The Center for Reproductive Medicine and Infertility, Weill Medical College of Cornell University, 505 East 70th Street, HT-336, New York, NY 10021, USA
1 To whom correspondence should be addressed. e-mail: gdpalerm{at}med.cornell.edu
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Abstract |
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Key words: embryo development/germinal vesicle transplantation/ICSI/in vitro maturation/nuclear transfer
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Introduction |
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Aneuploidy in aged oocytes occurs at a specific maturational step, first meiosis, and is due to non-disjunction. This results from a dysfunction of the ooplasm which is unable to provide the molecular structural components necessary to generate a normal meiotic spindle. Specific mechanisms that underlie this may be related to mitochondrial DNA mutations (Keefe et al., 1995; Barritt et al., 2000
; Schon et al., 2000
), oxidative stress (Tarín, 1995
) or abnormalities in the ovarian microcirculation (Gaulden, 1992
; Van Blerkom et al., 1995
; Van Blerkom, 1996
). All of these age-related aberrations can lead to impaired mitochondrial metabolism and so incomplete chromosomal segregation. In fact, a lower mitochondrial membrane potential is associated with an age-related higher incidence of meiotic spindle abnormalities during oocyte maturation (Battaglia et al., 1996
; Volarcik et al., 1998
; Wilding et al., 2003
).
Germinal vesicle transplantation (GVT) has been proposed as a treatment for correction of age-related oocyte aneuploidy (Zhang et al., 1999). A putatively normal GV nucleus transplanted into a younger ooplast would have the right molecular and structural components to build a normal meiotic spindle, allowing proper segregation of chromosomes between the constructed oocyte and the first polar body (PB) (Tsai et al., 2000
). It remains difficult to prove the feasibility of this concept, however, due to the absence of animal models that simulate human oocyte aneuploidy. Thus far, in only a small number of human oocytes it has been possible to prove that placement of an aged GV nucleus into a younger ooplasm reduces occurrence of aneuploidy (Zhang et al., 1999
; Takeuchi et al., 2001
; Palermo et al., 2002
).
In the hope of establishing such a model in which to test this idea, we have induced mitochondrial damage in GV oocytes and attempted to rescue them by replacing the ooplasm through GVT (Palermo et al., 2002). The results demonstrate that the GVT oocytes can overcome maturational arrest and undergo first meiotic division with normal chromosomal segregation.
A further step to be assessed is the ability of the GVT oocytes to undergo fertilization and normal development (Li et al., 2001; Takeuchi et al., 2001
). Although micromanipulation of oocytes requires cumulus cell removal, granulosa cells are crucial for oocyte maturation and for a successful completion of meiosis and full-term development (Niwa et al., 1976
; Shcroeder and Eppig, 1984
; Vanderhyden and Armstrong, 1989
; Flood et al., 1990
; Cecconi et al., 1996
). To avoid this problem, a sequential cytoplasmic transfer supplying in vivo matured cytoplasm either at metaphase II (MII) stage (Kono et al., 1996
; Bao et al., 2000
; Liu et al., 2003
) or at pronuclear stage (Liu et al., 2003
) was considered necessary.
In some cases, mammalian GV oocytes are able to undergo spontaneous in vitro maturation once isolated from unstimulated ovaries (Pincus and Enzmann, 1935; Edwards, 1965
), and this has become the source of immature oocytes for GVT (Takeuchi et al., 1999
). However, although they mature at a standard rate, the resulting MII oocytes display an oolemma the fragility of which renders ICSI extremely challenging (Liu et al., 2000
; Palermo et al., 2002
). Despite of this handicap we have been able to obtain a fertilization rate of up to
40%, but the very low number developing a blastocyst (Takeuchi et al., 2002
) discouraged us from assessing the potential of these non-primed oocytes for normal development.
It has been shown that prior gonadotrophin stimulation enhances the ability of oocytes matured in vitro to develop into normal embryos (Schroeder and Eppig, 1989), and the presence of FSH in the medium is known to promote the development of mouse GV oocytes (Merriman et al., 1998
; Anderiesz et al., 2000
). However, because these studies utilized cumulus-invested oocytes, there is no definitive information as to the effect of gonadotropin-priming on fertilizability or development of cumulus-denuded GVT oocytes, nor whether normal offspring can be obtained directly from GVT oocytes without cytoplasmic supplementation (Kono et al., 1996
; Bao et al., 2000
).
In this study, we assessed the effect on future development of gonadotrophin-priming prior to collection of GV oocytes. In order to assess their ability to undergo full-term development after spontaneous maturation, oocytes transplanted with GV nuclei were subjected to ICSI and transferred to pseudopregnant female mice, with non-manipulated GV oocytes matured in vitro after denudation serving as controls. To assess the genetic constitution of the resulting embryos, karyotyping was performed randomly at the 2-cell stage. The fertility status of the first generation pups was also assessed.
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Materials and methods |
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Gamete collection
Full-grown GV oocytes of 75 µm diameter (Bao et al., 2000
) from 711 week B6D2F1 female mice were retrieved either from non-stimulated ovaries or those treated by pregnant mares serum gonadotrophin (PMSG; Sigma, St Louis, MO, USA) 48 h prior to the procedure. Cumulus cells were removed by repeated aspiration through a hand-drawn pipette. In order to prevent spontaneous germinal vesicle breakdown, oocytes were cultured in Waymouths medium (MB752/1; Invitrogen, Carlsbad, CA, USA) supplemented with a phosphodiesterase inhibitor (0.2 mmol/l 3-isobutyl-1-methylxanthine; IBMX; Sigma), 0.23 µmol/l pyruvate (Sigma) and 5% fetal bovine serum (Invitrogen) until the GVT procedure. Mature MII oviductal oocytes were collected from the same mouse strain after PMSG and HCG treatment as described previously (Takeuchi et al., 1999
). After cumulus removal, MII oocytes were incubated in a CZB medium (Chatot et al., 1990
) until ICSI. Spermatozoa were obtained from caudae epididymides of mature male mice of the same strain and kept in CZB medium for at least 1 h, prior to injection.
Nuclear transplantation and oocyte maturation in vitro
All the micromanipulation and electrofusion procedures were performed under an inverted microscope equipped with a hydraulic micomanipulator as described previously (Takeuchi et al., 1999). Enucleation was conducted in a medium supplemented with 25 µg/ml of cytochalasin B (Sigma) and was performed with a 25 µm glass cylindrical pipette after making a slit in the zona pellucida with a microneedle. Isolated GV karyoplasts were transferred into enucleated oocytes of the same maturational stage. Each grafted oocyte was manually aligned between two microelectrodes, and cell fusion was subsequently induced by direct electrical current (Takeuchi et al., 1999
).
Oocytes reconstituted successfully were rinsed and cultured in IBMX-free Waymouths medium for 14 h to allow nuclear maturation. Oocyte maturation was judged according to the appearance of a single PB. Non-manipulated cumulus-free GV oocytes served as controls.
Piezo-ICSI and fertilization assessment
The reconstituted oocytes after in vitro maturation were injected with a single dissected sperm head by the aid of a Piezo actuator (PiezoDrill; Burleigh Instruments, Inc., Fishers, NY, USA and PMM-150FU, PrimeTech, Ibaragi, Japan), essentially as reported previously (Wakayama and Yanagimachi, 1998). Briefly, a single spermatozoon was aspirated into an injection pipette of
5 µm inner diameter. The sperm head and tail were separated by applying a single or a few piezo pulses to the neck region, then the head alone was injected. Approximately 56 h after the injection, the oocytes surviving injection were evaluated for the presence of two distinct pronuclei (PN) and a clear second PB, the criteria of normal fertilization (Figure 1). Oocytes with an extruded second PB and the presence of at least one PN were designated as activated oocytes.
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Pre- and post-implantation development of nuclear transplanted oocytes after ICSI
Fertilized oocytes were incubated further in KSOMAA medium for up to 96 h in order to assess embryonic cleavage compared with that of control oocytes matured in vitro.
Some experimental embryos at the 2-cell stage were surgically transferred into oviducts of CD-1 foster mothers that had been mated with a vasectomized male of the same strain during the previous night. Because the initial part of this study revealed a significantly impaired blastocyst formation rate in PMSG non-primed oocytes, only embryos derived from stimulated ovaries were utilized for this experiment. Some 2-cell stage embryos derived from oocytes matured in vivo, or those matured in vitro without GVT, were transferred to pseudopregnant females as controls.
Data analysis
The 2-test was utilized to identify differences in cell survival, maturation, fertilization, blastocyst formation and live birth rates observed between the different groups. Significance was set at P = 0.05. All statistical computations were conducted using StatView (SAS Institute, Inc., Cary, NC, USA). Only significant differences are noted in the text and/or tables.
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Results |
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Effect of PMSG priming on fertilization and embryonic cleavage after ICSI
Fertilization characteristics and embryonic cleavage of GVT oocytes are summarized in Table I. While cell survival (74.0 versus 80.8%) and oocyte activation per surviving oocyte (92.9 versus 95.2%) were not different between the unprimed and stimulated oocytes, respectively, more oocytes from PMS stimulated ovaries were fertilized normally (63.5 versus 39.6%; P < 0.01). A beneficial effect of priming was also confirmed for the rate of blastocyst formation (31.8% versus 7.9; P < 0.01) (Figure 2). A fertilization rate of 64.5% (40 out of 62) and a blastocyst formation rate of 35.0% (14 out of 40) observed in non-manipulated in vitro matured control oocytes obtained from PMSG-stimulated ovaries were not different from those of PMSG-primed GVT oocytes.
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Discussion |
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Mouse offspring can be obtained from in vitro matured oocytes more readily by ICSI than by conventional in vitro insemination, demonstrating the ability of direct injection to bypass the eventual changes in the zona pellucida and oolemma during in vitro maturation (Yamazaki et al., 2001). Thus, in accordance with the high fertilization rates of in vitro matured oocytes isolated from preantral follicles (Liu et al., 2002
), ICSI appears to be the best way to fertilize oocytes matured in vitro, particularly when deprived of their cumulus cells.
With ICSI we were able to obtain a consistent fertilization rate of 65%, compared with the previous 35% with in vitro matured denuded oocytes after standard insemination (Schroeder and Eppig, 1984). Only when cumulus cells were present did the fertilization rate reach 73% in in vitro matured oocytes (Schroeder and Eppig, 1984
), indicating cumulus cell-induced modifications of the sperm membrane that in our GVT oocytes are obviously missing but are successfully bypassed by ICSI. In the present study, >90% of the oocytes surviving the ICSI procedure showed signs of activation regardless of gonadotrophin priming. The large majority had a normal fertilization rate, while the remainder displayed a single PN. The relatively higher incidence of abnormal fertilization was due to a failure of the sperm nucleus to decondense in the still immature cytoplasm (Lee et al., 2003
). Oocytes matured in vitro may lack cytoplasmic factors required for male PN formation, such as glutathione (Funahashi et al., 1995
) or ATP (Collas and Poccia, 1998
), which are found in MII but not GV ooplasm (Maeda et al., 2000
). It is possible that a subgroup matured in vitro had reduced levels of sperm decondensing factor(s) due to suboptimal maturation. Therefore, it is likely that despite the obvious nuclear maturity, oocytes may not yet complete maturation of their cytoplasm, and exhibit an asynchronous maturation between nucleus and cytoplasm, with consequent abnormal sperm nuclear decondensation.
Here, we have demonstrated that gonadotrophin stimulation enhances fertilization after ICSI and later cleavage of GVT oocytes. Once granulosa cells of preantral follicles experience the stimulations of FSH and develop estrogen responsiveness, many proliferate rapidly, so ensuring the formation of the preovulatory follicle (Rao et al., 1978). Both the proliferation and differentiation of granulosa cells are facilitated by the increasing intracellular level of cyclin D2 under FSH regulation (Robker and Richards, 1998
). Appropriate proliferation and terminal differentiation of granulosa cells are critical for normal folliculogenesis and, therefore, for organized oogenesis.
It is clear that FSH priming can rescue a cohort of small antral follicles destined to undergo atresia during the follicular phase of a natural cycle (Schramm and Bavister, 1994), probably through inhibition of granulosa cell apoptosis (Braw and Tsafriri, 1980
; Hsueh et al., 1994
). Gonadotrophins may do so by regulating ovarian glutathione (GSH) synthesis by modulating glutamate cystein ligase subunit expression (Luderer et al., 2001
), since a GSH precursor, N-acetyl cystein, inhibits apoptosis as effectively as the FSH in cultured antral follicles (Tilly and Tilly, 1995
), and high concentrations of GSH in mature oocytes are critical for fertilization and early embryo development (Calvin et al., 1986
; Perreault et al., 1988
; Gardiner and Reed, 1995
). Thus gonadotropin priming enhances overall oocyte development simply by recruiting more synchronous non-apoptotic oocytes through GSH synthesis. Although in a recent report it has been shown that FSH priming has no beneficial effect on in vitro maturation of human oocytes (Lin et al., 2003
), they were cultured with an intact cumulus oophorus, exposed to HCG in vivo 36 h prior to retrieval, and were obtained from women with polycystic ovaries. Thus, culture conditions of the oocytes in the above work were different from those of the current study. Therefore, the beneficial effects of gonadotropin stimulation on the developmental competence of mouse oocytes may become more pronounced when maturation occurs in the absence of cumulus cells.
Although the replacement with younger cytoplasm appeares to be a promising approach to correction of age-related aneuploidy (Zhang et al., 1999; Takeuchi et al., 2001
; Palermo et al., 2002
), this remains to be proven. The cumbersome procedure in the mouse, a species known for its difficulty in surviving the ICSI procedure, together with the shortage of spare human oocytes have contributed to the premature decision to offer the procedure to couples suffering from age-related infertility (J.Grifo, ASRM Annual Meeting 1998). This approach, together with a less invasive procedure called cytoplasmic transfer (Barritt et al., 2001
), has prompted the Food and Drug Administration intervention and consequent regulations (Zoon, 2001
).
The ability to generate offspring from immature mouse oocytes in a direct manner provides a feasible model in which to study nuclear transplantation and to examine whether this may offer a way of correcting oocyte aneuploidy. In addition, the occurrence of full-term development without additional cytoplasmic infusions avoids the further complication of foreign mtDNA heteroplasmy (Cummins, 2001; 2002
) and its transmission to future generations (Van Blerkom et al., 1998
; Barritt et al., 2001
; St John, 2002
). Furthermore, the epigenetics of the reconstituted oocytes and derived conceptuses should be investigated, since altered DNA methylation patterns have been observed in offspring produced by nuclear transplantation (Reik et al., 1993
; 2001
; Humpherys et al., 2001
; Hawes et al., 2002
). A final step to assess the efficacy of GVT in prevention of oocyte aneuploidy would be reproduction of a system that simulates the damage present in oocytes isolated from older women. This may be, for example, accomplished by specifically altering membrane potentials of mitochondria (Palermo et al., 2002
; Takeuchi et al., 2003
).
In conclusion, we demonstrate that mouse GVT oocytes can undergo in vitro maturation, fertilization and full-term development without supplementation with mature ooplasm. Oocyte survival after ICSI, fertilization and embryo development did not differ from that observed in non-manipulated, in vitro matured oocytes.
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Acknowledgements |
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Submitted on September 1, 2003; accepted on November 24, 2003.