. Immature oocytes retrieved from infertile women undergoing IVF treatment are commonly used for research
709 purposes. Most of these oocytes mature in vitro after 24 h of in-vitro culture. These in-vitro matured oocytes from infertile patients could therefore be a potential cost-effective source of ooplasm or karyoplasts for spindle transfer. If the cytoplasm, spindle apparatus of in-vitro matured oocytes could support the development of spindle transfer embryos, the exogenous hormone treatment of donor or recipient patients is unnecessary. Such treatments are costly and can cause severe health problems. It is unclear, however, whether in-vitro matured human ooplasm or karyoplasts are capable of supporting the development of reconstructed oocytes. To study the effects of in-vitro matured oocytes (ooplasm and karyoplast, respectively) on spindle transfer efficiency, reciprocal spindle transfer between in-vivo matured and invitro matured human oocytes was conducted, and the reconstructed oocytes were parthenogenetically activated rather than fertilized with donor sperm to avoid the generation of human embryos. The in-vitro development of parthenogenetic embryos was examined to evaluate the developmental potential of reconstructed oocytes with differently sourced ooplasm and SCC.
Materials and methods Oocyte donation, experimental design and ethical approval Patients undergoing intracytoplasmic sperm injection were routinely asked to donate their immature oocytes for research at the Reproductive Medicine Center, First Affiliated Hospital, Sun Yat-sen University. More than 95% of them agreed to donate their immature oocytes for research and provided written informed consent before treatment. Therefore, invitro matured oocytes were available almost every day for various studies in our centre, including the present study of spindle transfer. Mature oocytes were donated by azoospermic couples who had been diagnosed with severe spermatogenic failure before IVF treatment. The couples were fully informed and aware that they were at high risk of sperm failing to inseminate their oocytes. Between 2009 and 2014, six such couples were diagnosed with severe Spermatogenic Failure. After biopsy on both sides of the testis, and an extensive search under the microscope, one of the couples decided to cryopreserve their oocytes for possible future use. The other five couples chose to undergo intrauterine insemination with donated sperm in an attempt to achieve pregnancy (the women were young and did not have any infertility factors) and donated all of their oocytes for spindle transfer research. Written informed consent was obtained before spindle transfer. All of the patients followed a protocol using gonadotrophin-releasing hormone agonist and Gonal-F (Gonal-F; Merck Serono, The Netherlands) for ovarian stimulation (Khoudja et al., 2013). Oocyte retrieval was carried out 34–36 h after the administration of 10,000 IU HCG (Ovidrel; Merck Serono, The Netherlands). Oocytes lacking a polar body were considered immature (germinal vesicle and metaphase I oocytes) after stripping for intracytoplasmic sperm injection (ICSI) on the day of oocyte retrieval (day 0). Only the oocytes remaining at the metaphase I stage were used for in-vitro maturation.
710 The study groups for reciprocal spindle transfer were defined by the sources of the donor ooplasm and SCCs as follows: in vivo/in vivo (group 1), in vivo/in vitro (group 2), in vitro/in vivo (group 3), and in vitro/in vitro (group 4). The reconstructed oocytes from each group were artificially activated, and the parthenogenetic embryos were cultured to the blastocyst stage to study the development of spindle transfer embryos and the spindle transfer efficiency. The present study was approved by the Research Ethics Committee of the First Hospital of Sun Yat-sen University (Approval Reference Number: 2012-268, Issue date: 10 April 2012).
In-vitro maturation The in-vitro maturation culture medium consisted of tissue culture medium 199 (TCM 199; Sigma) supplemented with 10% serum protein substitute (Sage In-vitro Fertilization, Inc. Trumbull, USA), 50,000 IU/L penicillin (Sigma), 50 mg/l streptomycin (Sigma), 25 mmol/l sodium pyruvate (Sigma), 75 IU/l recombinant FSH (Gonal-F; Merck Serono, The Netherlands), and 150 IU/l HCG (Ovidrel; Merck Serono, The Netherlands). Immature oocytes were cultured in a humidified atmosphere of 6% CO2 in air at 37°C. The oocyte maturational status was evaluated after 15 h of in-vitro culture. Oocytes were considered to have reached metaphase II and therefore to have ‘matured in vitro’ if they extruded a polar body after 15 h of in-vitro culture (day 1) and were then used for spindle transfer. Oocytes remaining immature after 15 h of in-vitro culture were considered incompetent for maturation and were discarded.
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Oocyte preparation and spinal transfer manipulation The oocyte preparation before spindle transfer was carried out as previously described with slight modifications (Mai et al., 2007). Briefly, the oocytes were denuded enzymatically by brief exposure of the cumulus–oocyte complexes to 80 IU/ml hyaluronidase (SAGE In-Vitro Fertilization, Inc. Trumbull, USA) followed by mechanical denudation about 1–5 h after oocyte collection. The oocytes with the first polar body were defined as in vivo-matured oocytes and were immediately ready for spindle transfer, whereas immature MI oocytes (without germinal vesicle and polar body) were allowed to mature overnight and were subjected to spindle transfer after maturation in vitro (day 1). A total of 237 oocytes were used in the present study (71 oocytes matured in vivo and 166 oocytes matured in vitro). Oocytes of different origin (in-vivo or in-vitro matured) were placed in separate drops of G-MOPS (Vitrolife; Vitrolife Sweden AB Göteborg, Sweden) containing 10 µg/ml cytochalasin B (Sigma) and covered with mineral oil (Vitrolife; Vitrolife Sweeden AB Göteborg, Sweden). Enucleation of the oocytes was carried out using a piezo drill and SpindleView Imaging System (CRI-Inc. co, UK) at 37°C. Pipettes with an internal diameter of 10 µm were used for the removal and injection of the SCC between different oocytes. The meiotic metaphase plate was aspirated into a pipette and then gently withdrawn from the cytoplasm. After removal of the recipient SCC, the donor SCC (karyoplast) was directly injected into the recipient ooplasm (Figure 1). All of the manipulations were performed on a 37°C heated stage of a Nikon TE 2000U inverted
Figure 1 Spindle-chromosome complex (SCC) transfer between in-vitro matured and in-vivo matured human oocytes and visualization of the SCC in human metahphase II oocytes using the SpindleView imaging system during enucleation and reconstruction. (A) In-vitro matured human metaphase II oocytes; the spindle and its position are indicated by the arrowhead; (B) and (E), enucleation of in-vitro and in-vivo matured human oocytes. Removed SCCs were intact and pulled into the pipettes as indicated by the arrow; (C) removed SCC from in-vitro matured oocyte was injected directly into enucleated oocytes matured in vivo to obtain a reconstructed spindle transfer oocyte. The arrow indicates the donor SCC; (D) in-vivo matured human metaphase II oocytes; the spindle and its position are indicated by the arrowhead; (F) immunofluorescent staining of a reconstructed oocyte to show the structure and distribution of microtubules and chromosomes. Red = DNA; green = tubulin; arrow = SCC; arrowhead = first polar body; star = cumulus cells. Scale bar: 50 µm.
Spindle transfer of human oocytes microscope using Narishige micromanipulators (Yu et al., 2007). Three Piezoelectric microinjection (PEM) parameters (pulse frequency, pulse intensity and pulse duration) that influence the strength of the piezoelectric pulses were chosen for spindle transfer: PEM542 (speed 5 Hz, intensity 4, 2 s) and PEM 332 (speed 3 Hz, intensity 3, 2 s). PEM542 was the common set-up used for breaking the zona pellucida, and PEM 332 was used to break the oocyte membrane. To avoid the toxic effects of mercury, Fluorinert (FC77, sigma) was used in the piezoelectric system to assist the removal and injection of SCCs (Yu et al., 2007).
Observation of premature activation of spindle transfer reconstructed oocytes To detect the occurrence of premature activation of spintletransfer reconstructed oocytes, 23 reconstructed oocytes resulting from spindle-transfer manipulation of in-vitro matured oocytes and 15 control oocytes matured in vitro were placed into in-vitro culture within the living-cell workstation of a Leica microscope (DMI 6000B, Germany) for 24 h without artificial activation and dyed with H33342 (10 µg/ml). The second polar body extrusion and pronucleus formation were defined as the premature activation of ST oocytes. The reconstructed oocytes were carefully monitored every 30 min through continuous photography (Andor iXon-885, EM-CCD, Co).
Artificial activation and embryo culture After spindle transfer manipulation, the reconstructed oocytes were immediately transferred back into G-IVF (Vitrolife; Vitrolife Sweeden AB Göteborg, Sweden) at 37°C. The oocytes were parthenogenetically activated by incubation in 7.5 µM ionomycin (I3909, Sigma, St Louis, MO, USA) for 10 min followed by incubation in 2 mM 6-dimethylamino purine (6DMAP; d2629, Sigma, St Louis, MO, USA) for 4 h. After activation, the reconstructed embryos were cultured in microdrops of IVF cleavage medium (G1; Vitrolife, Vitrolife Sweeden AB Göteborg, Sweeden) at 37°C in a humidified atmosphere of 6% CO2. Intact, non-enucleated oocytes were activated to act as parthenogenetic controls. On day 3, spindle trannsfer embryos at the six- to eight-cell stages were transferred to blastocyst medium and further cultured to day 6 at 37°C in a humidified atmosphere of 6% CO2.
Immunostaining of reconstructed oocytes Spindle transfer oocytes were fixed in 4% formaldehyde in phosphate buffered saline (PBS; pH 7.4) for 30 min at room temperature or overnight at 4°C, permeabilized in 0.5% v/v Triton X-100 for 30 min at room temperature and blocked with 10% v/v normal goat serum for 1 h. The oocytes were then incubated overnight at 4°C with a fluorescein isothiocyanate (FITC)-conjugated mouse monoclonal antibody against alphatubulin (Sigma F2168) diluted 1:200 in 10% normal goat serum and phosphate buffered saline. The oocytes were then washed three times at room temperature in phosphate buffered saline
711 and stained with 10 µg/ml propidium iodide for 20 min. The oocytes and embryos were mounted in 10 µl of anti-fade solution (80% glycerol and 0.4% n-propyl gallate in phosphate buffered saline; Sigma) and compressed with a cover slip. The image was examined under an Olympus immunofluoresence microscope using the Lucia FISH software (Lucia Cytogenetics, Laboratory Imaging, S. R. O., Za Drahou 171/17 CZ10200 Praha 10).
Data analysis The differences in the survival rate and activation rate of the reconstructed oocytes between the groups were analysed by Student’s t-test. The rates of spindle transfer embryos that developed to the two-cell stage, eight-cell stage and blastocyst stage were compared by the 2 × 2 and 4 × 2 contingency tables using Fisher’s exact test. P < 0.05 was considered statistically significant.
Results Oocytes, spindle transfer, and embryos Immature metaphase I oocytes (166) were collected from 63 women (mean age, 32.4; range, 27–40) who were receiving ovarian stimulation and ICSI treatment. After 15 h of invitro culture, 145 MI oocytes extruded the first polar body, resulting in a maturation rate of 87.3%. Most of these invitro matured oocytes used for reciprocal spindle transfer between in-vivo and in-vitro matured oocytes displayed clearly visible, normal-shaped spindles (Figure 1), as determined using the Spindle View imaging system (64/72 [88.9%]) (Table 1), and this rate is similar to that of obtained with the oocytes matured in vivo (47/49 [95.9%]) (Table 1). Thirty-eight oocytes matured in vitro were used to assess premature activation of spindle transfer oocytes (23 spindle transfer oocytes and 15 control oocytes) (Table 2). Five couples undergoing ICSI donated 71 in-vivo matured oocytes (patient age 32–38 years). The development of reconstructed spindle transfer embryos was compared between the four groups of reconstructed oocytes with in-vitro or in-vivo matured ooplasm and karyoplasts and intact controls (Table 1). The survival rate of the reconstructed oocytes after spindle transfer manipulation was not significantly different between the four groups (76.9–82.4%). The activation rate (71.4–85.0%) was also similar between the groups. Parthenogenetic embryos at the onecell stage displayed one large pronucleus (Figure 2). No significant difference were found in the cleavage rates between the four groups (Table 1). The in-vitro development of spindle transfer embryos to the eight-cell stage and blastocyst formation (day 6) did not differ significantly between the in-vivo–in-vivo (60% and 40.0%, respectively) and in-vivo–in-vitro (57.1% and 35.7%, respectively) groups. This result showed that parthenogenetic spindle transfer embryos with in-vitro matured SCC displayed similar developmental potential compared with those with in-vivo matured SCC reconstructed with in-vivo matured ooplasm. When in-vitro and in-vivo matured SCC were transferred into in-vitro matured ooplasm, only 12.5% and 11.1% spindle
Intact oocytes without manipulation
Between rows of the same column: different letters (aVS b), P < 0.05. In vivo/in vivo group: in-vivo matured spindle-chromosome complex was transferred into in-vivo matured ooplasm. In vivo/in vitro group: in vitro-matured spindle-chromosome complex was transferred into in-vivo matured ooplasm. In vitro/in vivo group: in-vivo matured spindle-chromosome complex was transferred into in-vitro-matured ooplasm. In vitro/in vitro group: in vitro-matured spindle-chromosome complex was transferred into in-vitro matured ooplasm. Control group: parthenogenetic oocytes (in-vivo or in-vitro matured) without spindle transfer manipulation.
8 (40.0)a 5 (35.7)a 0 (0)b 0 (0)b 10 (45.5)a 1 (2.9)b 10 (50.0)a 5 (35.7)a 1 (5.6)b 0 (0)b 10 (45.5)a 1 (2.9)b 17 (85.0) 10 (71.4) 14 (77.8) 26 (81.3) 16 (72.7) 30 (85.7) Spindle transfer oocytes
In vivo/in vivo In vivo/in vitro In vitro/in vivo In vitro/in vitro In-vivo matured oocyte In-vitro matured oocyte
26 17 21 39 22 35
20 (76.9) 14 (82.4) 18 (85.7) 32 (82.1) / /
15 (75.0) 10 (71.4) 14 (77.8) 24 (75.0) 15 (68.2) 22 (62.9)
12 (60.0)a 8 (57.1)a 2 (11.1)b 4 (12.5)b 12 (54.5)a 5 (14.3)b
Blastocysts n (%) Morulas n (%) Eight-cell embryos n (%) Two-cell embryos n (%) Activated n (%) Reconstructed oocytes n (%) Oocytes n Group with the source of ooplasm/spindle
In-vitro development of parthenogenetic spindle transfer embryos. Table 1
5 (25.0)a 3 (21.4)a 0 (0)b 0 (0)b 7 (31.8)a 1 (2.9)b
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transfer embryos developed to the eight-cell stage in the two groups, respectively, and no spindle transfer embryos developed to the blastocyst stage, with no difference detected between the groups. Furthermore, the development rate of parthenogenetic spindle transfer embryos with in-vitro matured SCC and in-vivo matured ooplasm was as high as that obtained with intact parthenogenetic controls (in-vivo matured oocytes without spindle transfer). Most of these parthenogenetic spindle transfer embryos displayed an expanded blastocoel on day 6 (Table 1 and Figure 2C). In contrast, the in-vitro development of parthenogenetic spindle transfer oocytes to the eight-cell stage differed significantly between the groups with differently sourced ooplasm. The rates of embryo development to the eightcell stage with in-vitro matured ooplasm, including SCC matured in vitro (12.5%) and in vivo (11.1%), were statistically lower than that of embryos with in-vivo matured ooplasm (in vivo/in vivo, 60.0%; in vivo/in vitro, 57.1%; P < 0.05). Moreover, most of the parthenogenetic spindle transfer embryos with in-vitro matured ooplasm (in vitro/in vitro and in vitro/in vivo groups) were arrested at the eight-cell stage, regardless of whether the SCC was derived from in-vivo or in-vitro-matured oocytes. The development rate of spindle transfer embryos with in-vitro matured ooplasm was similar to that of in-vitro matured parthenogenetic intact controls without spindle transfer (Table 1). This result indicates that the impaired development of spindle transfer embryos was not caused by spindle transfer manipulation but rather by the incompetence of donor ooplasm matured in vitro. The premature activation rate of reconstructed oocytes was low in the present study, even after 24 h of in-vitro culture (Figure 3). Only two spindle transfer oocytes had formed 1PN (2/23 [8.7%]), which is comparable to that of control oocytes without spindle transfer manipulation (Table 2).
Efficiency of spindle transfer using the direct injection method for oocyte reconstruction The reconstruction rate and activation rate of spindle transfer embryos with ooplasm and SCC from different sources are shown in Table 1. More than 81% (84/103) of the manipulated oocytes survived the injection procedure, which is a higher rate than that reported by other researchers for oocytes reconstructed using the electro-fusion method (73%, Tachibana et al., 2009). The reconstructed embryos in all four groups were activated with high efficiency (Table 1), which did not differ from that of intact parthenogenetic control oocytes. These results demonstrated the high efficiency of the direct injection method.
Distribution of microtubules and chromosomes in spindle transfer oocytes Five spindle transfer oocytes derived from in-vitro matured oocytes were immunofluorescently stained to investigate the distribution of microtubules and chromosomes. All of these oocytes displayed normal spindle morphology (Figure 1F).
Spindle transfer of human oocytes Table 2
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Occurrence of premature activation of oocytesa.
Spindle transfer oocytes Control
2h n/n (%)
4h n/n (%)
8h n/n (%)
24 h n/n (%)
0/23 (0) 0/15 (0)
0/23 (0) 0/15 (0)
0/23 (0) 0/15 (0)
2/23 (8.7) 0/15 (0)
a
Spindle transfer oocytes: the spindles of three to five in-vitro matured oocytes were removed at one time and then randomly transferred back to the oocyte cytoplasm; control: in-vitro matured oocytes without any manipulation were used as control.
Figure 2 In-vitro development of parthenogenetic embryos derived from spinle transfer oocytes. (A) Spindle transfer embryo with one large pronucleus after activation; (B) seven-cell-stage spindle transfer embryos (day 3); (C) parthenogenetic spindle transfer blastocyst (day 6); (D) arrested spindle transfer embryos (day 5). Scale bar: 50 µm.
Discussion In a standard IVF cycle, about 10–20% of the retrieved oocytes will be in the metaphase I or germinal vesicle stage (Chian and Tan, 2002). Many of these immature oocytes are not needed in IVF clinics, and have therefore become a valuable source of study material. In the present study, such oocytes (MI stage) were matured in vitro and used for reciprocal spindle transfer between in-vivo matured and in-vitro matured human oocytes. Most of these oocytes extruded the first PB (i.e. reached maturity) during the first 15 h of invitro culture (maturation time range, 4–15 h). When the spindle apparatus from in-vitro matured oocytes was transferred into in-vivo matured ooplasm, the activation and blastocyst formation rates were similar to those observed with in-vivo matured SCC transferred into in-vivo matured ooplasm. This result demonstrated that the SCC derived from in-vitro matured oocytes could support the development of reconstructed oocytes and therefore be
efficiently used for spindle transfer. Therefore, future spindle transfer for patients with mtDNA disorders may not require the use of exogenous hormones to acquire a fully in-vivo matured SCC from such patients for transfer, simplifying the treatment. When in-vitro matured ooplasm was used to reconstruct oocytes with SCCs matured in vitro or in vivo, the development of embryos to the eight-cell stage was significantly lower than that obtained for embryos with in-vivo matured ooplasm (Table 1). Moreover, all of the embryos in these spindle transfer groups arrested before reaching the blastocyst stage. In the control group (intact oocytes without spindle transfer), only one in-vitro matured oocyte developed to the blastocyst stage after being parthenogenetically activated (2.9%), which is a significantly lower rate than that obtained with invivo matured oocytes (31.8%) (Table 1), demonstrating the lower developmental competence of in-vitro matured oocytes compared with that of in-vivo matured oocytes. Although several reports have documented blastocyst development or live birth achieved from oocytes matured in vitro (Chian and Tan, 2002; Liu et al., 2003), these were different from the leftover immature oocytes from patients undergoing ICSI used in this study. To carry out ICSI, the immature oocytes in the present study had been stripped of cumulus cells before being placed into maturation medium. The cytoplasmic maturation may be affected by a lack of cumulus cells surrounding immature oocytes, which modulate nuclear and cytoplasmic maturation by both physical cell-cell contact and the combined actions of paracrine factors (Kimura et al., 2007). McElroy et al. (2010) observed the ability of intact, cumulusfree, in-vitro matured oocytes to develop to the blastocyst stage after parthenogenetic activation. These researchers reported that all parthenotes cultured in common in-vitro maturation supplementation media arrested before the blastocyst stage, but only three parthenogenetic blastocysts (5.9%) were produced when in-vitro maturation medium was specifically supplemented with a complex combination of ovarian paracrine/autocrine factors. On the basis of this finding, it is not surprising to expect a markedly lower spindle transfer efficiency when in-vitro matured cytoplasm from cumulusfree oocytes is used. With the exception of the high frequencies of aneuploidy (complex), in-vitro matured, cumulus-free oocytes may not have acquired the factors and machinery necessary for complete cytoplasmic maturation. In this study, specifically for spindle transfer treatment, in-vitro matured ooplasm may lack adequate mitochondria and mtDNA copies or have failed to form proper mitochondrial distribution patterns, affecting the development of reconstructed oocytes (Nishi et al., 2003; Smith and Alcivar, 1993; Stojkovic et al., 2001). Therefore,
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Figure 3 Visualization of reconstructed oocytes by live fluorescent DNA staining at various time intervals after spindle transfer. (A) Intact mature metaphase II oocytes; (B): enucleated metaphase II oocytes (the inserted picture shows the removed SCC, scale bar: 20 µm); (C–E) reconstructed oocytes/embryos at 2 h (C); 4 h (D); and 8 h (E) after spindle transfer (scale bar: 50 µm). The DNA was visualized by live blue fluorescent staining.
this type of oocyte would not be considered potential spindle recipients to correct pathogenetic mtDNA mutations at the current time. With improvements in the in-vitro culture conditions and in-vitro maturation technology, however, it is possible that in-vitro matured ooplasm could be used in the future. Although much progress has been made in recent spindle transfer studies, it is difficult to further increase spindle transfer efficiency to validate its hypothetical application in
clinical treatments. For example, the spindle-chromosome apparatus in methapse II oocytes is an extremely sensitive structure that can easily be perturbed by physical or chemical manipulations. Paull et al. (2013) reported that the manipulation of SCC during spindle transfer procedure frequently induces the premature activation of oocytes, resulting in karyotype abnormalities. These researchers observed that the use of a relatively immature SCC could decrease the occurrence
Spindle transfer of human oocytes of the early activation of oocytes through electrofusion methods (Paull et al., 2013). The underlying reasons are not clear at the present time, although this phenomenon may be related to the decreased kinase target phosphorylation observed in fully matured SCCs caused by oocyte reconstruction procedures. The careful observation of the morphology of ST oocytes (polar body extrusion and pronuclear formation) is critical for the discrimination between normal and prematurely activated spindle transfer oocytes. Using living cell workstation microscopy, the extrusion of the second polar body and pronucleus formation at different time points after spindle transfer were carefully observed in this study. In the present study, the reconstructed oocytes had a low rate of premature activation, which was not significantly different from that of unmanipulated control oocytes, regardless of whether the SCCs were matured in vitro or in vivo (Table 2). In our study, SCC was directly injected into ooplasm without any fusion procedure to reconstruct oocytes. The low occurrence of the premature activation of oocytes found in this study may be related to the omission of fusion manipulation. The possible association between the fusion method and the premature activation of oocytes, however, needs to be further investigated using different oocyte reconstruction methods. With the new design of the piezo-driven cell injector, a small piezo stack is sufficient to perform the cell injection process. With proper selection of the excitation frequency and amplitude, harmful lateral tip oscillations of the injector pipette could be reduced to a satisfactory degree (Yu et al., 2007). In the present study, the survival rates or successful reconstruction rates of oocytes after spindle transfer manipulation were greater than 75% in each group, demonstrating the safety and efficiency of the direct injection method for the reconstruction of oocytes with the piezo-driven cell injector. In the present study, the second polar body (PB2) extrusion was deliberately suppressed when spindle transfer oocytes were activated to obtain parthenogenetic embryos for the analysis of their developmental potential. The chromosomal aneuploid rate of spindle transfer embryos may therefore be much lower than that obtained with other methods because the second meiosis did not occur. In conclusion, our results show that in vitro-matured karyoplasts (SCC) can effectively support the development of reconstructed oocytes. Therefore, patients with mtDNA disorders may not require the use of exogenous hormones to acquire fully in-vivo matured oocytes and their SCC for spindle transfer, simplifying the treatment if spindle transfer is to become a practical method to address maternally inherited oxidative phosphorylation disease. In contrast, a human oocyte that matured entirely in vivo is currently the appropriate gamete for the cytoplasmic donor because the in-vitro matured ooplasm did not support the development of reconstructed oocytes using the currently available technology. This finding may change if the physiological basis for the poor developmental performance of completed in-vitro maturation is better understood and can be addressed in vitro.
Acknowledgements This study was supported by the grants from the Key Laboratory of Guangdong Province, the priming scientific
715 research foundation for the junior teachers of medicine in Sun Yat-sen University (10ykpy14; 12ykpy23), the National Natural Science Foundation of China (81100472).
References Bai, Z.D., Liu, K., Wang, X.Y., 2006. Developmental potential of aged oocyte rescued by nuclear transfer following parthenogenetic activation and in vitro fertilization. Mol. Reprod. Dev. 73, 1448– 1453. Bao, S., Ushijima, H., Hirose, A., Aono, F., Ono, Y., Kono, T., 2003. Development of bovine oocytes reconstructed with a nucleus from growing stage oocytes after fertilization in vitro. Theriogenology 59, 1231–1239. Chian, R.C., Tan, S.L., 2002. Maturational and developmental competence of cumulus-free immature human oocytes derived from stimulated and intracytoplasmic sperm injection cycles. Reprod. Biomed. Online 5, 125–132. Craven, L., Tuppen, H.A., Greggains, G.D., Harbottle, S.J., Murphy, J.L., Cree, L.M., Murdoch, A.P., Chinnery, P.F., Taylor, R.W., Lightowlers, R.N., Herbert, M., Turnbull, D.M., 2010. Pronuclear transfer in human embryos to prevent transmission of mitochondrial DNA disease. Nature 465, 82–85. Cummins, J., 1998. Mitochondrial DNA in mammalian reproduction. Rev. Reprod. 3, 172–182. Fulka, H., 2004. Distribution of mitochondria in reconstructed mouse oocytes. Reproduction 127, 195–200. Hecht, N.B., Liem, H., Kleene, K.C., Distel, R.J., Ho, S.M., 1984. Maternal inheritance of the mouse mitochondrial genome is not mediated by a loss or gross alteration of the paternal mitochondrial DNA or by methylation ofthe oocyte mitochondrial DNA. Dev. Biol. 102, 452–461. Khoudja, R., Li, T., Ding, C., Xu, Y., Liu, Y., Zhou, W., Zhou, C., 2013. Effect of co-incubation of oocytes with a decreasing number of spermatozoa on embryo quality. Reprod. Biomed. Online 26, 353– 359. Kimura, N., Hoshino, Y., Totsukawa, K., Sato, E., 2007. Cellular and molecular events during oocyte maturation in mammals: molecules of cumulus-oocyte complex matrix and signalling pathways regulating meiotic progression. Soc. Reprod. Fertil. Suppl. 63, 327–342. Liu, J., Lu, G., Qian, Y., Mao, Y., Ding, W., 2003. Pregnancies and births achieved from in vitro matured oocytes retrieved from poor responders undergoing stimulation in in vitro fertilization cycles. Fertil. Steril. 80, 447–449. Mai, Q., Yu, Y., Li, T., Wang, L., Chen, M.J., Huang, S.Z., Zhou, C., Zhou, Q., 2007. Derivation of human embryonic stem cell lines from parthenogenetic blastocysts. Cell Res. 17, 1008–1019. McElroy, S.L., Byrne, J.A., Chavez, S.L., Behr, B., Hsueh, A.J., Westphal, L.M., Pera, R.A., 2010. Parthenogenic blastocysts derived from cumulus-free in vitro matured human oocytes. PLoS ONE 5, e10979. Nishi, Y., Takeshita, T., Sato, K., Araki, T., 2003. Change of the mitochondrial distribution in mouse ooplasm during in vitro maturation. J. Nippon Med. Sch. 70, 408–415. Paull, D., Emmanuele, V., Weiss, K.A., Treff, N., Stewart, L., Hua, H., Zimmer, M., Kahler, D.J., Goland, R.S., Noggle, S.A., Prosser, R., Hirano, M., Sauer, M.V., Egli, D., 2013. Nuclear genome transfer in human oocytes eliminates mitochondrial DNA variants. Nature 493, 632–637. Shitara, H., Kaneda, H., Sato, A., Inoue, K., Ogura, A., Yonekawa, H., Hayashi, J.I., 2000. Selective and continuous elimination of mitochondria microinjected into mouse eggs from spermatids, but not from liver cells, occurs throughout embryogenesis. Genetics 156, 1277–1284.
716 Smith, L.C., Alcivar, A.A., 1993. Cytoplasmic inheritance and its effects on development and performance. J. Reprod. Fertil. Suppl. 48, 31–43. Solano, A., Playan, A., Lopez-Perez, M.J., Montoya, J., 2001. Genetic diseases of the mitochondrial DNA in humans. Salud Publica Mex. 43, 151–161. Stojkovic, M., Machado, S.A., Stojkovic, P., Zakhartchenko, V., Hutzler, P., Gonçalves, P.B., Wolf, E., 2001. Mitochondrial distribution and adenosine triphosphate content of bovine oocytes before and after in vitro maturation: correlation with morphological criteria and developmental capacity after in vitro fertilization and culture. Biol. Reprod. 64, 904–909. Sutovsky, P., Moreno, R.D., Ramalho-Santos, J., Dominko, T., Simerly, C., Schatten, G., 1999. Ubiquitin tag for sperm mitochondria. Nature 402, 371–372. Sutovsky, P., van Leyen, K., McCauley, T., Day, B.N., Sutovsky, M., 2003. Degradation of paternal mitochondria after fertilization: implications for heteroplasmy, assisted reproductive technologies and mtDNA inheritance. Reprod. Biomed. Online 8, 21–33. Tachibana, M., Sparman, M., Sritanaudomchai, H., Ma, H., Clepper, L., Woodward, J., Li, Y., Ramsey, C., Kolotushkina, O., Mitalipov, S., 2009. Mitochondrial gene replacement in primate offspring and embryonic stem cells. Nature 461, 367–372. Tachibana, M., Amato, P., Sparman, M., Gutierrez, N.M., TippnerHedges, R., Ma, H., Kang, E., Fulati, A., Lee, H.S., Sritanaudomchai, H., Masterson, K., Larson, J., Eaton, D., SadlerFredd, K., Battaglia, D., Lee, D., Wu, D., Jensen, J., Patton, P., Gokhale, S., Stouffer, R.L., Wolf, D., Mitalipov, S., 2013. Human embryonic stem cells derived by somatic cell nuclear transfer. Cell 153, 1228–1238. Takeuchi, T., Gong, J., Veeck, L.L., Rosenwaks, Z., Palermo, G.D., 2001. Preliminary findings in germinal vesicle transplantation of immature human oocytes. Hum. Reprod. 16, 730–736.
C Ding et al. Trounson, A., 2001. Nuclear transfer in human medicine and animal breeding. Reprod. Fertil. Dev. 13, 31–39. Van Blerkom, J., 2004. The role of mitochondria in human oogenesis and preimplantation embryogeneisis: engines of metabolism, ionic regulation and developmental competence. Reproduction 128, 269–280. Van Blerkom, J., 2008. Mitochondria as regulatory forces in oocytes, preimplantation embryos and stem cells. Reprod. Biomed. Online 16, 553–569. Wakayama, S., Thuan, N.V., Kishigami, S., Ohta, H., Mizutani, E., Hikichi, T., Miyake M., Wakayama T., 2004. Production of offspring from one-day-old oocytes stored at room temperature. J. Reprod. Dev. 50, 627–637. Wallace, D.C., Singh, G., Lott, M.T., Hodge, J.A., Schurr, T.G., Lezza, A.M., Elsas, L.J., 2nd, Nikoskelainen, E.K., 1988. Mitochondrial DNA mutation associated with Leber’s hereditary optic neuropathy. Science 242, 1427–1430. Wang, M.K., Chen, D.Y., Liu, J.L., Li, G.P., Sun, Q.Y., 2001. In vitro fertilisation of mouse oocytes reconstructed by transfer of metaphase II chromosomes results in live births. Zygote 9, 9–14. Yu, Y., Ding, C., Wang, E., Chen, X., Li, X., Zhao, C., Fan, Y., Wang, L., Beaujean, N., Zhou, Q., Jouneau, A., Ji, W., 2007. Piezoassisted nuclear transfer affects cloning efficiency and may cause apoptosis. Reproduction 133, 947–954.
Declaration: The authors report no financial or commercial conflicts of interest.
Received 14 April 2014; refereed 20 August 2014; accepted 27 August 2014.
w w w. s c i e n c e d i r e c t . c o m w w w. r b m o n l i n e . c o m
ARTICLE
Effects of in-vitro or in-vivo matured ooplasm and spindle-chromosome complex on the development of spindle-transferred oocytes Chenhui Ding1, Tao Li1, Yanhong Zeng, Pingping Hong, Yanwen Xu, Canquan Zhou * Reproductive Medicine Center, First Affiliated Hospital of Sun Yat-sen University, 58 Zhongshan Road II, Guangzhou, Guangdong 510080, China * Corresponding author. E-mail address: [email protected] (C Zhou).
1
Tao Li and Chenhui Ding contributed equally to this paper.
Chenhui Ding has been working in the IVF lab at the Reproductive Medicine Center, First Affiliated Hospital of Sun Yat-sen University since 2009. He received his PhD in Zoology from the Kunming institute of Zoology, Chinese Academy of Sciences in 2006 and completed his post-doctoral studies in reproductive biology at the Institute of Zoology, Chinese Academy of Sciences in 2010. Current interests include medical laboratory science and most aspects of reproductive sciences, including sperm biology, early embryos development and genome reprogramming.
To study the effects of in-vitro matured ooplasm and spindle-chromosome complex (SCC) on the development of spindletransferred oocytes, reciprocal spindle transfer was conducted between in-vivo and in-vitro matured oocytes. The reconstructed oocytes were divided into four groups according to their different ooplasm sources and SCC, artificially activated and cultured to the blastocyst stage. Oocyte survival, activation and embryo development after spindle transfer manipulation were compared between groups. Survival, activation, and cleavage rates of reconstructed oocytes after spindle transfer manipulation did not differ significantly among the four groups. The eight-cell stage embryo formation rates on day 3 and the blastocyst formation rate on day 6 were not significantly different between the in-vitro and in-vivo matured SCC groups when they were transplanted into in-vivo matured ooplasm. The rate of eight-cell stage embryo formation with in-vitro matured ooplasm was significantly lower (P < 0.05) than that of embryos with in-vivo matured ooplasm, and none of the embryos developed to the blastocyst stage. Therefore, SCC matured in vitro effectively supported the in-vitro development of reconstructed oocytes. Ooplasm matured in vitro, however, could not support the development of reconstructed oocytes, and may not be an appropriate source of ooplasm donation for spindle transfer.
Abstract
© 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. KEYWORDS: in-vitro maturation, mtDNA, oocytes, parthenogenetic activation, spindle transfer, spindle-chromosome complex
http://dx.doi.org/10.1016/j.rbmo.2014.08.012 1472-6483/© 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.
Spindle transfer of human oocytes
Introduction Mature human oocyte contains between 19,000 and 25,000 mitochondria based on electron microscopic morphometry, and the estimated mtDNA copies per oocyte range from 90,000 and about 150,000, according to measurements of the mitochondrial gene ATPase 6 (Van Blerkom, 2004, 2008). In contrast, sperm cells only contain between 10 and 700 copies of mtDNA (Hecht et al., 1984; Shitara et al., 2000), and sperm mtDNA is eliminated quickly after fertilization by ubiquitination followed by selective digestion by endonucleases (Sutovsky et al., 1999, 2003). Therefore, mtDNA is transmitted entirely through the maternal line via the mitochondria contained in the ooplasm. Since the first report of diseases caused by mtDNA mutations in 1988 (Wallace et al., 1988), more than 150 mtDNA mutations associated with serious human disorders have been identified. These disorders include myopathies, neurodegenerative diseases, diabetes, cancer, and infertility (Solano et al., 2001). The transfer of the nuclear genome into an enucleated oocyte containing normal mitochondria is probably the first choice to effectively prevent the transmission of mtDNA disorders (Tachibana et al., 2009). Although prenatal genetic diagnosis can select embryos with a reduced mutation load, variation between blastomeres in single embryos limits the effectiveness of such screening. Spindle transfer, essentially a modified cloning technique, transfers the meiotic spindle and attached chromosomes (spindle-chromosome complex, SCC) from one mature oocyte to another to select for a cytoplasm or mtDNA background. This technology has been used to generate both cattle and mice after subsequent fertilization (Bai et al., 2006; Bao et al., 2003; Wakayama et al., 2004; Wang et al., 2001), and has generated live monkeys (Macaca mulatta) after sperm injection (Tachibana et al., 2009). Similar techniques, such as germinal vesicle transfer and pronuclear transfer, have also been used to prevent the transmission of mtDNA from one generation to the next (Craven et al., 2010; Cummins, 1998; Fulka, 2004; Takeuchi et al., 2001; Trounson, 2001). Potential problems could arise, however, as the transferred germinal vesicle or pronuclear transfer is still obviously surrounded by mitochondria, which will also be carried over into the donor ooplasm. As pronuclear transfer also involves the destruction of a zygote, usage may be restricted because of ethical and moral considerations. Recent studies using donated human in-vivo matured oocytes for spindle transfer after ovarian hyperstimulation treatment are promising. Tachibana et al. (2009, 2013) demonstrated the feasibility and outcomes of spindle transfer with human oocytes donated by healthy volunteers on the basis of their prior studies in a monkey model. They recruited seven volunteers who underwent ovarian stimulation. A total of 106 in-vivo matured metaphase II (MII) oocytes were retrieved in their study (Tachibana et al., 2013). Although these pioneering works are encouraging, more studies with human oocytes are needed to further optimize spindle transfer protocols and ensure that these procedures are safe. Donated human oocytes matured in vivo are difficult to acquire, hindering the further development of spindle transfer techniques and its possible use in preventing inherited mtDNA diseases. Immature oocytes retrieved from infertile women undergoing IVF treatment are commonly used for research
709 purposes. Most of these oocytes mature in vitro after 24 h of in-vitro culture. These in-vitro matured oocytes from infertile patients could therefore be a potential cost-effective source of ooplasm or karyoplasts for spindle transfer. If the cytoplasm, spindle apparatus of in-vitro matured oocytes could support the development of spindle transfer embryos, the exogenous hormone treatment of donor or recipient patients is unnecessary. Such treatments are costly and can cause severe health problems. It is unclear, however, whether in-vitro matured human ooplasm or karyoplasts are capable of supporting the development of reconstructed oocytes. To study the effects of in-vitro matured oocytes (ooplasm and karyoplast, respectively) on spindle transfer efficiency, reciprocal spindle transfer between in-vivo matured and invitro matured human oocytes was conducted, and the reconstructed oocytes were parthenogenetically activated rather than fertilized with donor sperm to avoid the generation of human embryos. The in-vitro development of parthenogenetic embryos was examined to evaluate the developmental potential of reconstructed oocytes with differently sourced ooplasm and SCC.
Materials and methods Oocyte donation, experimental design and ethical approval Patients undergoing intracytoplasmic sperm injection were routinely asked to donate their immature oocytes for research at the Reproductive Medicine Center, First Affiliated Hospital, Sun Yat-sen University. More than 95% of them agreed to donate their immature oocytes for research and provided written informed consent before treatment. Therefore, invitro matured oocytes were available almost every day for various studies in our centre, including the present study of spindle transfer. Mature oocytes were donated by azoospermic couples who had been diagnosed with severe spermatogenic failure before IVF treatment. The couples were fully informed and aware that they were at high risk of sperm failing to inseminate their oocytes. Between 2009 and 2014, six such couples were diagnosed with severe Spermatogenic Failure. After biopsy on both sides of the testis, and an extensive search under the microscope, one of the couples decided to cryopreserve their oocytes for possible future use. The other five couples chose to undergo intrauterine insemination with donated sperm in an attempt to achieve pregnancy (the women were young and did not have any infertility factors) and donated all of their oocytes for spindle transfer research. Written informed consent was obtained before spindle transfer. All of the patients followed a protocol using gonadotrophin-releasing hormone agonist and Gonal-F (Gonal-F; Merck Serono, The Netherlands) for ovarian stimulation (Khoudja et al., 2013). Oocyte retrieval was carried out 34–36 h after the administration of 10,000 IU HCG (Ovidrel; Merck Serono, The Netherlands). Oocytes lacking a polar body were considered immature (germinal vesicle and metaphase I oocytes) after stripping for intracytoplasmic sperm injection (ICSI) on the day of oocyte retrieval (day 0). Only the oocytes remaining at the metaphase I stage were used for in-vitro maturation.
710 The study groups for reciprocal spindle transfer were defined by the sources of the donor ooplasm and SCCs as follows: in vivo/in vivo (group 1), in vivo/in vitro (group 2), in vitro/in vivo (group 3), and in vitro/in vitro (group 4). The reconstructed oocytes from each group were artificially activated, and the parthenogenetic embryos were cultured to the blastocyst stage to study the development of spindle transfer embryos and the spindle transfer efficiency. The present study was approved by the Research Ethics Committee of the First Hospital of Sun Yat-sen University (Approval Reference Number: 2012-268, Issue date: 10 April 2012).
In-vitro maturation The in-vitro maturation culture medium consisted of tissue culture medium 199 (TCM 199; Sigma) supplemented with 10% serum protein substitute (Sage In-vitro Fertilization, Inc. Trumbull, USA), 50,000 IU/L penicillin (Sigma), 50 mg/l streptomycin (Sigma), 25 mmol/l sodium pyruvate (Sigma), 75 IU/l recombinant FSH (Gonal-F; Merck Serono, The Netherlands), and 150 IU/l HCG (Ovidrel; Merck Serono, The Netherlands). Immature oocytes were cultured in a humidified atmosphere of 6% CO2 in air at 37°C. The oocyte maturational status was evaluated after 15 h of in-vitro culture. Oocytes were considered to have reached metaphase II and therefore to have ‘matured in vitro’ if they extruded a polar body after 15 h of in-vitro culture (day 1) and were then used for spindle transfer. Oocytes remaining immature after 15 h of in-vitro culture were considered incompetent for maturation and were discarded.
C Ding et al.
Oocyte preparation and spinal transfer manipulation The oocyte preparation before spindle transfer was carried out as previously described with slight modifications (Mai et al., 2007). Briefly, the oocytes were denuded enzymatically by brief exposure of the cumulus–oocyte complexes to 80 IU/ml hyaluronidase (SAGE In-Vitro Fertilization, Inc. Trumbull, USA) followed by mechanical denudation about 1–5 h after oocyte collection. The oocytes with the first polar body were defined as in vivo-matured oocytes and were immediately ready for spindle transfer, whereas immature MI oocytes (without germinal vesicle and polar body) were allowed to mature overnight and were subjected to spindle transfer after maturation in vitro (day 1). A total of 237 oocytes were used in the present study (71 oocytes matured in vivo and 166 oocytes matured in vitro). Oocytes of different origin (in-vivo or in-vitro matured) were placed in separate drops of G-MOPS (Vitrolife; Vitrolife Sweden AB Göteborg, Sweden) containing 10 µg/ml cytochalasin B (Sigma) and covered with mineral oil (Vitrolife; Vitrolife Sweeden AB Göteborg, Sweden). Enucleation of the oocytes was carried out using a piezo drill and SpindleView Imaging System (CRI-Inc. co, UK) at 37°C. Pipettes with an internal diameter of 10 µm were used for the removal and injection of the SCC between different oocytes. The meiotic metaphase plate was aspirated into a pipette and then gently withdrawn from the cytoplasm. After removal of the recipient SCC, the donor SCC (karyoplast) was directly injected into the recipient ooplasm (Figure 1). All of the manipulations were performed on a 37°C heated stage of a Nikon TE 2000U inverted
Figure 1 Spindle-chromosome complex (SCC) transfer between in-vitro matured and in-vivo matured human oocytes and visualization of the SCC in human metahphase II oocytes using the SpindleView imaging system during enucleation and reconstruction. (A) In-vitro matured human metaphase II oocytes; the spindle and its position are indicated by the arrowhead; (B) and (E), enucleation of in-vitro and in-vivo matured human oocytes. Removed SCCs were intact and pulled into the pipettes as indicated by the arrow; (C) removed SCC from in-vitro matured oocyte was injected directly into enucleated oocytes matured in vivo to obtain a reconstructed spindle transfer oocyte. The arrow indicates the donor SCC; (D) in-vivo matured human metaphase II oocytes; the spindle and its position are indicated by the arrowhead; (F) immunofluorescent staining of a reconstructed oocyte to show the structure and distribution of microtubules and chromosomes. Red = DNA; green = tubulin; arrow = SCC; arrowhead = first polar body; star = cumulus cells. Scale bar: 50 µm.
Spindle transfer of human oocytes microscope using Narishige micromanipulators (Yu et al., 2007). Three Piezoelectric microinjection (PEM) parameters (pulse frequency, pulse intensity and pulse duration) that influence the strength of the piezoelectric pulses were chosen for spindle transfer: PEM542 (speed 5 Hz, intensity 4, 2 s) and PEM 332 (speed 3 Hz, intensity 3, 2 s). PEM542 was the common set-up used for breaking the zona pellucida, and PEM 332 was used to break the oocyte membrane. To avoid the toxic effects of mercury, Fluorinert (FC77, sigma) was used in the piezoelectric system to assist the removal and injection of SCCs (Yu et al., 2007).
Observation of premature activation of spindle transfer reconstructed oocytes To detect the occurrence of premature activation of spintletransfer reconstructed oocytes, 23 reconstructed oocytes resulting from spindle-transfer manipulation of in-vitro matured oocytes and 15 control oocytes matured in vitro were placed into in-vitro culture within the living-cell workstation of a Leica microscope (DMI 6000B, Germany) for 24 h without artificial activation and dyed with H33342 (10 µg/ml). The second polar body extrusion and pronucleus formation were defined as the premature activation of ST oocytes. The reconstructed oocytes were carefully monitored every 30 min through continuous photography (Andor iXon-885, EM-CCD, Co).
Artificial activation and embryo culture After spindle transfer manipulation, the reconstructed oocytes were immediately transferred back into G-IVF (Vitrolife; Vitrolife Sweeden AB Göteborg, Sweden) at 37°C. The oocytes were parthenogenetically activated by incubation in 7.5 µM ionomycin (I3909, Sigma, St Louis, MO, USA) for 10 min followed by incubation in 2 mM 6-dimethylamino purine (6DMAP; d2629, Sigma, St Louis, MO, USA) for 4 h. After activation, the reconstructed embryos were cultured in microdrops of IVF cleavage medium (G1; Vitrolife, Vitrolife Sweeden AB Göteborg, Sweeden) at 37°C in a humidified atmosphere of 6% CO2. Intact, non-enucleated oocytes were activated to act as parthenogenetic controls. On day 3, spindle trannsfer embryos at the six- to eight-cell stages were transferred to blastocyst medium and further cultured to day 6 at 37°C in a humidified atmosphere of 6% CO2.
Immunostaining of reconstructed oocytes Spindle transfer oocytes were fixed in 4% formaldehyde in phosphate buffered saline (PBS; pH 7.4) for 30 min at room temperature or overnight at 4°C, permeabilized in 0.5% v/v Triton X-100 for 30 min at room temperature and blocked with 10% v/v normal goat serum for 1 h. The oocytes were then incubated overnight at 4°C with a fluorescein isothiocyanate (FITC)-conjugated mouse monoclonal antibody against alphatubulin (Sigma F2168) diluted 1:200 in 10% normal goat serum and phosphate buffered saline. The oocytes were then washed three times at room temperature in phosphate buffered saline
711 and stained with 10 µg/ml propidium iodide for 20 min. The oocytes and embryos were mounted in 10 µl of anti-fade solution (80% glycerol and 0.4% n-propyl gallate in phosphate buffered saline; Sigma) and compressed with a cover slip. The image was examined under an Olympus immunofluoresence microscope using the Lucia FISH software (Lucia Cytogenetics, Laboratory Imaging, S. R. O., Za Drahou 171/17 CZ10200 Praha 10).
Data analysis The differences in the survival rate and activation rate of the reconstructed oocytes between the groups were analysed by Student’s t-test. The rates of spindle transfer embryos that developed to the two-cell stage, eight-cell stage and blastocyst stage were compared by the 2 × 2 and 4 × 2 contingency tables using Fisher’s exact test. P < 0.05 was considered statistically significant.
Results Oocytes, spindle transfer, and embryos Immature metaphase I oocytes (166) were collected from 63 women (mean age, 32.4; range, 27–40) who were receiving ovarian stimulation and ICSI treatment. After 15 h of invitro culture, 145 MI oocytes extruded the first polar body, resulting in a maturation rate of 87.3%. Most of these invitro matured oocytes used for reciprocal spindle transfer between in-vivo and in-vitro matured oocytes displayed clearly visible, normal-shaped spindles (Figure 1), as determined using the Spindle View imaging system (64/72 [88.9%]) (Table 1), and this rate is similar to that of obtained with the oocytes matured in vivo (47/49 [95.9%]) (Table 1). Thirty-eight oocytes matured in vitro were used to assess premature activation of spindle transfer oocytes (23 spindle transfer oocytes and 15 control oocytes) (Table 2). Five couples undergoing ICSI donated 71 in-vivo matured oocytes (patient age 32–38 years). The development of reconstructed spindle transfer embryos was compared between the four groups of reconstructed oocytes with in-vitro or in-vivo matured ooplasm and karyoplasts and intact controls (Table 1). The survival rate of the reconstructed oocytes after spindle transfer manipulation was not significantly different between the four groups (76.9–82.4%). The activation rate (71.4–85.0%) was also similar between the groups. Parthenogenetic embryos at the onecell stage displayed one large pronucleus (Figure 2). No significant difference were found in the cleavage rates between the four groups (Table 1). The in-vitro development of spindle transfer embryos to the eight-cell stage and blastocyst formation (day 6) did not differ significantly between the in-vivo–in-vivo (60% and 40.0%, respectively) and in-vivo–in-vitro (57.1% and 35.7%, respectively) groups. This result showed that parthenogenetic spindle transfer embryos with in-vitro matured SCC displayed similar developmental potential compared with those with in-vivo matured SCC reconstructed with in-vivo matured ooplasm. When in-vitro and in-vivo matured SCC were transferred into in-vitro matured ooplasm, only 12.5% and 11.1% spindle
Intact oocytes without manipulation
Between rows of the same column: different letters (aVS b), P < 0.05. In vivo/in vivo group: in-vivo matured spindle-chromosome complex was transferred into in-vivo matured ooplasm. In vivo/in vitro group: in vitro-matured spindle-chromosome complex was transferred into in-vivo matured ooplasm. In vitro/in vivo group: in-vivo matured spindle-chromosome complex was transferred into in-vitro-matured ooplasm. In vitro/in vitro group: in vitro-matured spindle-chromosome complex was transferred into in-vitro matured ooplasm. Control group: parthenogenetic oocytes (in-vivo or in-vitro matured) without spindle transfer manipulation.
8 (40.0)a 5 (35.7)a 0 (0)b 0 (0)b 10 (45.5)a 1 (2.9)b 10 (50.0)a 5 (35.7)a 1 (5.6)b 0 (0)b 10 (45.5)a 1 (2.9)b 17 (85.0) 10 (71.4) 14 (77.8) 26 (81.3) 16 (72.7) 30 (85.7) Spindle transfer oocytes
In vivo/in vivo In vivo/in vitro In vitro/in vivo In vitro/in vitro In-vivo matured oocyte In-vitro matured oocyte
26 17 21 39 22 35
20 (76.9) 14 (82.4) 18 (85.7) 32 (82.1) / /
15 (75.0) 10 (71.4) 14 (77.8) 24 (75.0) 15 (68.2) 22 (62.9)
12 (60.0)a 8 (57.1)a 2 (11.1)b 4 (12.5)b 12 (54.5)a 5 (14.3)b
Blastocysts n (%) Morulas n (%) Eight-cell embryos n (%) Two-cell embryos n (%) Activated n (%) Reconstructed oocytes n (%) Oocytes n Group with the source of ooplasm/spindle
In-vitro development of parthenogenetic spindle transfer embryos. Table 1
5 (25.0)a 3 (21.4)a 0 (0)b 0 (0)b 7 (31.8)a 1 (2.9)b
C Ding et al. Expanded/hatched blastocysts n (%)
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transfer embryos developed to the eight-cell stage in the two groups, respectively, and no spindle transfer embryos developed to the blastocyst stage, with no difference detected between the groups. Furthermore, the development rate of parthenogenetic spindle transfer embryos with in-vitro matured SCC and in-vivo matured ooplasm was as high as that obtained with intact parthenogenetic controls (in-vivo matured oocytes without spindle transfer). Most of these parthenogenetic spindle transfer embryos displayed an expanded blastocoel on day 6 (Table 1 and Figure 2C). In contrast, the in-vitro development of parthenogenetic spindle transfer oocytes to the eight-cell stage differed significantly between the groups with differently sourced ooplasm. The rates of embryo development to the eightcell stage with in-vitro matured ooplasm, including SCC matured in vitro (12.5%) and in vivo (11.1%), were statistically lower than that of embryos with in-vivo matured ooplasm (in vivo/in vivo, 60.0%; in vivo/in vitro, 57.1%; P < 0.05). Moreover, most of the parthenogenetic spindle transfer embryos with in-vitro matured ooplasm (in vitro/in vitro and in vitro/in vivo groups) were arrested at the eight-cell stage, regardless of whether the SCC was derived from in-vivo or in-vitro-matured oocytes. The development rate of spindle transfer embryos with in-vitro matured ooplasm was similar to that of in-vitro matured parthenogenetic intact controls without spindle transfer (Table 1). This result indicates that the impaired development of spindle transfer embryos was not caused by spindle transfer manipulation but rather by the incompetence of donor ooplasm matured in vitro. The premature activation rate of reconstructed oocytes was low in the present study, even after 24 h of in-vitro culture (Figure 3). Only two spindle transfer oocytes had formed 1PN (2/23 [8.7%]), which is comparable to that of control oocytes without spindle transfer manipulation (Table 2).
Efficiency of spindle transfer using the direct injection method for oocyte reconstruction The reconstruction rate and activation rate of spindle transfer embryos with ooplasm and SCC from different sources are shown in Table 1. More than 81% (84/103) of the manipulated oocytes survived the injection procedure, which is a higher rate than that reported by other researchers for oocytes reconstructed using the electro-fusion method (73%, Tachibana et al., 2009). The reconstructed embryos in all four groups were activated with high efficiency (Table 1), which did not differ from that of intact parthenogenetic control oocytes. These results demonstrated the high efficiency of the direct injection method.
Distribution of microtubules and chromosomes in spindle transfer oocytes Five spindle transfer oocytes derived from in-vitro matured oocytes were immunofluorescently stained to investigate the distribution of microtubules and chromosomes. All of these oocytes displayed normal spindle morphology (Figure 1F).
Spindle transfer of human oocytes Table 2
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Occurrence of premature activation of oocytesa.
Spindle transfer oocytes Control
2h n/n (%)
4h n/n (%)
8h n/n (%)
24 h n/n (%)
0/23 (0) 0/15 (0)
0/23 (0) 0/15 (0)
0/23 (0) 0/15 (0)
2/23 (8.7) 0/15 (0)
a
Spindle transfer oocytes: the spindles of three to five in-vitro matured oocytes were removed at one time and then randomly transferred back to the oocyte cytoplasm; control: in-vitro matured oocytes without any manipulation were used as control.
Figure 2 In-vitro development of parthenogenetic embryos derived from spinle transfer oocytes. (A) Spindle transfer embryo with one large pronucleus after activation; (B) seven-cell-stage spindle transfer embryos (day 3); (C) parthenogenetic spindle transfer blastocyst (day 6); (D) arrested spindle transfer embryos (day 5). Scale bar: 50 µm.
Discussion In a standard IVF cycle, about 10–20% of the retrieved oocytes will be in the metaphase I or germinal vesicle stage (Chian and Tan, 2002). Many of these immature oocytes are not needed in IVF clinics, and have therefore become a valuable source of study material. In the present study, such oocytes (MI stage) were matured in vitro and used for reciprocal spindle transfer between in-vivo matured and in-vitro matured human oocytes. Most of these oocytes extruded the first PB (i.e. reached maturity) during the first 15 h of invitro culture (maturation time range, 4–15 h). When the spindle apparatus from in-vitro matured oocytes was transferred into in-vivo matured ooplasm, the activation and blastocyst formation rates were similar to those observed with in-vivo matured SCC transferred into in-vivo matured ooplasm. This result demonstrated that the SCC derived from in-vitro matured oocytes could support the development of reconstructed oocytes and therefore be
efficiently used for spindle transfer. Therefore, future spindle transfer for patients with mtDNA disorders may not require the use of exogenous hormones to acquire a fully in-vivo matured SCC from such patients for transfer, simplifying the treatment. When in-vitro matured ooplasm was used to reconstruct oocytes with SCCs matured in vitro or in vivo, the development of embryos to the eight-cell stage was significantly lower than that obtained for embryos with in-vivo matured ooplasm (Table 1). Moreover, all of the embryos in these spindle transfer groups arrested before reaching the blastocyst stage. In the control group (intact oocytes without spindle transfer), only one in-vitro matured oocyte developed to the blastocyst stage after being parthenogenetically activated (2.9%), which is a significantly lower rate than that obtained with invivo matured oocytes (31.8%) (Table 1), demonstrating the lower developmental competence of in-vitro matured oocytes compared with that of in-vivo matured oocytes. Although several reports have documented blastocyst development or live birth achieved from oocytes matured in vitro (Chian and Tan, 2002; Liu et al., 2003), these were different from the leftover immature oocytes from patients undergoing ICSI used in this study. To carry out ICSI, the immature oocytes in the present study had been stripped of cumulus cells before being placed into maturation medium. The cytoplasmic maturation may be affected by a lack of cumulus cells surrounding immature oocytes, which modulate nuclear and cytoplasmic maturation by both physical cell-cell contact and the combined actions of paracrine factors (Kimura et al., 2007). McElroy et al. (2010) observed the ability of intact, cumulusfree, in-vitro matured oocytes to develop to the blastocyst stage after parthenogenetic activation. These researchers reported that all parthenotes cultured in common in-vitro maturation supplementation media arrested before the blastocyst stage, but only three parthenogenetic blastocysts (5.9%) were produced when in-vitro maturation medium was specifically supplemented with a complex combination of ovarian paracrine/autocrine factors. On the basis of this finding, it is not surprising to expect a markedly lower spindle transfer efficiency when in-vitro matured cytoplasm from cumulusfree oocytes is used. With the exception of the high frequencies of aneuploidy (complex), in-vitro matured, cumulus-free oocytes may not have acquired the factors and machinery necessary for complete cytoplasmic maturation. In this study, specifically for spindle transfer treatment, in-vitro matured ooplasm may lack adequate mitochondria and mtDNA copies or have failed to form proper mitochondrial distribution patterns, affecting the development of reconstructed oocytes (Nishi et al., 2003; Smith and Alcivar, 1993; Stojkovic et al., 2001). Therefore,
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Figure 3 Visualization of reconstructed oocytes by live fluorescent DNA staining at various time intervals after spindle transfer. (A) Intact mature metaphase II oocytes; (B): enucleated metaphase II oocytes (the inserted picture shows the removed SCC, scale bar: 20 µm); (C–E) reconstructed oocytes/embryos at 2 h (C); 4 h (D); and 8 h (E) after spindle transfer (scale bar: 50 µm). The DNA was visualized by live blue fluorescent staining.
this type of oocyte would not be considered potential spindle recipients to correct pathogenetic mtDNA mutations at the current time. With improvements in the in-vitro culture conditions and in-vitro maturation technology, however, it is possible that in-vitro matured ooplasm could be used in the future. Although much progress has been made in recent spindle transfer studies, it is difficult to further increase spindle transfer efficiency to validate its hypothetical application in
clinical treatments. For example, the spindle-chromosome apparatus in methapse II oocytes is an extremely sensitive structure that can easily be perturbed by physical or chemical manipulations. Paull et al. (2013) reported that the manipulation of SCC during spindle transfer procedure frequently induces the premature activation of oocytes, resulting in karyotype abnormalities. These researchers observed that the use of a relatively immature SCC could decrease the occurrence
Spindle transfer of human oocytes of the early activation of oocytes through electrofusion methods (Paull et al., 2013). The underlying reasons are not clear at the present time, although this phenomenon may be related to the decreased kinase target phosphorylation observed in fully matured SCCs caused by oocyte reconstruction procedures. The careful observation of the morphology of ST oocytes (polar body extrusion and pronuclear formation) is critical for the discrimination between normal and prematurely activated spindle transfer oocytes. Using living cell workstation microscopy, the extrusion of the second polar body and pronucleus formation at different time points after spindle transfer were carefully observed in this study. In the present study, the reconstructed oocytes had a low rate of premature activation, which was not significantly different from that of unmanipulated control oocytes, regardless of whether the SCCs were matured in vitro or in vivo (Table 2). In our study, SCC was directly injected into ooplasm without any fusion procedure to reconstruct oocytes. The low occurrence of the premature activation of oocytes found in this study may be related to the omission of fusion manipulation. The possible association between the fusion method and the premature activation of oocytes, however, needs to be further investigated using different oocyte reconstruction methods. With the new design of the piezo-driven cell injector, a small piezo stack is sufficient to perform the cell injection process. With proper selection of the excitation frequency and amplitude, harmful lateral tip oscillations of the injector pipette could be reduced to a satisfactory degree (Yu et al., 2007). In the present study, the survival rates or successful reconstruction rates of oocytes after spindle transfer manipulation were greater than 75% in each group, demonstrating the safety and efficiency of the direct injection method for the reconstruction of oocytes with the piezo-driven cell injector. In the present study, the second polar body (PB2) extrusion was deliberately suppressed when spindle transfer oocytes were activated to obtain parthenogenetic embryos for the analysis of their developmental potential. The chromosomal aneuploid rate of spindle transfer embryos may therefore be much lower than that obtained with other methods because the second meiosis did not occur. In conclusion, our results show that in vitro-matured karyoplasts (SCC) can effectively support the development of reconstructed oocytes. Therefore, patients with mtDNA disorders may not require the use of exogenous hormones to acquire fully in-vivo matured oocytes and their SCC for spindle transfer, simplifying the treatment if spindle transfer is to become a practical method to address maternally inherited oxidative phosphorylation disease. In contrast, a human oocyte that matured entirely in vivo is currently the appropriate gamete for the cytoplasmic donor because the in-vitro matured ooplasm did not support the development of reconstructed oocytes using the currently available technology. This finding may change if the physiological basis for the poor developmental performance of completed in-vitro maturation is better understood and can be addressed in vitro.
Acknowledgements This study was supported by the grants from the Key Laboratory of Guangdong Province, the priming scientific
715 research foundation for the junior teachers of medicine in Sun Yat-sen University (10ykpy14; 12ykpy23), the National Natural Science Foundation of China (81100472).
References Bai, Z.D., Liu, K., Wang, X.Y., 2006. Developmental potential of aged oocyte rescued by nuclear transfer following parthenogenetic activation and in vitro fertilization. Mol. Reprod. Dev. 73, 1448– 1453. Bao, S., Ushijima, H., Hirose, A., Aono, F., Ono, Y., Kono, T., 2003. Development of bovine oocytes reconstructed with a nucleus from growing stage oocytes after fertilization in vitro. Theriogenology 59, 1231–1239. Chian, R.C., Tan, S.L., 2002. Maturational and developmental competence of cumulus-free immature human oocytes derived from stimulated and intracytoplasmic sperm injection cycles. Reprod. Biomed. Online 5, 125–132. Craven, L., Tuppen, H.A., Greggains, G.D., Harbottle, S.J., Murphy, J.L., Cree, L.M., Murdoch, A.P., Chinnery, P.F., Taylor, R.W., Lightowlers, R.N., Herbert, M., Turnbull, D.M., 2010. Pronuclear transfer in human embryos to prevent transmission of mitochondrial DNA disease. Nature 465, 82–85. Cummins, J., 1998. Mitochondrial DNA in mammalian reproduction. Rev. Reprod. 3, 172–182. Fulka, H., 2004. Distribution of mitochondria in reconstructed mouse oocytes. Reproduction 127, 195–200. Hecht, N.B., Liem, H., Kleene, K.C., Distel, R.J., Ho, S.M., 1984. Maternal inheritance of the mouse mitochondrial genome is not mediated by a loss or gross alteration of the paternal mitochondrial DNA or by methylation ofthe oocyte mitochondrial DNA. Dev. Biol. 102, 452–461. Khoudja, R., Li, T., Ding, C., Xu, Y., Liu, Y., Zhou, W., Zhou, C., 2013. Effect of co-incubation of oocytes with a decreasing number of spermatozoa on embryo quality. Reprod. Biomed. Online 26, 353– 359. Kimura, N., Hoshino, Y., Totsukawa, K., Sato, E., 2007. Cellular and molecular events during oocyte maturation in mammals: molecules of cumulus-oocyte complex matrix and signalling pathways regulating meiotic progression. Soc. Reprod. Fertil. Suppl. 63, 327–342. Liu, J., Lu, G., Qian, Y., Mao, Y., Ding, W., 2003. Pregnancies and births achieved from in vitro matured oocytes retrieved from poor responders undergoing stimulation in in vitro fertilization cycles. Fertil. Steril. 80, 447–449. Mai, Q., Yu, Y., Li, T., Wang, L., Chen, M.J., Huang, S.Z., Zhou, C., Zhou, Q., 2007. Derivation of human embryonic stem cell lines from parthenogenetic blastocysts. Cell Res. 17, 1008–1019. McElroy, S.L., Byrne, J.A., Chavez, S.L., Behr, B., Hsueh, A.J., Westphal, L.M., Pera, R.A., 2010. Parthenogenic blastocysts derived from cumulus-free in vitro matured human oocytes. PLoS ONE 5, e10979. Nishi, Y., Takeshita, T., Sato, K., Araki, T., 2003. Change of the mitochondrial distribution in mouse ooplasm during in vitro maturation. J. Nippon Med. Sch. 70, 408–415. Paull, D., Emmanuele, V., Weiss, K.A., Treff, N., Stewart, L., Hua, H., Zimmer, M., Kahler, D.J., Goland, R.S., Noggle, S.A., Prosser, R., Hirano, M., Sauer, M.V., Egli, D., 2013. Nuclear genome transfer in human oocytes eliminates mitochondrial DNA variants. Nature 493, 632–637. Shitara, H., Kaneda, H., Sato, A., Inoue, K., Ogura, A., Yonekawa, H., Hayashi, J.I., 2000. Selective and continuous elimination of mitochondria microinjected into mouse eggs from spermatids, but not from liver cells, occurs throughout embryogenesis. Genetics 156, 1277–1284.
716 Smith, L.C., Alcivar, A.A., 1993. Cytoplasmic inheritance and its effects on development and performance. J. Reprod. Fertil. Suppl. 48, 31–43. Solano, A., Playan, A., Lopez-Perez, M.J., Montoya, J., 2001. Genetic diseases of the mitochondrial DNA in humans. Salud Publica Mex. 43, 151–161. Stojkovic, M., Machado, S.A., Stojkovic, P., Zakhartchenko, V., Hutzler, P., Gonçalves, P.B., Wolf, E., 2001. Mitochondrial distribution and adenosine triphosphate content of bovine oocytes before and after in vitro maturation: correlation with morphological criteria and developmental capacity after in vitro fertilization and culture. Biol. Reprod. 64, 904–909. Sutovsky, P., Moreno, R.D., Ramalho-Santos, J., Dominko, T., Simerly, C., Schatten, G., 1999. Ubiquitin tag for sperm mitochondria. Nature 402, 371–372. Sutovsky, P., van Leyen, K., McCauley, T., Day, B.N., Sutovsky, M., 2003. Degradation of paternal mitochondria after fertilization: implications for heteroplasmy, assisted reproductive technologies and mtDNA inheritance. Reprod. Biomed. Online 8, 21–33. Tachibana, M., Sparman, M., Sritanaudomchai, H., Ma, H., Clepper, L., Woodward, J., Li, Y., Ramsey, C., Kolotushkina, O., Mitalipov, S., 2009. Mitochondrial gene replacement in primate offspring and embryonic stem cells. Nature 461, 367–372. Tachibana, M., Amato, P., Sparman, M., Gutierrez, N.M., TippnerHedges, R., Ma, H., Kang, E., Fulati, A., Lee, H.S., Sritanaudomchai, H., Masterson, K., Larson, J., Eaton, D., SadlerFredd, K., Battaglia, D., Lee, D., Wu, D., Jensen, J., Patton, P., Gokhale, S., Stouffer, R.L., Wolf, D., Mitalipov, S., 2013. Human embryonic stem cells derived by somatic cell nuclear transfer. Cell 153, 1228–1238. Takeuchi, T., Gong, J., Veeck, L.L., Rosenwaks, Z., Palermo, G.D., 2001. Preliminary findings in germinal vesicle transplantation of immature human oocytes. Hum. Reprod. 16, 730–736.
C Ding et al. Trounson, A., 2001. Nuclear transfer in human medicine and animal breeding. Reprod. Fertil. Dev. 13, 31–39. Van Blerkom, J., 2004. The role of mitochondria in human oogenesis and preimplantation embryogeneisis: engines of metabolism, ionic regulation and developmental competence. Reproduction 128, 269–280. Van Blerkom, J., 2008. Mitochondria as regulatory forces in oocytes, preimplantation embryos and stem cells. Reprod. Biomed. Online 16, 553–569. Wakayama, S., Thuan, N.V., Kishigami, S., Ohta, H., Mizutani, E., Hikichi, T., Miyake M., Wakayama T., 2004. Production of offspring from one-day-old oocytes stored at room temperature. J. Reprod. Dev. 50, 627–637. Wallace, D.C., Singh, G., Lott, M.T., Hodge, J.A., Schurr, T.G., Lezza, A.M., Elsas, L.J., 2nd, Nikoskelainen, E.K., 1988. Mitochondrial DNA mutation associated with Leber’s hereditary optic neuropathy. Science 242, 1427–1430. Wang, M.K., Chen, D.Y., Liu, J.L., Li, G.P., Sun, Q.Y., 2001. In vitro fertilisation of mouse oocytes reconstructed by transfer of metaphase II chromosomes results in live births. Zygote 9, 9–14. Yu, Y., Ding, C., Wang, E., Chen, X., Li, X., Zhao, C., Fan, Y., Wang, L., Beaujean, N., Zhou, Q., Jouneau, A., Ji, W., 2007. Piezoassisted nuclear transfer affects cloning efficiency and may cause apoptosis. Reproduction 133, 947–954.
Declaration: The authors report no financial or commercial conflicts of interest.
Received 14 April 2014; refereed 20 August 2014; accepted 27 August 2014.
