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BOR - Papers in Press, published online ahead of print November 3, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.032391
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BIOLOGY OF REPRODUCTION 72, 584–592 (2005)
DOI: 10.1095/biolreprod.104.032391
© 2005 by the Society for the Study of Reproduction, Inc.

Effect of Treating Induced Mitochondrial Damage on Embryonic Development and Epigenesis

Takumi Takeuchi, Queenie V. Neri, Yukiko Katagiri, Zev Rosenwaks, and Gianpiero D. Palermo1

The Center for Reproductive Medicine and Infertility, Weill Medical College of Cornell University, New York, New York 10021


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Germinal vesicle transplantation (GVT) has been proposed as a possible treatment to correct age-related oocyte aneuploidy caused by dysfunctional ooplasm. How healthy ooplasm regulates normal meiosis and subsequent development has yet to be elucidated, but impaired mitochondrial metabolism may be attributable to incomplete segregation of the oocyte chromosomes. In the present study, after ooplasmic mitochondrial damage by photoirradiating chloromethyl-X-rosamine, examination of the oocyte nuclei's ability to survive after transfer into healthy ooplasts was performed. To assess their fertilizability and potential for development, GVT oocytes were fertilized by intracytoplasmic sperm injection (ICSI) and transferred to foster mice. Condition of the offspring at birth was assessed, and epigenetic analysis was performed. Photosensitization consistently inhibited oocyte maturation. However, after GVT of photosensitized nuclei into healthy ooplasts, 67.2% were reconstituted, and 76.2% of these matured normally, with an overall rate of 51.2%, much higher than that (6.0%) in the mitochondrially injured oocytes. After ICSI, 65.8% (52/79) of GVT oocytes were fertilized normally, and 21.1% (11/52) eventually reached the blastocyst stage. The transfer of 132 two-cell GVT embryos into the oviducts of pseudopregnant females resulted in 17 apparently healthy live offspring. For some key developmental genes, a high level of expression was identified in the GVT and "rescue"-derived fetal adnexa. Thus, one can induce in oocyte mitochondria a photosensitization-based type of damage, which consistently inhibits GV breakdown, meiotic spindle formation, chromosomal segregation, and polar body extrusion. Germinal vesicle transplanted and rescued oocytes were able to undergo maturation, fertilization, and embryonic cleavage and, ultimately, to develop to term. This approach may provide a model with which to study the age-related ooplasmic dysfunction seen in human oocytes.

fertilization, gamete biology, gene regulation, genomic imprinting, germinal vesicle transplantation, meiosis, mitochondrial photosensitization, oocyte development, ooplasmic dysfunction


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The fecundity of women is affected negatively by maternal age because of a higher incidence of oocyte aneuploidy [1–3], which appears during the first meiotic division, as a result of predivision or nondisjunction of chromatids [4]. The segregation of meiotic chromosomes/chromatids depends on their correct binding to the fibers of the meiotic spindle [5]. The dynamics of spindle function remain unclear, but the polymerization of spindle microtubules appears to depend on ATP [6, 7]. The major organelles responsible for ATP production are mitochondria, which are distributed in particular patterns during the maturational stages and early zygote development [6, 8, 9]. Mitochondrial DNA may undergo point mutations and deletions, and they may replicate abnormally or, more importantly, experience the effects of an underproduction of ATP, resulting in respiratory chain defects [10]. As in other cell types, mutations in mitochondrial DNA accumulate with age [11–13], and a similar situation has been encountered in aged oocytes and their surrounding granulosa cells [14– 16]. In accord with this "mitochondrial aging" hypothesis [15], it has been demonstrated that a lower mitochondrial membrane potential is associated with an age-related higher frequency of abnormal meiotic spindle formation in mature human oocytes [17–19].

In aged ovaries, cytoplasmic damage can be induced by abnormalities of the microcirculation [20]. As serial ovulations and follicular atresia induce scarring of the ovarian cortex, this scarring is progressively substituted by connective tissue, thereby creating a greater distance between the follicular content and the perifollicular capillary bed [21, 22]. In this situation, both follicular hypoxia and a related production of oxygen free radicals have been linked to the ooplasmic damage observed with age [9, 23, 24]. Germinal vesicle transplantation (GVT) has been reported to prevent or treat the age-related cytoplasmic dysfunction responsible for oocyte aneuploidy [25–27], but to our knowledge, no experimental model of mitochondrial damage directly linked to chromosomal imbalance has been reported that is analogous to the situation in humans.

In addition to oocyte aging, oxidizing agents and, at high dosages, drugs such as chloramphenicol (a potent inhibitor of mitochondrial peptidyl transferase) [28] or diazepam (a tranquilizer and anticonvulsant) can disturb the spatiotemporal distribution of mitochondria during oocyte maturation. Binding of the latter drug to peripheral-type benzodiazepine receptors on the mitochondrial membrane may affect the availability of ATP and calcium homeostasis [29], ultimately leading to aneuploidy [9, 24, 30, 31]. Another drug, chloromethyl-X-rosamine (CMXRos), a mitochondrion-specific fluorescent probe, recently has been shown to photosensitize living mitochondria after treated cells are excited by ionizing radiation [32]. In that connection, we have proposed mitochondrial photoinduced damage as a method through which to provoke ooplasmic dysfunction leading to aneuploidies similar to those observed with age [33].

In the present study, the effect of CMXRos-mediated mitochondrial photosensitization on the functional state of the ooplasm as monitored by the normal completion of the first meiotic division was investigated. To confirm mitochondrial damage, confocal imaging and ultrastructural analysis were performed on random oocytes. In an attempt to restore the ability to mature normally, germinal vesicle (GV) nuclei from treated oocytes were transplanted into healthy enucleated cytoplasts. Karyotyping was performed to control for any possible effects of the micromanipulation procedures involved. Manipulated oocytes were then fertilized by intracytoplasmic sperm injection (ICSI) and cultured to the blastocyst stage, after which the "rescued" embryos were transferred to pseudopregnant mice. General conditions of the offspring were assessed, and epigenetic analysis on some embryo developmental genes was performed, with the results compared to data for fetuses conceived in vivo.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Collection of Gametes

The experimental protocol was approved by the Institutional Animal Care and Use Committee of Weill Medical College of Cornell University. Mouse oocytes and spermatozoa were obtained from B6D2F1 strain mice (age, 7–11 wk) as described previously [34]. To prevent spontaneous GV breakdown (GVBD), oocytes were first cultured in Waymouth medium (MB752/1; Invitrogen, Carlsbad, CA) supplemented with a phosphodiesterase inhibitor (0.2 mM 3-isobutyl-1-methylxanthine [IBMX]; Sigma Chemical Co., St. Louis, MO), 0.23 µM sodium pyruvic acid (Sigma), and 5% fetal bovine serum (FBS; Invitrogen).

Induction of Mitochondrial Damage

Oocytes were cultured in Waymouth-IBMX containing 500 nM of CMXRos (MitoTracker Red; Molecular Probes, Eugene, OR) for approximately 30 min, followed by rinsing (three times) in Waymouth-IBMX and exposure to epifluorescent illumination for 10 sec. In preliminary experiments, we compared the effect of exposure time on oocyte nuclear maturation while maintaining a steady concentration of CMXRos. We identified a loading dosage of 500 nM CMXRos with a 10-sec photoirradiation time as the ideal combination to consistently achieve inhibition of nuclear maturation (Fig. 1). Excitation of the fluorochrome was performed on an inverted microscope (Olympus IX-70; New York/New Jersey Scientific, Inc., Middlebush, NJ) equipped with an epifluorescent attachment (100-W mercury burner) with a Texas Red filter (excitation wavelength, 560/55 nm; emission wavelength, 645/75 nm) at 200x magnification. Control oocytes were exposed either to CMXRos alone or to photoirradiation alone.



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  		FIG. 1. Inhibitory effect of photoirradiation on oocyte maturation. The CMXRos-loaded GV-stage oocytes were photoirradiated for 0 sec (n = 88), 5 sec (n = 73), 10 sec (n = 85), 20 sec (n = 58), and 30 sec (n = 52), respectively. All values are expressed as a percentage (mean ± SD) of the GV-stage oocytes that matured to MII stage. Different letters indicate significant difference (P < 0.001)

Function, Distribution Patterns, and Morphology of Damaged Mitochondria

To assess any implied changes in the membrane potential and distribution patterns of the mitochondria, fluorescent patterns in photosensitized oocytes were studied under an epifluorescent or a confocal laser-scanning microscope (LSM510; Zeiss, Oberkochen, Germany). Mitochondrial damage typical of the initial stage of cell apoptosis was characterized by more dilated, aggregated, and less homogeneously distributed fluorescent signals [32].

To confirm the conclusions based on fluorescent signals, the morphology and distribution pattern of the mitochondria photosensitized in eggs were assessed by transmission-electron microscopy. Two hours following their photosensitization, a group of GV-stage oocytes was fixed for 1 h in 2% glutaraldehyde in 0.1 M cacodylate buffer at room temperature and overnight at 4°C, then rinsed in buffer, postfixed in 2% osmium tetroxide for 1 h, dehydrated through increasing concentrations of ethanol up to 100%, and embedded in Spurr resin. Thick plastic sections (1 µm) were cut and stained with toluidine blue in borate buffer. Ultrathin sections (60 nm) stained with uranyl acetate and lead citrate were studied in a JEOL 100S transmission-electron microscope [35].

Assessment of Spindle Structure by Immunofluorescent Study

In the oocytes maturing to the metaphase II (MII) stage, the shape and size of the meiotic spindle as well as chromosomal alignment were imaged by ß-tubulin and nuclear DNA staining as previously described [36–38]. Oocytes were fixed in 4% paraformaldehyde in Dulbecco PBS (Invitrogen) containing 0.1% polyvinyl alcohol (PVA; Sigma) for 40 min at 37°C followed by treatment with 0.2% Triton X-100 (Sigma) in PBS-PVA for 40 min. The fixed oocytes were washed twice in PBS-PVA for 15 min each and then stored at least overnight in PBS-PVA supplemented with 1% BSA at 4°C. After a further two washes as described above, oocytes were blocked with 10% normal goat serum and incubated with anti-ß-tubulin antibody (1:200; Sigma) for 1 h at 37°C, followed by three washes and subsequent labeling with fluorescein isothiocyanate-conjugated anti-mouse immunoglobulin G (1:100; Sigma) for 1 h. Samples were washed and incubated with 10 µg/ml of propium iodide (Molecular Probes) for 15 min to counterstain nuclear DNA. The oocytes were observed using a fluorescent microscope (Olympus BX-61; New York/New Jersey Scientific) with an appropriate filter set. The fluorescent images were captured and analyzed with an imaging software (CytoVision; Applied Imaging, San Jose, CA).

GVT and Oocyte Maturation In Vitro

The micromanipulation and electrofusion procedures were carried out in a plastic Petri dish on the heated stage of an inverted microscope equipped with hydraulic micromanipulators (model MM-188 and MO-109; Narishige USA, Inc., East Meadow, NY), and microinjectors (model I.M.-6 and SRY-15; Narishige USA) in under dim light [26, 27]. The GVT was performed after approximately 2 h of photoirradiation. Then, GV-stage karyoplasts were transferred into enucleated oocytes in combinations where 1) neither had been photosensitized (control); 2) or only the karyoplast; or 3) only the ooplast had been photosensitized (Figs. 2–5).



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  		FIG. 2. Effect of CMXRos loading and/or photoirradiation on oocyte maturation. The chart shows the in vitro maturation rates among the four different groups. The CMXRos-loaded and photoirradiated (photosensitized) oocytes showed a remarkable impairment in maturation rate (P = 0.0001), whereas neither CMXRos loading nor photoirradiation individually affected oocyte maturation rate



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  		FIG. 5. A grafted oocyte consisting of a photosensitized karyoplast and an intact cytoplast as depicted by (a) bright-field and (b) fluorescence microscopy. Bar = 50 µm

Successfully reconstituted oocytes were rinsed and cultured in IBMX-free Waymouth medium for 14–16 h to allow nuclear maturation, which was judged according to the extrusion of a defined polar body (PB). Some of the oocytes that had matured were processed for karyotyping by staining chromosome spreads with Giemsa as previously described [26].

Oocyte Development and Transfer

Oocytes matured in vitro with the different treatment combinations of karyoplast plus cytoplast (Fig. 3, B and C) were subjected to piezo-ICSI using isolated sperm heads and then cultured for 96 h in KSOM medium to assess fertilization rates and embryo development [34]. Oocytes that underwent in vitro maturation and were not manipulated after removal of the surrounding cumulus cells served as controls. Two-cell embryos after rescue GVT (GVTr) were transferred surgically via a lateral back body-wall incision into oviducts of CD-1 foster mothers mated with a vasectomized male of the same strain [34]. In addition, some two-cell ICSI embryos derived from oocytes matured in vitro were transferred to pseudopregnant females as controls.



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  		FIG. 3. In vitro maturation rates of oocytes treated by GVT. A) A group of GVT oocytes in which a GV-stage karyoplast isolated from an intact oocyte was transplanted into an enucleated, nonphotosensitized oocyte. B and C) GVT oocytes in which reciprocal transplantation was performed between photosensitized and intact oocytes

Epigenetic Assessment on GVTr-Derived Offspring

Processing of specimens for RNA extraction and qualification Fetuses and their adnexa generated by nuclear transfer (NT) rescue of photoirradiated oocytes also were processed for RNA isolation. The fetuses were delivered at Day 19–20 by cesarean section. Similarly aged fetuses obtained by in vivo conception served as controls. Tissues were dissected in Dulbecco PBS (Invitrogen) with sterile microforceps and needles. Total RNA was isolated from tissue by Absolutely RNA Microprep Kit (Stratagene, La Jolla, CA), including a DNase treatment step. Elution buffer was warmed to 60°C to increase the RNA yield. The RNA was stored at –80°C until use, with a small portion, ranging between 1 and 5 µl, being used for the quantification and qualification analyses.

Quantitative reverse transcription-polymerase chain reaction Primers were custom-designed by OligoPerfect Designer software (Invitrogen) with a final primer concentration of 200 nM, a melting temperature of 60°C, and a salt concentration of 73.8 mM. The design was based on mRNA sequences obtained from GenBank: Gapdh (accession no. NM008084) served as an endogenous control, and the assayed genes were Igf2 (accession no. NM010514), H19 (accession no. AK003142), and Igf2r (accession no. NM010515). Analysis was performed using an ABI Prism 7900HT (Applied Biosystems, Foster City, CA). Reverse transcription (RT) and quantitative polymerase chain reaction (qPCR) were performed using SuperScript III Platinum Two-Step qRT-PCR Kit with SYBR Green (Invitrogen). Real-time PCR was performed using the ABI PRISM 7900 sequence detector (Applied Biosystems), in which templates were amplified up to 45 cycles with denaturation at 95°C for 15 sec, annealing at 55°C for 30 sec, and extension at 72°C for 30 sec. The qPCR results were plotted by the Sequence Detection System Analysis Software (Version 2.0; Applied Biosystems). Gene expression was reported as the percentage calculated on the cycle threshold against Gapdh considered to represent 100% expression. Samples were always analyzed in replicates.

Data Analysis

For the different comparisons involving oocyte survival, reconstitution, maturation, embryo development, and gene expression analysis, a chi-square test was used. A two-tailed test was used to assess significance levels and considered at 5% probability. Statistical comparisons are reported in the text and tables only when significance was reached. All statistical computations were conducted using StatView 512+ (BrainPower, Inc., Calabasas, CA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Influence of Photosensitization on Membrane Potential, Morphology, and Distribution Patterns of Oocyte Mitochondria

As seen in the fluorescent and the confocal laser-scanning microscopes at 2 h after photoirradiation, 49 of 51 photosensitized GV-stage oocytes displayed clustering and swelling of their mitochondria and a reduced intensity of fluorescent signals (Fig. 6, B2). These mitochondrial features were confirmed in ultrastructural analysis of photosensitized oocytes arrested at the GV stage, with more aggregated or dilated/swollen mitochondria in the photosensitized oocytes than in the controls (Fig. 6, C2). Some of the photosensitized oocytes displayed blebbing (Fig. 6, A2) of the plasma membrane or even degeneration (22% and 2.4%, respectively) during the 14–16 h of the in vitro culture period.



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  		FIG. 6. Intact GV-stage oocyte after MitoTracker loading (column 1) and after photoirradiation (column 2) depicted by different imaging techniques: bright-field (A), laser-scanning confocal (B), and transmission-electron microscopy (C). The cellular damage can be demonstrated by the membrane blebbing (A2), whereas at the organelle level, swelling and clustering of mitochondria induced by photosensitization are visible (B2 and C2). Bar = 50 µm (A and B) and 5 µm (C)

Effect of Mitochondrial Photosensitization on Oocyte Maturation In Vitro

Mitochondrial photosensitization interfered significantly (P < 0.001) with the oocyte maturation process. Neither exposure to CMXRos alone nor irradiation alone affected in vitro maturation rates, whereas photosensitization significantly inhibited oocyte maturation (Fig. 2). Whereas 86% of intact controls matured to the MII stage, only 6% of the experimental group (CMXRos-loaded and photoirradiated) did so after photosensitization.

Effect of Mitochondrial Photosensitization on Meiotic Spindle

Among the oocytes suffering from maturational arrest following photosensitization, the meiotic spindle configuration was compromised, with consequent chromosomal misalignment in 44.8% (13/29). This percentage was remarkably higher than that in the intact oocytes serving as control (8.6% [3/35], P < 0.01) (Fig. 7).



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  		FIG. 7. Immunofluorescent images of meiotic spindles and chromosomes of MII-stage oocytes. a) A barrel-shaped spindle with normal chromosomal alignment in a control oocyte, ß-tubulins (green), and chromosomes (red) are depicted. b) Misalignment of chromosomes in a photosensitized oocyte. Bar = 50 µm

In Vitro Maturation of the Reconstituted Oocytes Following GVT

As described in Figure 3, of the oocytes receiving nonphotosensitized GVs (Row A), 85% (45/53) survived, with 95% (43/45) of those maturing normally. By contrast, when oocyte reconstitution involved a photosensitized karyoplast (Row B) or a photosensitized ooplast (Row C), most showed extensive cell lysis after electrofusion (P < 0.01) (Fig. 3). Oocytes successfully reconstituted by transfer of a photosensitized karyoplast to an intact ooplast (GVTr) (Row B) underwent maturation to the MII stage, as reflected by extrusion of the first PB, and at a significantly higher rate than that of oocytes reconstituted with photosensitized ooplasts (Row C) (76.2% of 299 and 20.9% of 43 of the reconstituted oocytes, respectively). When photosensitized karyoplasts were transplanted into nonirradiated ooplasts, the maturation rate was significantly greater than that in intact photosensitized oocytes (P < 0.001) (Figs. 2 and 3). Cytogenetic analysis was performed successfully in 21 of 33 reconstituted (rescued) oocytes that had matured in vitro. All but one (20/21) had a normal haploid chromosomal complement (Fig. 8a).



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  		FIG. 8. a) A normal haploid set of chromosomes from a rescued, in vitro-matured oocyte. b) A foster mother and two offspring generated from rescued oocytes. Bar = 10 µm

Fertilization and Embryonic Cleavage of the Reconstituted Oocytes

The rates of fertilization after ICSI and the in vitro development of mitochondrially impaired oocytes up to 96 h—intact, photosensitized, and after GVTr—are shown in Table 1. In the GVTr group, 65.8% were fertilized normally by ICSI, and 13.9% developed as blastocysts. The rescued oocytes developed at a rate similar to that of the nontreated oocytes matured in vitro, whereas the few zygotes derived from nonmanipulated, in vitro-matured, photosensitized oocytes did not develop.


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  		TABLE 1. Embryo development of cumulus-denuded, in vitro-matured, photosensitized, and germinal vesicle-transplanted "rescued" oocytes

Postimplantation Development of the GVT Oocytes

When a total of 132 two-cell embryos derived from the rescued oocytes was transferred to 10 recipients, 17 live offspring were obtained at Day 20 of pregnancy either by spontaneous delivery or by cesarean section (Fig. 8b). This live-birth rate was comparable to that of control, nonmanipulated, GV-stage oocytes (Table 2).


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  		TABLE 2. Postimplantation development of conceptuses derived from rescue germinal vesicle transplantation

Epigenetic Assessment on GVTr-Derived Offspring

Three fetuses generated by spontaneous conception were delivered at Day 19 by cesarean section with fetal weights of 1.4, 1.3, and 1.8 g. The respective placental tissues weighed 0.13, 0.12, and 0.15 g. Fetuses generated from photoirradiated oocytes rescued by NT were delivered at Day 20 by cesarean section. Their weights were 2.0 and 1.4 g for fetus 1 and fetus 2, respectively. The placental tissues weighed at 0.51 g for fetus 1 and 0.20 g for fetus 2.

Once RT-qPCR was run, linear and logarithmic plotting (Fig. 9, A and B) revealed that expression of Gapdh, the internal control, was consistent in all samples. Although a trend toward higher expression was observed for all genes analyzed, a significant difference was found in expression of Igf2 (P = 0.01) and H19 (P = 0.0006) in GV-transferred oocyte placentae (Fig. 9C).



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  		FIG. 9. Gene expression plotted as cycle threshold in relation to amplicon content. A) Linear plotting of the control gene (Gapdh) and study genes (Igf2, H19, and Ifg2r). B) Logarithmic plotting of control gene (Gapdh) and study genes (Igf2, H19, and Ifg2r). C) Gene expression of mouse GVTr and in vivo-derived placentae. Superscripts indicate the following: a versus b, chi-square test, 2 x 2, 1 df, effect of nuclear transplantation on fetal Igf2 expression, P = 0.01; c versus d, chi-square test, 2 x 2, 1 df, effect of nuclear transplantation on fetal H19 expression, P = 0.0006


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study demonstrates that photosensitization-induced damage of mouse oocyte mitochondria consistently inhibits GVBD, meiotic spindle formation (Fig. 7b), chromosomal segregation, and PB extrusion. Maturational arrest induced by such mitochondrial damage, which we believe may serve as a model for age-related ooplasmic dysfunction, has been reversed successfully by nuclear transplantation, as evidenced not only by oocyte nuclear maturation but also by in vitro and in vivo embryo development. The present study also demonstrates that normal meiosis can still take place where a nucleus isolated from a mitochondrially damaged oocyte is exposed to healthy ooplasm, in that GVTr oocytes are able to undergo maturation, fertilization, and in some cases, development to term.

The reciprocal transplantation of individual karyo- and cytoplasts has shown that the procedure itself does not impair the rate of maturation when GV-stage nuclei were transferred to nonirradiated oocytes as we previously reported [26, 27, 34]. On the other hand, meiosis usually was compromised when a normal nucleus was transferred to a photosensitized ooplast. Whenever maturation did occur, it was at an extremely low rate (~6%), with fertilization also being low (17%). However, although some cleavage occurred, none of the treated oocytes reached the blastocyst stage. On the other hand, situated in a healthy ooplasm, a photoirradiated nucleus was able to complete its maturation. However, this was somewhat less efficient than in the controls (P < 0.001)—perhaps because of the sensitized mitochondria or mitochondrial leakage of factors normally confined to the matrix but carried over with the residual ooplast [39]. This indicates that the procedure cannot be used for the treatment of mitochondria-related diseases until karyoplasts can be prepared without any residual cytoplasm and, so, some mitochondria [39].

When used as a photosensitizer, CMXRos was able to produce selective damage in ooplasmic mitochondria in a dose-related manner. The damage entails mitochondrial swelling because of an altered permeability of the inner membrane, its electrical depolarization, and consequent cytochrome c leakage into the cytosol together with the production of reactive oxygen species, leading to cell apoptosis [32, 40]. In our experience, the insult of photosensitization was reflected in a maturational blockage similar to that observed during aging of human oocytes [9, 15, 31].

The mitochondrial damage induced in the present study could be monitored only by the effect on the inhibition of nuclear maturation. In fact, it has not been possible in rodents to demonstrate a spontaneous occurrence of oocyte aneuploidy in relation to aging, even in selected CBA/CA or SAM (senescence-accelerated mouse) mouse strains [41, 42]. In these mice, a meaningful level of aneuploidy was unachievable because of the exponential decline in oocyte production, so we opted to monitor oocyte nuclear maturation as an indicator of cytoplasmic damage.

Importantly, the maturational arrest was reversed once the nucleus from oocytes with photosensitized cytoplasm was transplanted to an untreated ooplast. As a corollary of this, it has been demonstrated that mitochondrial infusion can increase the ATP content of the recipient-mouse, MII-stage oocytes [43] and that transfused mitochondria can survive throughout embryonic development and undergo replication [44]. The residual irradiated perinuclear cytoplasm apparently did not influence the efficacy of nuclear transplantation [26]. Thus, it seems to be possible that GVT could prevent oocyte aneuploidy—if, indeed, mitochondrial dysfunction is the sole cause of this—in the case of human oocytes as well [33].

It has been postulated that in vitro culture also may tend to induce epigenetic modifications in the genome of the preimplantation embryo [45]. Furthermore, analysis of multiple growth-related and imprinted genes in murine embryos cultured in a chemically defined medium with or without fetal calf serum revealed that culture in the presence of serum affects the regulation of imprinted genes [46]. These epigenetic alterations were not corrected during postimplantation development and were associated with aberrant fetal expression of imprinted genes and phenotypic abnormalities [47].

Recently, it was possible, to our knowledge for the first time, to link mRNA expression patterns with in vivo development of embryos derived in vitro [48]. As implied in discussing large offspring syndrome, birth weights of calves derived from in vitro-produced (IVP) embryos were significantly higher than normal. In general, the results support the hypothesis that early deviations in gene expression patterns are causally involved in this syndrome [48].

The data presented here support the hypothesis that the GVT environment and the NT procedure itself can have profound effects on mRNA expression patterns in embryos created this way. Possible explanations for the abnormal phenotypes frequently observed in offspring born after transfer of in vitro-fertilized and NT-derived embryos include reprogramming errors, epigenetic dysregulation during in vitro culture before their replacement, and in the case of the NT-derived embryos, undefined factors associated with the NT procedure per se. The common occurrence of aberrations in IVP or NT-derived embryos suggests that current in vitro production systems and/or cloning technology may lead to persistent alterations of gene expression patterns during development, perhaps as a result of changes in the methylation patterns [49], and accordingly, deregulation of gene expression appears to perturb placental and fetal growth [50, 51]. Interestingly, changes in a single imprinted gene do not seem to be sufficient to induce the significant overgrowth [52]. The widespread dysregulation of imprinted and nonimprinted genes in NT- and IVP-derived embryos that survived to term suggests that mammalian development can tolerate a substantial degree of epigenetic abnormality. Therefore, the molecular mechanisms underlying epigenetic reprogramming during early embryonic development require further investigation.

The insult of CMXRos was confined to the mitochondria, leaving nuclear DNA unharmed, as demonstrated by the ability of a photoirradiated nucleus to resume maturation after transplantation into an intact ooplast. The mitochondrial/cytoplasmic damage had a deleterious effect on the meiotic spindle similar to that observed in aged human oocytes. The GVTr is able to restore the ability of exposed nuclei to complete meiosis and provide embryo and full-term fetal development once inseminated. No significant epigenetic alterations were observed in fetal tissues generated by GVTr. In conclusion, because GVT can counteract ooplasmic damage, this may provide a way of avoiding that seen in age-related aneuploidy [33]. This procedure, however, is compromised by the limited availability of eggs in aging women; therefore, a definitive option would be to construct female gametes in an alternative manner, such as from a somatic cell [33, 53, 54].



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  		FIG. 4. Reciprocal exchange of nuclei. a) GV-stage karyoplasts from photosensitized and intact oocytes were reciprocally cross-transferred to the corresponding ooplast. b) The grafted oocyte during reconstitution and after 14–16 h in vitro culture


   ACKNOWLEDGMENTS
 
We are most grateful to Prof. J. Michael Bedford for editing the manuscript, Dr. Gayle Jones for her critical comment on the study design and selection of images, and Prof. Alan O. Trounson for contributing to the study planning and design.


   FOOTNOTES
 
1 Correspondence: Gianpiero D. Palermo, The Center for Reproductive Medicine and Infertility, Weill Medical College of Cornell University, 505 East 70th Street, HT-336, New York, New York 10021. FAX: 212 746 8860; gdpalerm{at}med.cornell.edu Back

Received: 20 May 2004.

First decision: 18 June 2004.

Accepted: 10 September 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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