The oxidizing agent tertiary butyl hydroperoxide induces disturbances in spindle organization, c-meiosis, and aneuploidy in mouse oocytes

It has been recently proposed that a concomitant generationof oxidative stress of oocytes with increasing maternal agemay be a major factor responsible for the age-related increasein aneuploid conceptions. As a preliminary step in the testingof this hypothesis, we need to confirm that oxidative stressin itself can induce errors in chromosome segregation. In orderto achieve this goal, germinal vesicle (GV)-stage mouse oocytesfrom unstimulated ICR and (C57BLxCBA) F₁ hybrid female micewere matured in vitro for 9 h for metaphase I (MI) oocytes or16 h for metaphase II (MII) oocytes in the presence of varyingconcentrations of the oxidizing agent tertiary-butyl hydroperoxide(tBH). MII oocytes from (C57BLxCBA) F₁ hybrid mice were fixedand C-banded for karyotyping analysis. MI and MII oocytes fromICR mice were fixed and stained with the DNAfluorescent probe4',6-diamidino-2-phenylindole (DAPI) to detect abnormalitiesin chromosomal distribution. Meiosis I and meiosis II spindlesfrom ICR mice were visualized by confocal immunofluorescencemicroscopy. Data from these experiments demonstrate that in-vitroexposure of mouse oocytes to tBH during meiosis I reduces thelength (pole-to-pole distance) and width (diameter at the equatorof the spindle) of meiosis I and meiosis II spindles. This reductionis associated with an increase in the percentage of oocytesshowing chromosome scattering and clumping on the MII plate,and of aneuploidy (hyperhaploidy) in MII oocytes. However, tBHat the concentrations used in the present study has only a minimalnegative effect on the frequency of meiotic maturation. Theseresults suggest that oxidative stress during meiotic maturationin vitro may induce chromosomal errors that are undetectablein the living oocyte and whose developmental consequences maybecome manifest after fertilization. aneuploidy/meiosis/mouse oxidative stress/spindle/tertiary butyl hydroperoxide     

Distinct microtubule and chromatin characteristics of human oocytes after failed in-vivo and in-vitro meiotic maturation

BACKGROUND: While a complete failure of meiotic maturation following hCG administration is rare during IVF cycles, cases arise in which patients repeatedly display a high incidence of failure to complete maturation to metaphase II (MII) in vivo. For the immature oocytes of such patients, our objectives were (i) to ask whether progression to MII could be supported in vitro, and (ii) to define their microtubule/chromatin properties following in-vitro maturation (IVM). Together, these studies were aimed at augmenting our understanding of factors underlying meiotic arrest in the human. METHODS: Cases are presented here for two patients (A and B) producing oocytes that recurrently showed the inability to mature to metaphase II in vivo. Following IVM attempts, chromatin and microtubule characteristics were identified in those oocytes that remained arrested during meiosis I. RESULTS: In patient A, meiotically arrested oocytes exhibited clear defects in spindle and chromatin arrangements. In contrast, the majority of oocytes from patient B displayed normal MI and MII spindles with aligned chromosomes, although some oocytes exhibited indications for possible defects in cell cycle control. CONCLUSIONS: Together, these analyses illustrate two cases with oocytes exhibiting a common gross defect, that is meiotic maturation arrest, but revealing different aetiologies or manifestations as evidenced by the presence or absence of abnormal spindle/chromatin organization. This work reinforces the existence of intrinsic defects in oocytes of some patients, the molecular and cellular bases of which merit further investigation.     

Ageing-associated aberration in meiosis of oocytes from senescence-accelerated mice

BACKGROUND: The senescence-accelerated mouse (SAM) has been shown to exhibit ageing-associated mitochondrial dysfunction and oxidative stress, and early decline in fertility. METHODS: We compared meiotic progression of germinal vesicle oocytes between young (2-3 months) and old (10-14 months) SAM mice using triple immunostaining and fluorescence microscopy as well as Pol-Scope imaging. RESULTS: At 8-9 h of in-vitro maturation (IVM), most young SAM oocytes (86%, 32/37) were at meiosis I (MI) stage, with chromosomes aligned in the mid-region of MI spindles, whereas disrupted MI spindles and/or chromosome misalignments (45%, 18/40) and a few oocytes (20%, 8/40) with abnormal MII spindles were found in old SAM oocytes. At 15-17 h of IVM, old SAM oocytes, despite errors at MI stage, extruded a first polar body at an incidence of 88% (n = 85), which did not differ from that (92%, n = 106) of young SAM oocytes. However, oocytes from old SAM (64%, 32/50) showed aberrant MII, with chromosome misalignment and dispersal, in contrast to normal MII in most young SAM oocytes (87%, 65/75), showing chromosome alignment at the metaphase plate of MII spindles. Moreover, Pol-Scope imaging non-invasively detected disrupted or absent visible spindles and possibly aberrant chromosome alignment. CONCLUSIONS: Spindle disruption and/or chromosome misalignments at both MI and MII are associated with maternal ageing in the SAM mouse. Our findings also suggest that meiotic division lacks a competent checkpoint for spindle integrity and chromosome alignment during reproductive ageing-associated oocyte senescence.     

Caspase‐2L,caspase‐9, and caspase‐3 during in vitro maturation and fragmentation of the mouse oocyte

Several studies have shown that apoptotic pathways control fragmentation of unfertilized ovulated oocyte, induced by doxorubicin. But very few have investigated the basis of this process, from prophase I to later stages. Our results revealed the presence of caspase‐2_L, caspase‐9, and caspase‐3 in their zymogen and cleaved forms in the oocyte before meiosis resumption. Caspase‐2_L and caspase‐9 were detected in the nucleus of GV‐oocytes in a distribution related to chromatin configuration. The inhibition of caspase activity by Z‐VAD‐fmk accelerated the transition from metaphase I to metaphase II, and caspase‐9 and caspase‐3 were detected along the meiotic spindle. Surprisingly, Western blot analysis revealed that the three cleaved caspases were present in similar amounts in healthy and fragmented oocytes and caspase inhibition did not prevent doxorubicin‐induced apoptosis. Our results suggest that, if cleaved, caspases may be dispensable for final oocyte death and they could be involved in regulating the maturation process. Developmental Dynamics 237:3892–3903, 2008. © 2008 Wiley‐Liss, Inc.     

A synthetic analogue of meiosis-activating sterol (FF-MAS) is a potent agonist promoting meiotic maturation and preimplantation development of mouse oocytes maturing in vitro

BACKGROUND: Follicular fluid-meiosis-activating sterol (FF-MAS) is a factor present in the pre-ovulatory follicle during the time of oocyte maturation. In mouse oocytes maturing in vitro, FF-MAS promotes the completion of meiotic maturation to metaphase II (MII) and improves competence to complete the 2-cell stage to blastocyst transition. We produced analogues of FF-MAS and selected three on the basis of potency to promote the resumption of meiosis by mouse oocytes maintained in meiotic arrest by hypoxanthine. The objective of this study was to determine whether these FF-MAS analogues also affect the quality of oocytes maturing in vitro with respect to the completion of meiotic maturation and augmenting the frequency of development to the blastocyst stage after fertilization in vitro. METHODS: Cumulus cell-enclosed oocytes were isolated from the small antral follicles of 18 or 20 day post-natal mice. These oocytes normally have a reduced competence to complete meiotic maturation and preimplantation embryo development. Oocytes were isolated at the germinal vesicle stage and matured in vitro using media supplemented with 0.1% ethanol, 1 micromol/l FF-MAS, or 0.1-10 micromol/l FF-MAS analogues ZK255884 (884), ZK255933 (933) and ZK255991 (991). Oocytes that progressed to MII were fertilized in vitro and the percentage developing to the 2-cell and blastocyst stages was determined. RESULTS: At 1 micromol/l, 991 and 933 increased the portion of oocytes progressing to MII, whereas the lowest dose of 991 and 884 was ineffective. Treatment of maturing oocytes with either 0.1 or 1 micromol/l 933 dramatically increased oocyte competence to complete preimplantation development. CONCLUSIONS: The synthetic analogue of FF-MAS, ZK255933, is a potent agonist that improves the quality of mouse oocytes matured in vitro. This compound may therefore have therapeutic value for treatment of oocytes from women undergoing therapy for infertility owing to poor oocyte quality.     

Diethylstilbestrol (DES)-induced cell cycle delay and meiotic spindle disruption in mouse oocytes during in-vitro maturation

Due to the growing amount of data related to the deleterious effects of the synthetic oestrogenic compound, diethylstilbestrol (DES), on the female reproductive system, we tested the potential effects of this compound on mouse oocytes. Controlled time- and dose-dependent in-vitro experiments were carried out on isolated cumulus-oocyte-complexes (COCs) to examine the meiotic spindle assembly and chromosome distribution. alpha-tubulin, chromosomes and F-actin were labelled and detected by confocal laser scanning microscope. COCs were exposed to varying doses of DES (5-30 micromol/l) from the germinal vesicle (GV) stage to the end of metaphase II (MII) when meiosis I and meiosis II is normally completed. Exposure to DES during meiosis I caused a dose-dependent inhibition of cell cycle progression. In comparison with controls, fewer oocytes reached metaphase I (MI) at low doses (5 micromol/l) of DES, while none of the oocytes reached MI in high doses (30 micromol/l). When COCs were exposed to high doses of DES during meiosis II, fragmentation of first meiotic spindle was detected, whereas lower doses caused loosening of the first and the second meiotic spindles. No microtubular abnormalities were detected either in GV-stage oocytes or in cumulus cells. The above data demonstrate that one mode of action of DES on mouse oocytes is a severe yet reversible deterioration of meiotic spindle microtubule organization during maturation.     

Epidermal growth factor combined with recombinant human chorionic gonadotrophin improves meiotic progression in mouse follicle-enclosed oocyte culture

Using a mouse early preantral follicle culture system, mature full grown oocytes, arrested in prophase I of meiosis, were produced after 12 days using a recombinant gonadotrophin-supplemented medium. This culture medium does not mimic the normal extracellular environment of the oocyte and might therefore modify meiotic regulation and more particularly progression to metaphase II (MII). The aim of this study was to optimize the treatment using recombinant stimulatory ligands which were known to induce germinal vesicle breakdown (GVBD) and completion of meiosis I, metaphase II (MII), namely recombinant follicle stimulating hormone (r-FSH), chorionic gonadotrophin (r-HCG) and epidermal growth factor (EGF). Full-grown intrafollicular oocytes could not resume meiosis when the 'ovulatory' stimulus was r-FSH, used at a 100 times higher dose than during culture. r-FSH did not increase progesterone production. When 1.5 IU/ml r-HCG was used as meiotic trigger, germinal vesicle breakdown was obtained in 95% of the oocytes 64% of which extruded a first polar body. r-HCG induced a dramatic increase in progesterone production. When EGF was administered as sole stimulus on day 12 to the attached follicle-enclosed oocytes, only doses > or =5 ng/ml could cause GVBD, although less effectively than r- HCG (45 versus 95%; P < 0.0001). Oocytes undergoing GVBD by the EGF pulse reached metaphase II at a rate of 54% (not significant versus r- HCG). EGF did not stimulate progesterone production. Addition of increasing doses of EGF (0.5; 5; 10; 50 ng/ml) to r-HCG did not increase the GVBD-rate, but EGF doses >5 ng/ml improved MI to MII transition (P=0.027), thereby improving the final yield of MII oocytes by 12.5%. These data show that up to a dose of 50 ng/ml, EGF on its own could only override the somatic inhibitory stimuli in less than half of the cultured follicles. However, in addition to HCG, EGF (25 ng/ml) had a stimulatory effect on completing the first meiotic division. It was concluded that, under the present culture conditions, EGF in combination with HCG provided optimal nuclear maturation.      

Characterization of susceptible chiasma configurations that increase the risk for maternal nondisjunction of chromosome 21

Recent studies of trisomy 21 have shown that altered levels of recombination are associated with maternal non-disjunction occurring at both meiosis I (MI) and meiosis II (MII). To comprehend better the association of recombination with nondisjunction, an understanding of the pattern of meiotic exchange, i.e. the exchange of genetic material at the four-strand stage during prophase, is required. We examined this underlying exchange pattern to determine if specific meiotic configurations are associated with a higher risk of non-disjunction than others. We examined the crossover frequencies of chromosome 21 for three populations: (i) normal female meiotic events; (ii) meiotic events leading to MI non-disjunction; and (iii) those leading to MII non-disjunction. From these crossover frequencies, we estimated the array of meiotic tetrads that produced the observed crossovers. Using this approach, we found that nearly one-half of MI errors were estimated to be achiasmate. The majority of the remaining MI bivalents had exchanges that clustered at the telomere. In contrast, exchanges occurring among MII cases clustered at the pericentromeric region of the chromosome. Unlike the single exchange distributions, double exchanges from the non-disjoined populations seemed to approximate the distribution in the normal population. These data suggest that the location of certain exchanges makes a tetrad susceptible to non- disjunction. Specifically, this susceptibility is associated with the distance between the centromere and closest exchange. This result challenges the widely held concept that events occurring at MII are largely independent of events occurring at MI, and suggests that all non-disjunction events may be initiated during MI and simply resolved at either of the two meiotic stages.      

In-vitro matured metaphase-I oocytes have a lower fertilization rate but similar embryo quality as mature metaphase-II oocytes after intracytoplasmic sperm injection.

About 4% of all the oocytes denuded prior to intracytoplasmic sperm injection (ICSI) are in metaphase-I (MI). Frequently, these oocytes achieve meiosis after a few hours of in-vitro culture and are available for ICSI on the day of oocyte retrieval. In this retrospective study, the aim was to evaluate the fertilization rate and the developmental capacity of these in-vitro matured MI oocytes. After controlled ovarian stimulation using human menopausal gonadotrophin (HMG) and human chorionic gonadotrophin (HCG) in 896 ICSI cycles, 1210 MI-to-MII-matured oocytes were injected approximately 4 h after in-vitro culture and 8803 MII oocytes were injected immediately, or later, after denudation. The fertilization rate of in-vitro matured oocytes was significantly lower than that of mature MII oocytes (52.7 and 70.8% respectively, P < 0.00l). Embryo quality was only slightly different as regards the numbers of good quality embryos: 47.4% good quality embryos were obtained in the in-vitro matured oocyte group, whereas 53.2% good quality embryos were obtained in the MII oocyte group (P < 0.05). The same proportions of excellent (5.7 and 7.0%, NS) and fair quality (17.6 and 15.3%, NS) embryos were obtained for in-vitro matured and mature oocytes respectively. Embryos derived from in-vitro matured oocytes were transferred only if they were of better quality or if there were not enough mature oocyte derived embryos available. Fifteen transfers involved only embryos derived from in-vitro matured oocytes: 11 single embryo transfers and four transfers of two embryos, resulting in one singleton pregnancy and the birth of a healthy baby. It may be concluded that in cycles with few MII oocytes it might be worthwhile to inject in-vitro matured MI oocytes in order to increase the number of embryos available for transfer.     

Start me up: cell signaling and the journey from oocyte to embryo in C. elegans.

Intercellular communication plays a pivotal role in regulating and coordinating oocyte meiosis and fertilization, key triggers for embryonic development. The nematode Caenorhabaditis elegans has emerged as an important experimental paradigm for exploring these fundamental reproductive processes and their regulation. The oocytes of most animal species arrest during meiotic prophase and complete meiosis in response to intercellular signaling in the process of meiotic maturation. Oocyte meiotic maturation is defined by the transition between diakinesis and metaphase of meiosis I and is accompanied by nuclear envelope breakdown and meiotic spindle assembly. As such, the meiotic maturation process is essential for completing meiosis and a prerequisite for successful fertilization. In C. elegans, the processes of meiotic maturation, ovulation, and fertilization are temporally coupled: sperm utilize the major sperm protein as a hormone to trigger oocyte meiotic maturation, and, in turn, the maturing oocyte signals its own ovulation, leading to fertilization. The powerful genetic screens possible in C. elegans have led to the identification of several sperm cell surface proteins that are required for the interaction and fusion of gametes at fertilization. The study of these proteins provides fundamental insights into fertilization mechanisms, their role in speciation, and their potential conservation across phyla. Signaling processes sparked by fertilization are required for meiotic chromosome segregation and initiating the embryonic program. Here we review recent advances in understanding how signaling mechanisms contribute to the oocyte-to-embryo transition in C. elegans.     

Okadaic acid, an inhibitor of protein phosphatase 1 and 2A, induces premature separation of sister chromatids during meiosis I and aneuploidy in mouse oocytes in vitro

Recent advances in understanding some of the molecular aspects of chromosome segregation during mitosis and meiosis provide a background for investigating potential mechanisms of aneuploidy in mammalian germ cells. Numerous protein kinases and phosphatases have important functions during mitosis and meiosis. Alterations in these enzyme activities may upset the normal temporal sequence of biochemical reactions and cellular organelle modifications required for orderly chromosome segregation. Protein phosphatases 1 (PP1) and 2A (PP2A) play integral roles in regulating oocyte maturation (OM) and the metaphase–anaphase transitions. Mouse oocytes were transiently exposed invitro to different dosages (0, 0.01, 0.1, or 1.0 μg/ml) of the PP1 and PP2A phosphatase inhibitor okadaic acid (OA) during meiosis I and oocytes were cytogenetically analyzed. Significant (p < 0.05) OA dose-response increases in the frequencies of metaphase I (MI) arrested oocytes, MI oocytes with 80 chromatids instead of the normal 20 tetrads, and anaphase I–telophase I (AI–TI) oocytes with two groups of an unequal number of chromatids were found. Analysis of MII oocytes revealed significant (p < 0.05) increases in the frequencies of premature sister chromatid separation, single-unpaired chromatids, and hyperploidy. Besides showing that OA is aneugenic, these data suggest that OA-induced protein phosphatase inhibition upsets the normal kinase–phosphatase equilibrium during mouse OM, resulting in precocious removal of cohesion proteins from chromosomes. This revised version was published online in July 2006 with corrections to the Cover Date.     

The outcome of ICSI of immature MI oocytes and rescued in vitro matured MII oocytes

BACKGROUND: The use of immature oocytes is limited to cases where these are the only available oocytes, and they are usually only microinjected with sperm after having undergone maturation in vitro. This study compares the outcome of injection of sperm into metaphase I oocytes immediately after their denudation (MI) performed 2 h after their retrieval, with the outcome of injection of sperm into rescued in vitro matured metaphase II (IVM MII) oocytes after their short incubation in routine laboratory conditions. METHODS: ICSI was performed on MI oocytes, rescued IVM MII oocytes and on MI oocytes that were incubated but failed to extrude their first polar body (arrested IVM MI). Fertilization and cleavage rates were compared with those achieved in mature metaphase II oocytes (MII). RESULTS: ICSI of MI oocytes showed impaired performance compared with ICSI of rescued IVM MII oocytes and MII oocytes, in terms of oocyte degeneration rate (11 versus 6 versus 4%; P < 0.0001), fertilization rate (28 versus 44 versus 68%; P < 0.0001) and multipronucleated fertilization (10 versus 4 versus 4%; P < 0.01). The cleavage rate was lower in rescued IVM MII oocytes compared with MII oocytes (86 versus 95%; P < 0.01). Arrested IVM MI oocytes showed similar results to those of MI oocytes but had a lower cleavage rate (72 versus 96%; P < 0.01). CONCLUSIONS: The injection of rescued IVM MII oocytes is preferred to the injection of MI oocytes.     

Knockdown of the cAMP-dependent protein kinase (PKA) Type Ialpha regulatory subunit in mouse oocytes disrupts meiotic arrest and results in meiotic spindle defects.

In mammalian oocytes, cyclic AMP-dependent protein kinase (PKA) is responsible for maintaining meiotic arrest. We examined the role of the predominant regulatory subunit, RIalpha in regulating PKA activity during mouse oocyte maturation by knocking down the protein levels using an RNA interference approach. In oocytes in which RIalpha protein was reduced to non-detectable levels, compensatory decreases were also observed in the RIIalpha and catalytic (Calpha) subunit levels. These oocytes resumed meiosis, despite culture under conditions that maintain elevated intracellular cAMP levels, suggesting that the remaining Calpha was not sufficient to maintain meiotic arrest. The resulting eggs, however, displayed meiotic spindle abnormalities and abnormal cleavage planes leading to extrusion of large polar bodies. These results demonstrate that RIalpha is required for regulating PKA activity in maturing oocytes and that compensatory upregulation of RII does not occur. Furthermore, we implicate PKA as a modulator of spindle morphology and function during meiosis.     

Aberrant spindle structures responsible for recurrent human metaphase I oocyte arrest with attempts to induce meiosis artificially

BACKGROUND In some couples, not all retrieved oocytes mature, even after prolonged in vitro culture. The underlying mechanisms are not known, although ionophore treatment may alleviate metaphase I (MI) arrest in some mouse strains. We attempted to induce first polar body (PB) extrusion and fertilization using assisted oocyte activation (AOA) after ICSI in maturation-resistant human MI oocytes. METHODS Four ICSI patients are described in this retrospective study. A pilot study tested the calcium ionophore ionomycin (10 μM) on donated MI oocytes from patients with a normal number of metaphase II (MII) oocytes. Subsequently, ionomycin was used to induce first PB extrusion in two patients showing maturation-resistant MI oocytes. AOA, by calcium injection and ionomycin exposure, was applied when mature oocytes were available. Oocytes were analysed by polarized microscopy and immunostaining. RESULTS Ionomycin induced the first PB extrusion in MI oocytes from patients with a normal number of retrieved MII oocytes, while extended in vitro culture failed to achieve the MII stage. Similarly, ionomycin induced first PB extrusion in one of two patients with recurrent maturation-resistant MI oocytes. Use of ICSI combined with AOA on MII oocytes matured in vitro or in vivo resulted in failed or abnormal fertilization with no further embryo cleavage potential. Highly abnormal spindle and chromosome configurations were observed in MI maturation-resistant oocytes, in contrast to control MI oocytes. CONCLUSIONS Ionophore induced first PB extrusion in MI oocytes from patients without maturation arrest but to a lower extent in maturation-resistant MI oocytes. Immunofluorescence staining and confocal analysis revealed, for the first time, highly abnormal spindle/chromosomal structures that may be responsible for this maturation arrest.     

Fluorescent study of chromatin and tubulin in apparently unfertilized human oocytes following ICSI

In this study we examined 138 oocytes which were meiotically mature and, on light microscopic examination, contained either no or one pronucleus following intracytoplasmic sperm injection (ICSI). Oocytes were fixed and simultaneously stained for chromatin (Hoechst 33258) and the spindle (alpha-tubulin antibody). In nine oocytes, no sperm nucleus was observed. The remaining oocytes were separated into two groups following staining; (i) oocytes which had remained at metaphase II after ICSI (n = 74); and (ii) oocytes in which resumption of meiosis was observed after ICSI (n = 55). In all oocytes in which sperm chromatin was absent no resumption of meiosis had occurred and therefore parthenogenetic activation by the process of ICSI seems to be a rare event. In 17 out of 74 (23%) oocytes which remained at metaphase II, staining identified premature chromosome condensation (PCC) of the sperm chromatin (G1-PCC). Sperm nuclear decondensation or further transformation of the sperm chromatin was observed in 56 out of 74 (76%) oocytes which remained at metaphase II after ICSI and in 46 out of 55 (84%) oocytes which had resumed meiosis, indicating that initiation of sperm decondensation is independent of the resumption of meiosis in the oocyte. In contrast, transition of the sperm nucleus beyond the decondensed stage only occurred in association with resumption of meiosis in the oocyte (no pronuclei in metaphase II oocytes). The presence of both male and female pronuclei in 53% of oocytes which had resumed meiosis indicates that changes in sperm chromatin beyond the initial decondensation stage are dependent on cytoplasmic conditions which also permit female pronuclear formation.      

The influence of postovulatory ageing on the retardation of mouse oocyte maturation and chromosome segregation induced by vinblastine

Certain compounds can induce ovulated metaphase I (MI) oocytes.To study if these MI oocytes can overcome this blockage, ICRmice were given human chorionic gonadotrophin and 0.6 mg/kgvinblastine sulfate (VBS). Their ovulated oocytes were collectedat 17, 19, 21, 23 and 25 h later. The results showed that thefrequencies of MI oocytes decreased, the proportions of diploidmetaphase II (MII) oocytes increased and the frequencies ofhyperploid MII oocytes did not significantly differ (P>0.05)among the five harvest or postovulatory times. We also foundthat the proportions of MII oocytes exhibiting premature anaphaseII and premature centromere separation increased with postovulatoryageing, and that these frequencies were consistently higherin controls than in the VBS groups. These findings indicatethat some of the oocytes blocked in MI can overcome this inhibitionand produce primarily diploid MII oocytes. The ultimate fateof ovulated MI and diploid oocytes on aneuploid production hingesupon the formation of a functioning meiotic spindle, which isaffected by both dosage and the specific chemical. ¹To whom correspondence should be addressed     

Turning it on and off: M-phase promoting factor during meiotic maturation and fertilization

Maturation (M-phase) promoting factor (MPF) plays a pivotal role in oocytes during their maturation. This review concentrates on its function at three important time-points. First, its activation during meiotic progression from prophase I arrest at germinal vesicle breakdown. Second, its role during the transition from meiosis I to meiosis II, a defining feature of meiosis involving segregation of homologous chromosomes. Third, maintenance of its activity at metaphase II arrest and the necessity for its destruction during oocyte activation. An understanding of how oocytes switch it on and turn it off underpins much of the basic cell biology of oocyte maturation.     

A confocal microscopy analysis of the spindle and chromosome configurations of human oocytes cryopreserved at the germinal vesicle and metaphase II stage

BACKGROUND: Routine oocyte cryopreservation remains an elusive technique in the wide range of assisted reproductive technologies available. This study examines the effect of a cryopreservation protocol on the spindle and chromosome configurations of human oocytes cryopreserved at the germinal vesicle (GV) and metaphase II (MII) stage. METHODS: GV oocytes were randomly assigned to one of three groups: (i) control oocytes matured in vitro to MII stage (n = 156); (ii) oocytes cryopreserved at the GV stage and then matured in vitro (n = 90); (iii) oocytes cryopreserved at the MII stage (n = 147). Following cryopreservation and in-vitro maturation, immunostaining of tubulin and chromatin was performed, before visualization using confocal microscopy. RESULTS: A statistically significant increase was observed in the survival rate in group 2 (73.3%, 66/90) compared to group 3 (55.7%, 82/147) (P < 0.007). Exposure of oocytes to the cryoprotective solutions without freezing had no effect on the structure of their second meiotic spindle. However, statistically significant differences were observed on both spindle and chromosome configurations of oocytes from group 2 (5.2 and 5.2% respectively) and group 3 (16.2 and 18.8% respectively) compared with group 1 oocytes (71.6 and 82.0% respectively) (P < 0.001 in all cases). CONCLUSIONS: The protocol followed results in high rates of survival and potential for in-vitro maturation, but has a deleterious effect on the organization of the meiotic spindle of human oocytes cryopreserved at both the GV and MII stages.     

Time-dependent capability of human oocytes for activation and pronuclear formation during metaphase II arrest

BACKGROUND: The purpose of this study was to investigate the fertilization rate and developmental potential of human oocytes in relation to the duration of their metaphase II (MII) arrest stage following the extrusion of the first polar body (1PB). METHODS: Immature metaphase I oocytes (MI; study oocytes, n = 468) that underwent meiotic maturation during brief in vitro culture and their matured in vivo, MII siblings (control oocytes, n = 3293) were subjected to ICSI. Fertilization and early cleavage were evaluated in both study and control groups. RESULTS: The overall fertilization rate was significantly lower in the oocytes matured in vitro than in those matured in vivo (42 versus 77%, P < 0.0001). A significant relationship was observed between oocyte activation potential and the length of MII arrest. The majority of study oocytes injected soon after PB extrusion remained unfertilized (64%; 98/154 oocytes). The proportion of normally activated oocytes that contained two pronuclei and two PBs gradually increased with prolonged time of MII arrest (43 and 61% at 2 and 3-6 h after 1PB extrusion). Significantly more embryos originating from the study than control oocytes were arrested soon after the first two cleavage divisions (39 and 17%; P < 0.0001) and exhibited multinucleated blastomeres (23 and 13%; P < 0.0001), which suggests the existence of chromosomal abnormalities. CONCLUSIONS: Human oocytes progressively develop the ability for full activation and normal development during the MII arrest stage.     

Maturation arrest of human oocytes as a cause of infertility: case report

Maturation arrest of human oocytes may occur at various stages of the cell cycle. A total failure of human oocytes to complete meiosis is rarely observed during assisted conception cycles. We describe here a case series of infertile couples for whom all oocytes repeatedly failed to mature during IVF/ICSI. Eight couples, all presenting with unexplained infertility, underwent controlled ovarian stimulation followed by oocyte retrieval and IVF/ICSI. The oocytes were stripped of cumulus cells prior to the ICSI procedure and their maturity status was defined. In each couple, oocyte maturation was repeatedly arrested at the germinal vesicle (GV) (n = 1), metaphase I (MI) (n = 4) and metaphase II (MII) (n = 3) stage. Oocyte maturation arrest may be the cause of infertility in some couples previously classified as having unexplained infertility. The recognition of oocyte maturation arrest as a specific medical condition may contribute to the characterization of the yet poorly defined entity currently known as 'oocyte factor'. The cellular and genetic mechanisms causing oocyte maturation arrest should be the subject of further investigation.