Hum. Reprod. Advance Access originally published online on September 6, 2006
Human Reproduction 2007 22(1):52-62; doi:10.1093/humrep/del345
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Developmental competence of human in vitro aged oocytes as host cells for nuclear transfer
1 Centre for Stem Cell Biology and Developmental Genetics, Institute of Human Genetics, University of Newcastle upon Tyne, UK 2 Department of Biochemistry, Dartmouth Medical School, Hanover, NH, USA 3 Principe Felipe, Centro de Investigación, C/E.P. Avda. Autopista del Saler, Valencia, Spain and 4 Newcastle Fertility Centre at Life, International Centre for Life, Newcastle upon Tyne, UK
5 To whom correspondence should be addressed at: Neuronal Survival Unit, Department of Experimental Medical Science, Wallenberg Neuroscience Center, Department of Physiological Sciences, Lund University, BMC A10, 221 84 Lund, Sweden. E-mail: Vanessa.Hall{at}med.lu.se
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Abstract |
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BACKGROUND: Improving human nuclear transfer (NT) efficiencies is paramount for the development of patient-specific stem cell lines, although the opportunities remain limited owing to difficulties in obtaining fresh mature oocytes. METHODS: Therefore, the developmental competence of aged, failed-to-fertilize human oocytes as an alternate cytoplasmic source for NT was assessed and compared with use of fresh, ovulation-induced oocytes. To further characterize the developmental potential of aged oocytes, parthenogenetic activation, immunocytochemical analysis of essential microtubule proteins involved in meiotic and mitotic division, and RT–PCR in single oocytes (n = 6) was performed to determine expression of oocyte-specific genes [oocyte-specific histone 1 (H1FOO), growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15), zygote arrest 1 (ZAR1)] and microtubule markers [nuclear mitotic arrest (NuMA), minus-end directed motor protein HSET and the microtubule kinesin motor protein EG5]. RESULTS: For NT, enucleation and fusion rates of aged oocytes were significantly lower compared with fresh oocytes (P < 0.05). Cleavage rates and subsequent development were poor. In addition, parthenote cleavage was low. Immunocytochemical analysis revealed that many oocytes displayed aberrant expression of NuMA and EG5, had disrupted meiotic spindles and tetrapolar spindles. One of the six oocytes misexpressed GDF9, BMP15 and ZAR1. Two oocytes expressed EG5 messenger RNA (mRNA), and HSET and NuMA were not detectable. RT-PCR of mRNA for oocyte specific genes and microtubule markers in single aged oocytes. CONCLUSIONS: Thus, aneuploidy and spindle defects may contribute to poor parthenogenetic development and developmental outcomes following NT.
Key words: gene expression/human/microtubule/oocyte/somatic cell nuclear transfer
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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In the developing field of human nuclear transfer (NT), access to oocytes is limited owing to ethical and regulatory guidelines. Limited numbers of fresh, ovulation-induced mature oocytes have been obtained for previous research following voluntary contributions from both fertile women and infertile couples seeking fertility-assisted treatment (Stojkovic et al., 2005a
). However, aged oocytes that fail to fertilize following infertility treatments are a more accessible and abundant source of oocytes that could be used as host cells for NT research. The use of failed-to-fertilize oocytes has not been fully explored in the field of human NT. Determining the developmental competence of these oocytes and their capacity to support nuclear reprogramming following NT is required.
Recently, NT was performed using aged, failed-to-fertilize human oocytes (obtained 16–18 hours post insemination) by direct injection of human female fibroblasts or male telomerized fibroblasts, which resulted in poor cleavage and early embryonic arrest (Lavoir et al., 2005). Evaluation of these NT embryos indicated high aneuploidy as determined by fluorescence in-situ hybridization (Lavoir et al., 2005). However, in the developing field of human NT, much optimization of the technique is required, and use of aged oocytes for NT in other animal models has been performed with some success. Earlier NT studies in the mouse, rabbit and cow used aged metaphase II (MII) oocytes as recipient cells rather than fresh MII oocytes because of their increased sensitivity to artificial activation stimulus (Collas and Robl, 1991; Cheong et al., 1994; Stice et al., 1994). In the cow, these aged oocytes were capable of supporting development following embryonic NT to the blastocyst stage and even support development to term (Stice et al., 1994). The aged oocytes were used in NT before the identification of the potent activator calcium ionophore A23187
[GenBank]
(CI) (Vincent et al., 1992). Furthermore, the ability to release cytosolic calcium following treatment with CI increases with oocyte age (Vincent et al., 1992). In rabbit NT, slightly aged oocytes (17 h post-ovulation induction/hCG injection) resulted in higher rates of activation when compared with freshly ovulated oocytes (13 h) (Cervera and Garcia-Ximenez, 2003), although fusion rates and in vitro development were reportedly lower in slightly aged oocytes (Cervera and Garcia-Ximenez, 2003). Similarly, the use of an electrical stimulus resulted in higher rates of activation in rabbit oocytes 22–24 h post-hCG injection compared with 16–18 h post-hCG (Fissore and Robl, 1992). This increased sensitivity may be because of a loss of cytostatic factor (CSF) (Hashimoto and Kishimoto, 1988), which regulates high levels of maturation-promoting factor within the oocyte, maintaining meiotic arrest (Sagata et al., 1989).
Parthenogenetic activation of aged oocytes appears to vary between species. An age-dependent response was not observed following electrical stimulation in porcine in vitro matured oocytes (Jolliff and Prather, 1997). Morulae/blastocyst development of parthenogenetically activated in vitro aged porcine oocytes (matured 48 h) was equal (14%) when compared with non-aged oocytes (matured 36 h). In contrast, studies have indicated human aged oocytes do not elicit a strong activation response to CI (Balakier and Casper, 1993; Rinaudo et al., 1997). Puromycin (PUR) appears to be a more potent activator in comparison (De Sutter et al., 1992; Balakier and Casper, 1993), although often results in the retention of the second polar body, resulting in karyotypical defects (De Sutter et al., 1994). CI activates oocytes and induces calcium oscillations by altering the oocyte’s membrane permeability, which in turn increases the level of intracellular stores of calcium. PUR is a protein synthesis inhibitor, which inhibits and destroys CSF within the oocyte and is thought to be more effective at inducing the resumption of meiosis in the human (Yamano et al., 2000). Parthenogenetic activation using a combination of CI and PUR in human aged and failed-to-fertilize ICSI (f-ICSI) oocytes resulted in pronuclear formation and extrusion of the second polar body in 80 and 30% of oocytes, respectively (Yamano et al., 2000).
Maternal ageing has also been correlated with an increased incidence of aneuploidy that occurs via meiotic non-disjunction (Battaglia et al., 1996) and meiotic errors (Liu and Keefe, 2004) in oocytes (reviewed in Hassold and Hunt, 2001). This may result from dysfunctional cytoplasm which could alter meiotic spindles (Battaglia et al., 1996) or may occur as a result of reduced cohesion between homologous chromosomes and chromatids in bivalents, resulting in trisomies (Wolstenholme and Angell, 2000; Hodges et al., 2005). Abnormal spindles within oocytes have also been detected in a high proportion of older women (age 40–45years), including both abnormal chromosomal alignment and altered microtubule matrices (Battaglia et al., 1996). Although removal of the meiotic spindle is performed during NT, research in non-human primate NT indicates that enucleation significantly depletes the ooplasm of nuclear mitotic arrest (NuMA) protein and the minus-end directed motor protein HSET (Simerly et al., 2003) and impairs NT embryo development (Simerly and Navara, 2003; Simerly et al., 2003). The role of microtubule proteins seems to be crucial in primate embryo development and needs to be further explored in the human model.
Maternal ageing also appears to affect gene expression patterns in oocytes. A recent expression-profiling study reported 5% of the total transcriptional profile of oocytes obtained from aged mice differed when compared with oocytes derived from young mice (530/11000 genes) (Hamatani et al., 2004). Many of these altered transcripts were related to mitochondrial function and oxidative stress. In addition, the level of expression of genes involved in chromatin structure, DNA methylation and genome stability was also altered. Many genes have been found to be misexpressed in mouse oocytes derived from aged animals, such as Bmpr2, a receptor of the oocyte-specific gene growth differentiation factor 9 (Gdf9) (Hamatani et al., 2004). Altered patterns of gene expression have also been attributed to in vitro culture effects (Wrenzycki et al., 2001a; Lonergan et al., 2006) and from embryo-assisted technologies such as NT (Daniels et al., 2000, 2001; Wrenzycki et al., 2001b, 2004; Humpherys et al., 2002; Smith et al., 2005). Therefore, the coupled effect of maternal ageing and in vitro culture on oocyte RNA may impair the capacity to support nuclear reprogramming and embryonic development following NT.
From recent research, a comparative assessment of different sources of human oocytes for NT suggested that failed-to-fertilize oocytes could not support nuclear reprogramming, and resultant embryos failed to cleave (Stojkovic et al., 2005a), which has been recently supported by Lavoir and colleagues (Lavoir et al., 2005). However, small numbers of oocytes were used in these studies, and NT techniques are continually being optimized. Therefore, an extension of our research knowledge on the use of aged, failed-to-fertilize oocytes for NT is paramount. To assess whether aged cytoplasts could support nuclear reprogramming, we performed NT using aged oocytes compared with fresh ovulation-induced oocytes. To further investigate the developmental competence of human in vitro aged oocytes, we performed a series of experiments. These included assessment of parthenogenetic development and expression patterns of essential microtubule proteins, HSET, NuMA and EG5, within the oocyte and the meiotic spindle, as well as evaluation of messenger RNA (mRNA) expression of oocyte-specific markers, growth differentiation factor (GDF9), oocyte-specific histone 1 (H1FOO), bone morphogenetic protein 15 (BMP15) and zygote arrest 1 (ZAR1) and microtubule markers in six single aged oocytes.
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Materials and methods |
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Patient consent and ethics approval
Patients undergoing infertility treatment at the Newcastle Fertility Centre at Life, Newcastle upon Tyne, UK, were asked whether they would be willing to donate either failed-to-fertilize or fresh oocytes following ovulation induction treatment, for somatic cell NT research. Detailed consent forms were distributed 2weeks before a decision was requested. Before the decision, the project outline and questions concerning the study were discussed with a non-biased nurse employed by the infertility centre. Following counselling before their treatment, some women agreed that if 12 oocytes were aspirated, they would give the next two aspirated oocytes to research. In addition, for acquisition of a human fibroblast cell line, consent from a healthy, female donor (age 37) was obtained for NT research. Oocytes and tissue were obtained from women undergoing treatment at Newcastle Fertility Centre at Life, Newcastle upon Tyne, UK. Procedures were approved by the Human Fertilization and Embryo Authority (HFEA) and by the Newcastle and North Tyneside Local Research Ethics committee.
Source of oocytes
Surplus and fresh ovulation-induced oocytes were obtained from consenting couples. Failed-to-fertilize oocytes were obtained 
48 h post-egg collection following either conventional insemination (IVF) or ICSI. Oocytes were recovered from women with varying infertility factors and were harvested from women of mean age 33. For these experiments, a total of 220 oocytes [48 fresh, 89 failed-to-fertilize IVF (f-IVF) and 83 f-ICSI] were obtained between March and December 2005 from 113 egg collections. Oocytes obtained immediately upon egg collection for use in NT were 38 h post-hCG.
Ovulation induction
Oocytes were obtained by ovulation-induction treatment, as described previously (Stojkovic et al., 2005a). Briefly, ovarian stimulation and down-regulation was achieved by administration of a daily dose of GnRH (800 µg SYNAREL® nasal spray; Pharmacia, Milton Keynes, UK), in addition to HMG (225 IU MENOPUR®; Ferring, M and S, Langley, Berks, UK) for 12 days. A dose of HCG (5000 or 10 000 IU NCG; Serono, Aubonne, Switzerland) was administered, and cumulus oocyte complexes were recovered via transvaginal aspiration 38 h post-hCG injection.
Insemination and culture of oocytes that failed to fertilize
Denuded MII stage oocytes were inseminated in either G-SPERMTM or G-FERTTM, then cultured in G1.3TM medium (Vitrolife, Kungsbacka, Sweden). Failure of fertilization was assessed on day 1 post-insemination by lack of visible pronuclei and failure to cleave by day 2. Failed-to-fertilize oocytes were obtained if couples had consented for research at day 2 post-insemination/48 h post-egg harvesting.
Human NT
Freshly ovulation-induced cumulus oocyte complexes were denuded using 0.1% hyaluronidase (Sigma, St Louis, MO, USA) in G-MOPSTM PLUS medium. All oocytes were incubated in 5 µg/ml Hoechst 33342 in G-MOPSTM PLUS and enucleated in 7.5 µg/ml cytochalasin B (Sigma) in G-MOPSTM PLUS. A slit in the zona pellucida was produced using the XYCloneTM laser system (Hamilton Thorne Biosciences, Beverly, MA, USA), and enucleation was confirmed following brief excitation by UV light.
Donor cells were a fibroblast-like (FIB-LIKE) cell line derived from a human embryonic stem cell (hESC) line (deposited in UK stem cell bank; 19 May 2004), hESC-NCL-1 (Stojkovic et al., 2004). In addition, donor cells were derived from undifferentiated hESC-NCL-1 cells (ESC) grown on mouse embryonic fibroblasts and a human epithelial fibroblast (FIB) cell line.
FIB-LIKE cells were isolated and cultured as previously described (Stojkovic et al., 2005c). Both FIB-LIKE and FIB cells used for NT were isolated from passages 4 and 8 and grown to confluency for 2–4 days to induce exit of the mitotic cell cycle. Confluent cells were dissociated by standard trypsinization (TrypLETM Select; Invitrogen, Carlsbad, CA, USA), centrifuged and re-suspended in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum (FCS) (Sigma) and 1% penicillin/streptomycin (Invitrogen).
ESCs were obtained from passage 63 for NT. Undifferentiated cell colonies were carefully lifted from feeder-free cultures (Stojkovic et al., 2005b) by mechanical disassociation, centrifuged and re-suspended into single-cell suspension by vigorous pipetting.
The human FIB cell line was isolated from a female healthy donor via skin biopsy (see Patient consent and ethics approval). Primary cells were grown to 70% confluency, passaged and maintained in a proliferative state until required for NT.
Single donor cells were injected into the perivitelline space and fused in mannitol fusion medium [0.28 M mannitol, 100 µM MgCl2, 50 µM CaCl2 and 0.01% human serum albumin (Sigma)]. Fusion was achieved with two consecutive electrofusion pulses of 0.75 kV of 15-µs duration, using a Multiporator (Eppendorf, Hamburg, Germany). Fused couplets were incubated in microdrops of G1.3TM under filtered, embryo-tested mineral oil (Sigma) at 37°C in a humidified incubator containing 5% CO2 in air for 2 h and artificially activated using 5 µM CI (Sigma) for 5 min followed by 2 mM 6-DMAP (Sigma) for 4 h. Reconstructed embryos were cultured in G1.3TM medium in incubator conditions described above and assessed for cleavage on day 2. Cleaved embryos were transferred into G2.3TM (Vitrolife) microdrops on day 3 and cultured in the same incubator conditions until day 5 of development.
Parthenogenetic activation
Oocytes obtained from f-IVF or f-ICSI were randomly assigned to either of two artificial activation regimes. Oocytes were activated using 5 µM CI for 5 min followed by either 2 mM 6-DMAP (f-IVF, n = 10; f-ICSI, n = 11) or 10 µg/ml PUR (Sigma) (f-IVF, n = 12; f-ICSI, n = 10) for 4 h at 37°C in 5% CO2 in humidified air. Activated oocytes were placed in a biphasic culture medium G1.3/G2.3 system, in microdrops under embryo-tested mineral oil. Embryos were assessed on day 2 post-activation and daily thereafter. Assessment of chromatin was performed by staining embryos with 5 µg/ml Hoechst (33342) (Sigma).
Immunocytochemical analysis
A total of 60 failed-to-fertilize oocytes (f-IVF, n = 30; f-ICSI, n = 30) were randomly selected for immunocytochemical analysis. A total of 20 oocytes (f-IVF, n = 10; f-ICSI, n = 10) were labelled for microtubule markers, NuMA, HSET and EG5 and double-labelled with 
-Tubulin. The outer zona pellucida was digested before fixation using 0.5% protease (Sigma). Fixation was performed using 100% methanol, and oocytes were double-immunolabelled using monoclonal anti--tubulin (Sigma) and either polyclonal anti-NuMA (Gaglio et al., 1995), anti-HSET (Mountain et al., 1999) or anti-EG5 (Mountain et al., 1999) at 1:1000 in blocking buffer, 5% FCS (Sigma; Lot:074K3396, USA) in calcium/magnesium-free phosphate-buffered saline for 30 min. Oocytes were then incubated with secondary antibodies including fluorescein isothiocyanate-conjugated donkey anti-mouse and rhodamine red-conjugated donkey anti-rabbit Immunoglobulin Gs at 1:50 and 1:100 dilutions (in blocking buffer), respectively, for 30 min. Oocytes were counterstained in Hoechst 33342 to label chromatin and mounted in glycerol on glass slides under coverslips (VWR, Poole, Dorset, UK).
Confocal microscopy
A laser-scanning microscope (LSM 510; Zeiss, Oberkochen, Germany), equipped with an argon/krypton laser and UV light, was used to acquire z-stack series images of immunostained oocytes. An average of 20 scans per oocyte was obtained. A separate scanned image using a Pseudo-DAPI filter and UV light was obtained. The image was overlaid over a selected single scanned image from the z-stack series.
Isolation of mRNA from single human oocytes
Six failed-to-fertilize oocytes (f-IVF, n = 3; f-ICSI, n = 3) were randomly selected for gene expression analyses. The zona pellucida was digested using 0.5% protease, and zona pellucida-free oocytes were placed individually into RNase-free microcentrifuge tubes containing lysis buffer [5 mM dithiothreitol, 0.8% IGEPAL (Sigma), 1 U/ml RNase OUT (Invitrogen) in diethylpyrocarbonate-treated Milli-Q water], before immersion into liquid nitrogen. Oocytes were stored short term at –80°C. Poly A+ RNA was isolated from total RNA of single oocytes using the Dynabeads mRNA DIRECTTM micro kit (Dynal Biotech, Smestad, Oslo, Norway), according to manufacturer’s instructions.
Amplification of cDNA from single human oocytes
Production of complementary DNA (cDNA) was achieved using the SMARTTM PCR cDNA synthesis kit (Becton Dickinson, Palo Alto, CA, USA). Double-stranded full-length cDNA was produced following reverse transcription and long-distance PCR (LD PCR), according to the manufacturer’s instructions. The cycle settings used for LD PCR were 30 cycles of 95°C for 25 s, 65°C for 30 s and 68°C for 7 min. The quality of cDNA was assessed by running 5 µl of cDNA on an ethidium bromide agarose gel. The remaining product was stored at –80°C.
Detecting mRNA transcripts in single human oocytes
The mRNA expression profiling in single oocytes was performed using RT–PCR to evaluate the expression of oocyte-specific markers, ZAR1, GDF9, H1FOO and BMP15, and microtubule markers, HSET, NuMA and EG5. Briefly, a master mix of 10x PCR buffer, 50 mM MgCl2, 10 mM dNTP mix, 5 U/ml Taq polymerase, 10 µM of both sense and antisense primer and 1 µl of amplified cDNA was added to each 25-µl reaction. Primers used were ACTIN, ZAR1, GDF9, H1FOO, BMP15, HSET, NuMA and EG5 (Table I). The positive control used for oocyte-specific markers was 0.5 ng FirstChoiceTM PCR-ready Human Ovary cDNA (Ambion, Austin, TX, USA) per reaction, and for microtubule markers, FIB-LIKE cell cDNA was used and prepared by standard reverse transcription. Cycling parameters included 5-min 94°C denaturation followed by 45 cycles, 1 min of each denaturation, annealing (Table I) and extension (74°C) and a final 15-min extension (74°C). The PCR was repeated at least twice for each gene of interest.
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Statistical analysis
Statistical analysis of oocyte-cleavage rates following artificial activation was analysed between each treatment group by using a standard two-tailed t-test with a confidence interval of 95%. This test was also used to analyse significant differences between NT efficiencies between treatments. Analysis was performed using GraphPad Prism 3.0TM software (San Diego, CA, USA), and a value of P < 0.05 was considered significant.
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Results |
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NT efficiencies of aged, failed-to-fertilize oocytes compared with fresh, ovulation-induced oocytes
It was evident during the process of NT that failed-to-fertilize oocytes were more difficult to enucleate and fuse compared with using fresh ovulation-induced human oocytes (Table II). Enucleation rates of f-IVF and f-ICSI oocytes were significantly reduced (P < 0.05) compared with fresh oocytes (Table II). In addition, fusion rates were significantly lower (P < 0.05) in the failed-to-fertilize treatments compared with the fresh oocytes, independent of cell type used. As a result, low numbers of reconstructed embryos were produced and cultured from f-IVF and f-ICSI treatments. Cleavage rates were poor across all treatments but were significantly higher when fresh oocytes were used as host cytoplasts. Cleavage rates were 0/6 (0%) and 9/32 (28%) for NT embryos cultured, reconstructed using aged and fresh oocytes, respectively. Interestingly, cleavage rates were highest in NT embryos produced from fresh oocytes and FIB-donor cells. Regardless of oocyte or donor-cell source, developing NT embryos were arrested at early stages of development and failed to form blastocysts. Specifically, one NT-cleaved embryo was produced from fresh oocytes, and a FIB-LIKE cell was arrested at the 4-cell stage (Figure 1A), and another oocyte was fragmented, arresting at the 8-cell stage (confirmed by Hoechst staining, not shown). One cleaved embryo was produced from a fresh oocyte, and ESC was also observed as fragmented (Figure 1B). Development of NT embryos reconstructed using fibroblasts was also limited, resulting in 1 x 2-cell, 1 x 3-cell, 1 x 6-cell and 4 x fragmented embryos (ranging from 5 to 10 cells) (Figure 1C and D).
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Parthenogenetic development of f-IVF and f-ICSI oocytes
The developmental outcomes across treatment groups were poor following artificial activation (Table III). The cleavage rate varied from 20 to 45% but was not significantly different between treatment groups. Fragmentation in a small proportion of oocytes was observed following activation within all treatment groups (Table III and Figure 2). No development was observed beyond the 6-cell stage (Figure 2). Hoechst staining of parthenogenetically activated oocytes indicated chromosomal aberrations such as condensed chromatin, fragmentation and chromosomal misalignment (Figure 3).
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Expression of NuMA protein in failed-to-fertilize, aged oocytes
Labelling of NuMA in f-IVF and f-ICSI oocytes indicated a large proportion of failed-to-fertilize oocytes aberrantly expressed NuMA. Localization of anti-NuMA was observed at the metaphase plate in 7/10 and 9/10 f-IVF and f-ICSI oocytes, respectively (Figure 4). In most cases, cytoplasmic asters (otherwise known as subcortical granules) were observed within the oocyte cytoplasm (e.g. Figure 4H). In one f-IVF and four f-ICSI oocytes, NuMA aligned to four centrosomal ends indicative of the presence of a tetrapolar spindle (Figure 4A, K, L, O and P) rather than a normal bipolar spindle (e.g. Figure 4I). In addition to this, unusual patterns of NuMA staining were observed in three f-IVF oocytes (Figure 4D, E and J) which consisted of large diffuse areas of NuMA within the cytoplasm which contained more punctuate, dense asters of NuMA not associated with the DNA. In addition, an unusual pattern of NuMA expression was observed in one f-ICSI oocyte, with diffuse NuMA and two distinct NuMA asters observed over the chromatin (Figure 4T). This observed staining is reminiscent of NuMA expression in germinal vesicle oocytes, although this oocyte had no visible nuclei and contained a single extruded polar body.
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Expression of HSET protein in failed-to-fertilize, aged human oocytes
The expression of HSET was cytoplasmic in all oocytes with the exception of two f-IVF oocytes (Figure 5A and B). In most oocytes, the pattern of HSET staining was in the form of small cytoplasmic asters, although condensed areas of HSET expression were also observed in a small proportion of f-IVF oocytes (Figure 5D, G and I).
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Expression of EG5 protein in failed-to-fertilize, aged human oocytes
Expression of the microtubule kinesin motor protein, EG5, was detected in all observed oocytes and was associated with the chromatin in only 6/10 f-IVF and 8/10 f-ICSI oocytes (Figure 6). Swollen Hoechst-stained nuclei were observed in three of the oocytes (Figure 6J, S and T). In each of these, EG5 expression was limited to cytoplasmic asters. It was possible to observe within some oocyte spindles, expression of EG5 localized along the microtubules (e.g. Figure 6K). Unusual patterns of EG5 expression were also detected in a small proportion of oocytes (Fig 6G and I).
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Expression of
-tubulin protein in failed-to-fertilize oocytesPeripheral staining of anti-
-tubulin was observed in most failed-to-fertilize oocytes analysed (Figures 4–
6), with the exception of oocytes depicted in Figures 4F and 6Q. Anti--tubulin was detected within two meiotic spindles (Figure 6K and Q).
RT–PCR of mRNA for oocyte-specific genes and microtubule markers in single aged oocytes
Oocyte-specific genes, GDF9, H1FOO, ZAR1 and BMP15, were examined in six single failed-to-fertilize oocytes (Figure 7). H1FOO was detected in all oocytes; however, an interesting larger PCR product (possible alternately spliced fragment) was detected in one f-IVF consistently. GDF9, ZAR1 and BMP15 were detected in only five of the six oocytes, with one f-ICSI oocyte not expressing these genes. Evaluation of mRNA expression of the microtubule marker EG5 indicated only two of the six oocytes expressed transcripts, and both HSET and NuMA transcripts were not detected. Interestingly, transcripts of HSET and NuMA were weak in positive control, FIB-LIKE cDNA, which may suggest that the transcripts are rare. Very weak band detected in f-ICSI3 is of incorrect size and therefore likely to be a non-specific product.
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Discussion |
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Access to large numbers of MII stage oocytes from fertile, young women is a major limiting factor for human NT. In contrast, aged oocytes that fail to fertilize from couples undergoing infertility treatment are a more accessible and abundant source. This study reports that aged, failed-to-fertilize oocytes are a poor source of oocytes for human NT. This may be due to a number of factors including, oocyte aneuploidy, disorganized meiotic spindles, aberrant expression of microtubule proteins and altered expression of crucial oocyte-specific genes. Interestingly, although cleavage occurred following NT using fresh, ovulation-induced oocytes, cleavage rates and ensuing development was also low, suggesting that further optimization of the NT technique in humans is required.
NT efficiencies were significantly lower when using aged, failed-to-fertilize oocytes compared with freshly ovulation-induced oocytes. This may be due to loss of membrane elasticity, which resulted in higher rates of lysis and poorer fusion rates. In most aged oocytes, the metaphase plate was oriented away from the extruded single polar body, which made enucleation more difficult. To successfully enucleate, an increased volume of cytoplasm was removed compared with freshly ovulation-induced oocytes. The cytoplasmic membrane had notably less elasticity in aged oocytes, and in many cases, attachment of the donor cell to the cytoplasm was inefficient. In addition, reconstructed embryos produced from aged, failed-to-fertilize oocytes failed to cleave. Unfortunately, reconstructed embryos produced from both aged and fresh oocytes failed to support development beyond early cleavage divisions. This is likely to be due to a lack of optimized NT methodology. Previous studies in primate NT suggest that the use of pre-MII oocytes improved NT efficiencies (Simerly et al., 2004), although the reasons for this are unknown, as animal NT is dependent on perfect cell cycle synchrony of interphase, MII arrested cytoplasts and G0/G1 donor cells. Further research is required to optimize human NT efficiencies and to support preimplantation development to the blastocyst stage. Perhaps the use of pre-MII oocytes in human NT may improve subsequent embryonic development.
Previous research has indicated in vitro aged oocytes can support blastocyst development in non-human species following NT (Collas and Robl, 1991; Cheong et al., 1994; Stice et al., 1994) and result in live offspring in the bovine (Stice et al., 1994). In this study, the developmental competence of human in vitro aged oocytes was evaluated through parthenogenetic activation, assessment of microtubule markers within the meiotic spindle, mRNA expression of oocyte-specific and microtubule markers in single oocytes and by comparing the use of aged oocytes with freshly ovulation-induced oocytes in NT.
Parthenogenetic activation rates of human in vitro aged oocytes were low. Most oocytes failed to cleave, and a smaller proportion entered mitotic arrest at the first cleavage division. Evaluation of oocyte DNA revealed chromosomal aberrations such as chromosome condensation and loss in most parthenogenetic embryos, which may be because of aberrant spindle structures. A high incidence of aneuploidy and/or loss of internal stores of calcium, resulting in inefficient calcium oscillations upon activation and/or mitotic arrest may also explain the ineffectiveness of parthenogenetic activation in aged oocytes. Post-ovulatory ageing of mammalian oocytes is associated with altered patterns of Ca2+ oscillations following insemination (Igarashi et al., 1997) and poor embryonic development (Igarashi et al., 2005). More recently, it has been demonstrated that the level of intracellular ATP is not readjusted following insemination in aged oocytes (Igarashi et al., 2005). Previous studies have also indicated that aged MII oocytes enter interphase more rapidly than freshly ovulated MII oocytes (Adenot et al., 1997). Similarly, altered protein synthesis has also been detected in aged oocytes (Howlett and Bolton, 1985). Initial cleavage rates of f-ICSI oocytes were relatively high (40 and 45% for Ca-I/PUR and Ca-I/DMAP, respectively) compared with f-IVF oocytes, which has also been shown in previous research (Yamano et al., 2000). Therefore, resultant mitotic arrest may be because of maternal chromatin damage rather than depleted stores of intracellular calcium in these oocytes.
Assessment of the spindle-pole protein, NuMA, indicated aberrant expression patterns and tetrapolar spindles (25%) in many failed-to-fertilize oocytes. NuMA was localized to the spindle in most oocytes analysed although was tethered to abnormal tetrapolar spindles in 25% of the oocytes observed. The minus-end directed motor protein, HSET, has previously been shown to localize between microtubules in the mammalian mitotic spindle and plays a critical role in meiotic spindle organization (Mountain et al., 1999; Simerly et al., 2004). Interestingly, in this study, it was not associated with the spindle in most (90%) aged, failed-to-fertilize human oocytes. Whether HSET is lost from the spindle because of prolonged in vitro culture and/or post-ovulatory ageing is not clear. Previous research has indicated that the motor kinesin protein, EG5, is localized both along the microtubules between the spindle poles and chromosomes of meiotic spindles in Xenopus oocytes (Houliston et al., 1994). In this study, EG5 was also localized along the spindle fibres and at the microtubule ends in most aged, failed-to-fertilize oocytes, although a number of perturbed patterns of expression indicate abnormal meiotic spindles. EG5 plays a crucial role in cell division, centrosome segregation (Blangy et al., 1995) and microtubule stability (Whitehead and Rattner, 1998), and its localization to centrosomes occurs via phosphorylation, which is regulated directly by p34cdc2 protein kinase (Blangy et al., 1995, 1997). It is possible that alterations in this pathway and the lack of EG5 phosphorylation could lead to mitotic arrest, as previous research has indicated that microinjection of anti-EG5 induces mitotic arrest and monastral spindles in HeLa cells (Blangy et al., 1995). This may consequentially result in impaired chromosome segregation and failure to cleave. Interestingly, -tubulin was expressed within the periphery or outer edge of the cytoplasm of most in vitro-aged human oocytes, which has not been previously reported. Whether this peripheral expression pattern in the outer cytoplasmic membrane is associated with in vitro culture and oocyte ageing is not known. In addition, whether this altered expression of -Tubulin is responsible for the lowered membrane elasticity is also unknown and should be further explored. The absence of -Tubulin within most spindles is also of concern. Previous research suggests that the amount of tubulin present within oocytes may also affect the formation of microtubule arrays (Harris and Clason, 1992). Recently, disorganized spindles have been detected within in vitro matured marmoset oocytes (Delimitreva et al., 2006), which suggests that primate oocyte spindle architecture may be sensitive to in vitro culture.
Previous studies in non-human primate NT suggest that the removal of the metaphase plate and associated microtubule proteins is detrimental to ensuing development (Simerly and Navara, 2003; Simerly et al., 2003). Disarrayed patterns of cortical microtubules have been detected in primate NT embryos (Simerly et al., 2004), although microtubule defects are not observed when primate donor cells are fused into bovine cytoplasts (Simerly et al., 2004). This inability to re-establish the microtubule matrix within the cytoplasm appears to be species-specific. The presence of cytasters within the cytoplasm appear to be linked by a series of mesh-like connections to the spindle, as assessed in PtK2 cells (kidney cells derived from the kangaroo rat) (De Brabander et al., 1986), and may play a role in reforming the spindle complex, although confirmation in other animal models is required.
Evaluation of the expression of oocyte-specific genes in individual aged oocytes indicated aberrant expression of genes could be detected. The oocyte-specific genes GDF9, BMP15 and ZAR1 were expressed in all oocytes with the exception of one f-ICSI oocyte. The growth factors, GDF9 and BMP15, are members of the transforming growth factor-
superfamily and are essential for follicular development, ovulation and corpus luteum formation (Juengel et al., 2004; McNatty et al., 2005). ZAR1 plays an important role in the oocyte-to-embryo transition, with null murine embryos arresting at the one-cell stage of development (Wu et al., 2003). The oocyte-specific gene, H1FOO, was expressed in all oocytes. This gene plays an essential role in follicular development. In one oocyte, a double band was detected which may represent a splice variant. In the mouse, two alternative variants H1foo and H1foo have recently been detected (Tanaka et al., 2005). These variants were discovered around the exon 4/intron 4 junction of the murine sequence. Interestingly, our human primers were designed to span intron 4. Sequencing of the upper band would clarify whether this is a splice variant of human H1FOO. The microtubule protein, EG5, was detected in only two of six oocytes. Whether mRNA had degraded or was simply aberrantly expressed is not known. Both HSET and NuMA transcripts were undetected in oocytes, possibly because of rarity of transcripts. Expression levels were very weak in the positive control tissue, FIB-LIKE cell cDNA. Comparative assessment of mRNA expression with fresh oocytes may have been useful, although ethical considerations made it unjustifiable to use fresh ovulation-induced oocytes for purposes other than NT because of their finite supply.
To conclude, our study has indicated that aged, failed-to-fertilize oocytes are a relatively inefficient source of oocytes to use in human NT. High levels of aneuploidy and aberrant spindles were observed in many of these oocytes, which may explain the failure to cleave and early mitotic arrest observed following parthenogenetic activation. In addition, aberrant expression patterns of a key microtubule marker and oocyte-specific genes occurred in some oocytes, which suggest that these oocytes have aberrant transcriptional profiles. NT efficiencies were also lower when using aged, failed-to-fertilize oocytes compared with fresh ovulation-induced oocytes and were unable to support cleavage and further development. However, embryonic development was still limited when using fresh ovulation-induced oocytes, which highlights the lack of optimization of the NT technique. These poor developmental outcomes suggest that there is an intrinsic need to investigate alternate NT protocols, in order to obtain human NT blastocysts. Progression of human NT is therefore dependent on alternate sources of oocytes obtained at the pre-metaphase stage of development. The ethical implications in harvesting fresh oocytes from fertile women will therefore be a critical factor for the development of human NT and the generation of patient-specific stem cell lines.
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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We thank the HFEA for permitting the research license and the local ethics committee for allowing us to perform research on oocytes for the purposes of human NT. We also acknowledge the kind donation of oocytes from all couples who consented to the research. This work was supported by One North East and the UK Department of Health (Life Knowledge Park).
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Submitted on March 3, 2006; resubmitted on July 24, 2006; accepted on August 1, 2006.
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