1 Department of Anatomy and Cellular Biology, Tufts University School of Medicine, Boston and 2 Department of Obstetrics and Gynecology and Reproductive Biology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA
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Abstract |
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Key words: chromatin/human/maturation/microtubule/oocyte
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Introduction |
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Likely reasons for the low efficacy of IVM in assisted reproduction include inadequacies of the culture media used. In addition, heterogeneity in reported maturation rates are probably due to intrinsic differences in oocytes recovered after ovarian stimulation from variously sized follicles in vivo that vary in their developmental capacity due to incomplete or abnormal growth. Meiotic competence acquisition and expression has been related to changes in oocyte growth, germinal vesicle chromatin organization, meiotic cell cycle status, and transcriptional activity in oocytes of several mammalian species (McGaughey et al., 1979; Wickramasinghe et al., 1991
; Schramm et al., 1993
; Fair et al., 1995
, 1996
). Markers of oocyte differentiation and meiotic cell cycle state include germinal vesicle chromatin patterns, mitotic phosphoprotein monoclonal-2 reactive protein (MPM-2) phosphorylation, and cytoplasmic microtubule organization (Wickramasinghe et al., 1991
; Wickramasinghe and Albertini, 1992
). Phosphorylation of histone-3, characteristic of metaphase (M-phase), is also a useful indicator of chromatin condensation in mitotic and meiotic cells (Hendzel et al., 1997
; Wei et al., 1999
; Carabatsos et al., 2000
). In rodent oocytes, the cell cycle expression of these markers is as follows: the G2 phase of the cell cycle, specifically the dictyate stage of prophase-1, is characterized by lack of phosphohistone 3 (PH3) reactivity, nuclear MPM-2, and interphase arrays of microtubules. In contrast, following formal entry into M-phase of the cell cycle, histone-3 is phosphorylated, MPM-2 becomes cytoplasmic, and microtubules are converted from a stable interphase state into dynamic polymers associated with condensed chromosomes (Wickramasinghe and Albertini, 1992
; Carabatsos et al., 2000
). Microtubule reorganization and stability is influenced at the transition of interphase to M-phase by several factors, including protein kinase activity, centrosome-based microtubule nucleation, and post-translational modifications of tubulin (Albertini, 1992
). The relative contribution of each of these factors has not been defined in human oocytes, despite the possibility that disruption of microtubule patterning might underlie failures in chromosome segregation or organelle allocation during later development (Van Blerkom et al., 1995
, 2000
). That human female gametes present an elevated risk of aneuploidy when compared with oocytes from other mammalian species further buttresses the need for additional study into the cell cycle-dependent changes in microtubule organization in human oocytes. To our knowledge, the baseline M-phase markers defined above (MPM-2, PH3, and microtubules) have not been analysed in human oocytes during meiotic competence acquisition and expression.
Available evidence indicates that culture systems adequately support nuclear maturation in human oocytes but fail to produce oocytes with cytoplasmic competency, thereby resulting in embryos with reduced developmental potential (Cha and Chian, 1998; Moor et al., 1998
; Trounson et al., 2001
). Cytoplasmic maturation encompasses a wide array of metabolic and structural modifications, including events that ensure the occurrence of normal fertilization, meiotic to mitotic cell cycle progression, and activation of pathways required for genetic and epigenetic programmes of preimplantation embryonic development (Eppig et al., 1994
; Eppig, 1996
; Heikinheimo and Gibbons, 1998
; Moor et al., 1998
; Trounson et al., 2001
). Therefore, three possibilities might underlie the limited success of human IVM: culture conditions, to date, are not supportive of expression of intrinsic developmental competency of oocytes; current IVM systems induce an asynchrony in the progression of nuclear and cytoplasmic maturation; or the oocytes utilized lack one or more of the components necessary for nuclear and cytoplasmic maturation and later embryonic development. The present study was designed to investigate the last possibility, which is that cytoplasmic maturity is deficient in in-vitro matured human oocytes. Specifically, cell cycle-dependent modifications in chromatin and microtubule patterning were studied in immature oocytes and during IVM in a defined culture medium.
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Materials and methods |
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Five experiments were evaluated: (i) GV chromatin patterning to identify what association, if any, the GV chromatin might have to meiotic competence; (ii) kinetic experiment to characterize the timing of meiotic progression; (iii) study of microtubules, with respect to their post-translational modification (acetylation) and the presence of phosphoproteins, to evaluate nuclear and cytoplasmic maturation during IVM; (iv) activation incidence to assess the ability of M-II oocytes to maintain meiotic arrest in vitro; and (v) taxol exposure to analyse microtubule dynamics in in-vivo matured human oocytes. The number of oocytes employed in each of these studies is shown in Table I; note that some oocytes were used in more than one study.
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Taxol exposure (Experiment 5)
Five to seven hours after retrieval, oocytes at various cell cycle stages were exposed to the microtubule stabilizing agent, taxol. A taxol solution (final concentration of 10 µmol/l) was prepared from a 1 mmol/l dimethylsulphoxide (DMSO) stock (stored at 20°C) in human tubal fluid medium (Irvine Scientific). Denuded oocytes were treated for 10 min at 37°C in taxol- or DMSO (control)-containing medium, prior to fixation and immunofluorescence analysis.
Processing of oocytes for immunofluorescence analysis (Experiments 15)
Oocytes were fixed and processed for microtubule detection, chromatin organization, and the presence of phosphoproteins as previously described (Messinger and Albertini, 1991; Cekleniak et al., 2001). Microtubules were labelled using either a monoclonal anti-
-tubulin and anti-
-tubulin mixture (Sigma Biosciences, St Louis, MO, USA), a rat monoclonal antibody against
-tubulin (YOL 34) (Kilmartin et al., 1982
), or a monoclonal anti-acetylated
-tubulin (Sigma Biosciences) at 1:100 final dilutions. Chromatin was detected using either a mouse monoclonal anti-histone H1 IgG (Leinco Technologies Inc., St Louis, MO, USA), or a rabbit polyclonal antibody directed against the PH3 mitosis marker (Upstate Biotechnology, Lake Placid, NY, USA), or Hoechst 33258 (Polysciences Inc., Warrington, PA, USA). To detect phosphoproteins, an MPM-2 antibody was used (Upstate Biotechnology). Affinity-purified fluoresceinated, Texas Red, or Cy-5 donkey anti-mouse, rat or rabbit IgG were used at a 1:500 final dilution (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Processed oocytes were mounted and analysed by conventional fluorescence or laser scanning confocal microscopy as previously described (Messinger and Albertini, 1991
; Cekleniak et al., 2001).
Statistical analysis
For the kinetic analysis (Experiment 2), all oocytes obtained from a given patient were treated as one experiment to eliminate interpatient variability. For every experiment, the average percentage of oocytes displaying a particular meiotic stage was measured and an overall mean percentage ± SEM was derived for each time point across all experiments. When comparing oocyte diameters across germinal vesicle chromatin patterns (Experiment 1), the mean ± SEM were reported; a non-parametric MannWhitney test was used to determine statistical differences between groups. For the GV chromatin pattern (Experiment 1) and activation studies (Experiment 4), proportions were compared using a two-tailed Z-test. P < 0.05 was considered significant.
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Results |
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To characterize the expression and to establish baseline cell cycle markers for meiotic arrest as defined above, histone-3 phosphorylation, M-phase phosphoproteins (detected by the MPM-2 monoclonal antibody) and microtubules were evaluated relative to chromatin patterns. PH3 reactivity was not associated with human oocytes arrested at prophase of meiosis-1, regardless of the GV chromatin pattern (Figure 1E, inset; n = 29). In all GV chromatin patterns examined, MPM-2 reactivity was confined to the nucleoplasm with prominent foci distributed throughout the nucleus (Figure 1E
; n = 36). Investigation of microtubule patterning showed that for all chromatin patterns, GV stage oocytes exhibited a dense interphase subcortical microtubule array (Figure 1F
; n = 133), consistent with an interphase cell cycle state. Under no circumstances were focused microtubule arrays observed. Indeed, all GV oocytes that failed to resume meiosis in culture retained these cell cycle properties.
Experiment 2: Kinetic analysis of IVM
To determine the rate of meiotic progression in P-1, meiotic stages were classified during IVM over a 24 h culture period. At the start of culture, the majority of oocytes were at the GV stage (97.6 ± 2.4%; mean ± SEM) with a minor fraction (one out of 15 oocytes) exhibiting signs of germinal vesicle breakdown (GVBD) (Figure 3). After 6 h in culture, 88.9% of oocytes had resumed meiosis with 61.1 ± 15.3% at GVBD and 27.8 ± 16.5% in prometaphase (PM)-1. At 12 h, 78.5% of oocytes were maturing with 19.0 ± 14.3% having undergone GVBD and 59.5 ± 17% being at PM-I/M-I. At 18 h, an approximately equivalent proportion of oocytes were in M-I (44.5 ± 22.2%) and telophase (T)-1 (49.9 ± 25.5%), with 66.7% (± 19.23) progressing to M-II by 24 h.
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The staining patterns for pH3 and microtubules for in-vitro matured human oocytes are shown in Figures 4 and 5. PH3 reactivity, first detected at GVBD (Figure 4A
), was maintained throughout meiotic progression until M-II arrest (Figures 45
, insets). However, the staining intensities for oocytes at GVBD and telophase I (T-I) were lower relative to those for other stages of meiotic progression. In GV and GVBD stage oocytes, interphase-like microtubule arrays were observed throughout the oocyte cortex (Figures 1F and 4A
). In contrast to the clear persistence of microtubule acetylation in cumulus cell microtubule-rich projections traversing the zona pellucida (Albertini et al., 2001
; Figure 4B
, arrows), GV and GVBD stage oocytes lacked detectable reactivity to antibodies specific for acetylated
-tubulin (Figure 4B
; GV, n = 39 and GVBD, n = 18). Between prometaphase and anaphase of meiosis-1, individual chromosome bivalents were located within the meiotic spindle, and no microtubules were observed in the ooplasm (Figure 4C
, E). Microtubules lacked acetylation at M-I (Figure 4D
; n = 17) while at anaphase-1, spindle microtubules were acetylated (Figure 4F
; n = 3). At T-I, oocytes displayed long microtubules, albeit in low densities, throughout the oocyte cortex alongside typical midbodies and spindle structures (Figure 5A
); only spindle microtubules were acetylated (Figure 5B
; n = 12). M-II oocytes possessed spindle-associated microtubules with chromosomes aligned at the spindle equator and no acetylated
-tubulin was detectable (Figure 5C
, D, inset; n = 16). In an effort to compare our in-vitro findings using IVM with in-vivo matured oocytes, microtubule acetylation was analysed in seven M-II oocytes fixed immediately at retrieval. None of these seven oocytes exhibited acetylated microtubules, consistent with the results reported above for in-vitro matured M-II oocytes. Also, oocytes that were in various stages of meiotic maturation following retrieval (n = 62; see Table I
, IVO) revealed total and acetylated microtubule staining patterns similar to those of in-vitro matured oocytes. In spontaneously activated oocytes (see below), only a small subset of cytoplasmic microtubules was acetylated (Figure 5E
, F).
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Discussion |
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Germinal vesicle chromatin patterns predict meiotic competency
Ovarian stimulation protocols generate predominantly mature M-II oocytes (Cha and Chian, 1998; Trounson et al., 2001
). However, germinal vesicle stage oocytes are frequently obtained, which, when fully stripped of cumulus cells and cultured in appropriate media, show a high incidence of resumption and completion of nuclear maturation, giving rise to M-II spindles that appear normal. Despite the many reports documenting meiotic competency, and even developmental potential of these cells (Cha and Chian, 1998
; Trounson et al., 2001
), no systematic effort has been undertaken to determine whether markers of cytoplasmic or nuclear maturation could predict the meiotic cell cycle efficiency of human GV stage oocytes retrieved during the course of ovarian stimulation. Developmental modification in chromatin organization, from a decondensed to a condensed perinucleolar disposition, has been reported in many mammals and is believed to correspond to a transition from a transcriptionally active to an inactive state near the end of the growth phase of oogenesis (Mattson and Albertini, 1990
; Zuccotti et al., 1995
; Bouniol-Baly et al., 1999
). In rodent oocytes, this modification occurs coincident with genomic imprinting (Kono et al., 1996
), transcriptional repression (Bouniol-Baly et al., 1999
; De La Fuente and Eppig, 2001
), and the acquisition of meiotic and embryonic competency (McGaughey et al., 1979
; Wickramasinghe et al., 1991
; Zuccotti et al., 1998
). Similar alterations in chromatin have been reported in oocytes of both primates (Schramm et al., 1993
) and bovine (Fair et al., 1996
). Our studies are the first to define the states of chromatin in human oocytes. Among the four classes of GV oocytes identified, three (B, C and D) were characterized by perinucleolar condensed chromatin (Figure 1
) with the type B pattern most closely resembling the `karyosphere structure' (Parfenov et al., 1989
). That type B (and perhaps others) represents a chromatin organization in a transcriptionally repressed state would be expected given the diminished [3H]uridine incorporation noted by one study (Parfenov et al., 1989
). Most striking, however, were changes observed in the distribution of GV classes prior to and following IVM (Figure 2
). While class B and D GV were consistently observed at the time of oocyte retrieval (25.0 and 2.2% respectively, n = 92), their incidence was significantly increased to 43.9 and 31.7% (n = 41) amongst the subpopulation of oocytes that failed to resume meiosis after 48 h in vitro (in a previous sample, 39.4% of oocytes remained as GV, n = 256) (Cekleniak et al., 2001
). Consistent with the idea that class B and D oocytes represent meiotically incompetent and cell cycle-arrested (G2) GV is the finding that these oocytes retain an extensive interphase network of microtubules with no PH3 reactivity following prolonged culture, further indicative of the failure to mount an MPF (M-phase or maturation promoting factor) response sufficient to depolymerize microtubules and condense chromosomes in anticipation of meiotic spindle assembly. To our knowledge, this is the first experimental demonstration that class C oocytes, the predominant type observed following retrieval, exhibit meiotic competence following culture; this conclusion is based on the observation that 59.8% of oocytes are type C before culture, 9.8% after culture, and from previous studies 60.6% of oocytes matured to M-II by 48 h (Figure 2
; Cekleniak et al., 2001). Further support for the idea that class C oocytes represent the meiotically competent fraction of oocytes is derived from our observation that these oocytes were larger in diameter than those in the other GV classes (Figure 2
), and therefore probably correspond to oocytes that achieved an advanced stage of growth during folliculogenesis. Conversely, class A GV oocytes were smallest in diameter and represented in similar proportions prior to and following culture, attesting to their classification as incompetent (13.0 and 14.6%, Figure 2
). This finding thus supports work in the bovine showing a relationship between oocyte diameter, follicle size, and IVM rates (Fair et al. 1995
). Two studies (McNatty et al., 1979
; Durinzi et al., 1995
) were consistent with the diameter of human oocytes reflecting their meiotic potential in vitro.
Unlike the chromatin state, evaluation of the other nuclear (MPM-2 positive foci, lack of pH3 epitope) and cytoplasmic (interphase microtubules) markers of G2 failed to demonstrate a relationship with meiotic competency, suggesting that this important aspect of nuclear remodelling deserves further study. The fact that nuclear state can be reversibly controlled with respect to meiotic competence expression makes this an attractive marker for manipulating human oocytes in culture.
Kinetic analysis of IVM
Given previous reports that in-vitro matured human oocytes are compromised in their ability to fertilize and support embryonic development, we additionally explored the kinetics and coordination of nuclear and cytoplasmic maturation in GV stage oocytes that exhibited meiotic progression under our culture conditions. Our analysis of 64 competent oocytes revealed a striking capacity to reinitiate and complete maturation to M-II (Figure 3). The culture system used in this study supported rapid progression of GV stage human oocytes to M-II by 24 h. Relative to previous in-vivo estimates (Edwards, 1965
) and studies from other laboratories (Cha and Chian, 1998
; Trounson et al., 2001
), the accelerated pace of meiotic progression was largely attributable to an enhancement in germinal vesicle breakdown since 88.9% of oocytes advanced through M-I by 6 h in culture compared with ~20% of oocytes over the same time interval (Cha and Chian, 1998
). Differing reports of rates of IVM are probably due to many factors including media composition, hormone/growth factor supplementation, the source of oocytes (unstimulated versus stimulated cycles), and whether or not cumulus cells are retained with the oocyte (Prins et al., 1987
; Gunnala et al., 1993
; Cha and Chian, 1998
; Goud et al., 1998
; Anderiesz et al., 2000
). Absence of cumulus cells and exposure to gonadotrophins have both been documented to accelerate meiotic maturation in vitro (Gomez et al., 1993
; Cha and Chian, 1998
; Goud et al., 1998
; Wynn et al., 1998
; Trounson et al., 2001
). Both of these variables are relevant to the culture conditions utilized here since we stripped cumulus cells prior to culture and added recombinant FSH and HCG (P-1) (Cekleniak et al., 2001
). In addition, P-1 medium is a simplified, glucose-free medium that could influence oocyte metabolism and other related processes. For example, removal of cumulus may compromise metabolic support required for the sustained activation of MPF that could alter microtubule dynamics and/or chromatin stability. Therefore, factors important in regulating the onset of oocyte maturation, the temporal aspects of meiotic progression, the completion (polar body extrusion) and maintenance of M-II state are not clearly defined and merit further attention given the propensity of human oocytes to become aneuploid.
M-Phase deficiencies in in-vitro matured human oocytes
Having documented enhanced nuclear progression in the above experiment, we next asked whether characteristic changes in microtubule dynamics were in any way compromised as a potential contributing factor to cytoplasmic immaturity. Activation of MPF, the driving force for progression through M-phase of animal cell cycles, results in significant post-translational modifications in histone-3, to ensure maintenance of compacted chromatin, and the presence of proteins that alter microtubule stability (Murray and Hunt, 1993; Hendzel et al., 1997
; Wei et al., 1999
). Deficiencies in the activation, amplification, or inactivation of MPF would be expected to offset the temporal and/or spatial parameters of cell cycle control that underlie the co-ordination of nuclear and cytoplasmic maturation of oocytes (Albertini and Carabatsos, 1998
; Pines, 1999
). Using immunodetection of phosphorylated histone-3 with an epitope-specific antibody (Wei et al., 1999
), we show timely modifications in histones commencing with diakinesis and ending with arrest at M-II (Figures 4 and 5
). However, this analysis revealed two critical junctures when loss of PH3 epitope occurred. At telophase-I and during parthenogenetic activation, we observed, respectively, partial reduction and complete loss of PH3. Thus, changes in H3 phosphorylation were correlated with chromatin decondensation of varying degrees. This chromatin-based indicator of failure to maintain M-phase could be explained by the transient inactivation of MPF known to occur during telophase of meiosis-1 as documented in other mammalian oocytes (Hashimoto and Kishimoto, 1988
; Fulka et al., 1992
; Wu et al., 1997
). In addition, the rapid and total loss of PH3 in in-vitro matured M-II oocytes that failed to maintain M-phase arrest could be due to an impaired c-mos/MAP kinase influence (Sagata, 1997
).
Further support for M-phase deficiencies derives from our analysis of microtubule patterns throughout the course of IVM. Again, at both telophase-I and in activated oocytes, we observed expression of prominent cytoplasmic microtubule arrays that would be an expected outcome from failure to sustain adequate levels of MPF or c-mos/MAP kinase. Whether such changes are a direct response to degradation of the cyclin B component of MPF, or a failure to sustain adequate levels of ATP to maintain cell cycle kinase activities, remain to be established. However, besides the appearance of interphase microtubules, this work also exemplified a direct post-translational modification of -tubulin in anaphase-1, telophase-I, and activated oocytes. The appearance of immunodetectable acetylated
-tubulin subunits within both spindle (Figures 4F and 5B
) and cytoplasmic (Figure 5F
) microtubules further attests to the transient, yet specific, nature of a biochemical modification in microtubules known to confer polymer stability (Webster and Borisy, 1989
; Bulinski and Gundersen, 1991
). It is intriguing to note that a persistent expression of acetylated microtubules has been observed throughout the course of meiotic maturation in mouse (de Pennart et al., 1988
; Can and Albertini, 1997
) and yet, as reported here, microtubule acetylation is limited to discrete stages of meiosis (anaphase-1, telophase-I) in human. Although microtubule acetylation patterns were consistent between our analysis of human oocytes matured under in-vivo and in-vitro conditions, it is difficult to ascertain the significance of restricted microtubule stability through acetylation. However, its limited occurrence would be a primary contributor to meiotic spindle defects predisposing human oocytes to meiotic non-disjunction. Further support for the notion that human oocytes lack those spindle-stabilizing forces expressed in species less prone to meiotic aneuploidy, is the apparent lack of spindle pole protein complexes (pericentrin,
-tubulin) observed in the present study.
It should be emphasized that the deficiencies alluded to above are representative of oocytes that failed to mature in vivo under standard conditions of ovarian stimulation. Alterations in cell cycle progression for both in-vitro and in-vivo (see below) matured human oocytes provides a baseline for future studies aimed at optimizing culture conditions to support maturation of oocytes obtained after reduced ovarian stimulation protocols. Finally, our studies have made rather novel use of the microtubule-stabilizing agent, taxol, to explore further the question of microtubule dynamics in in-vivo matured human oocytes. The application of exceedingly brief pulses of taxol revealed significant heterogeneity in the spatial and temporal responsiveness of oocytes to this test of tubulin assembly status (Figure 6, Table III
). While some M-I and M-II oocytes exhibited no response, others were readily coaxed into elaborating a cytoplasmic network that is most often associated with the oocyte cortex. This heterogeneity argues strongly for variability in individual oocytes to maintain an MPF-driven restriction of microtubule assembly to the spindle and generally lends further credence to the idea of M-phase deficiencies in human oocytes matured under in-vivo or in-vitro conditions.
Given the significant risk factors in human oocytes associated with the age-related increase in meiotic aneuploidy (Hunt and LeMaire-Atkins, 1998; Volarcik et al., 1998), coupled with the use of experimental culture conditions for IVM and IVF, and the need to develop adequate cryopreservation methods, further evaluations of human oocyte quality must focus on the coordination of nuclear and cytoplasmic maturation. These studies add yet another note of caution with respect to the use of human oocyte IVM, although they do offer direction for further studies involved with the manipulation and optimization of IVM for use in assisted reproduction.
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Acknowledgements |
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Notes |
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4 To whom correspondence should be addressed at: Department of Anatomy and Cellular Biology, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02111, USA. E-mail: david.albertini{at}tufts.edu
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References |
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Submitted on August 13, 2001; accepted on November 11, 2001.