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Reproductive Technology; |
Follicle Biology Laboratory,3 Vrije Universiteit Brussel, 1090 Brussels, Belgium
Scientific Institute of Public Health,4 Department of Clinical Biology, 1050 Brussels, Belgium
Eggcentris NV,5 BE-1731 Zellik, Belgium
ABSTRACT
Meiotic maturation of the oocyte is a timed sequence of events induced by the ovulatory LH surge. In vitro maturation of oocytes is known to alter the meiotic time course. This study documented the timing of meiosis in oocytes grown in vitro for 12 days, from the preantral follicle stage onward, and the influence of an oil overlay. In the oil-free culture, the stability of the metaphase II spindle was further explored to determine the postovulatory aging events. After the maturation stimulus, in vitro-grown oocytes were collected at 2-h intervals spanning the period of meiosis (0–18 h) and at 3-h intervals during early postovulatory aging (18–27 h). Stage of maturation was assessed both morphologically and by detailed spindle analysis and chromosome alignment. Results revealed that oil overlay did not impair the competence of cultured oocytes to proceed to meiosis II, but delayed meiosis I progression. Oil overlay during culture causes a different hormonal exposure of the follicles by a differential segregation into the oil overlay. The use of a progesterone receptor antagonist, however, did not induce a delay in meiotic progression. Aging effects in oil-free cultured follicles were detected 5 h after the establishment of the metaphase II spindle, comparable to their in vivo grown counterparts. The predominant effect of aging was an interphase-like appearance of the cytoskeleton. So an optimal time window for fertilization after in vitro follicular growth was determined to be 16–21 h after maturation induction.
aging, follicle, in vitro culture, meiosis, spindle
The use of an oil overlay is widely applied in in vitro maturation (IVM) and embryo culture. However, oil can contain impurities that cause reduced development in IVM and fertilization procedures [1–4]. Due to the differential segregation into oil, lypophilic substances like steroids and sterols are extracted from the culture medium [5]. Specifically, the lower progesterone concentration introduced by oil overlay was suggested to be the determining factor for a delay in the timing of oocyte maturation in IVM in pig [6]. Whether prolonged exposure to oil during follicular growth and oocyte maturation affects meiotic progression has never been investigated. There is little information in mice about the role of progesterone in the progression of meiosis, and although the initiation of meiosis in mice is generally suggested to be independent of progesterone [7], contradictory results continue to be published [8, 9].
Oocyte maturation is induced by the preovulatory gonadotropin surge and involves the resumption of the first meiotic division up to metaphase II (MII). Maturation requires a strictly timed coordination of events in both nuclear and cytoplasmic compartments [10]. The time course of successive nuclear stages of in vivo matured oocytes from stimulated cycles in adult mice has been well documented. The first visible signs of meiotic resumption are chromatin condensation, dissolution of the germinal vesicle (GV), and reorganization of the oocyte's cytoskeleton. They are first seen at 2 h after the hCG stimulation and are initiated in most oocytes resuming maturation by 4 h after hCG. The formation of the first metaphase spindle is completed by 8 h after stimulation. Just before ovulation of the mucified cumulus oocyte complex (COC), around 12 h after hCG, the first polar body (PB) is extruded, and an MII oocyte is ovulated [11–13]. The timing of meiosis, however, appears to be mouse strain dependent [14].
During IVM, however, the spontaneous meiotic process is described to be slower and shows more asynchrony, with oocytes starting and finalizing the meiotic process up to 6 h later than their in vivo-matured counterparts from hormonally stimulated cycles [13, 15]. When in vitro-cultured oocytes are used in studies for developmental competence, it is important to know the time frame in which meiosis proceeds, since oocytes show severe degenerative changes in their cytoskeleton and possibly gene expression associated with aging at metaphase II [16, 17].
The timing of the meiotic process is controlled by maturation-promoting factor (MPF), a complex composed of a CDC2A (also known as p34cdk1) and its regulatory subunit, CCNB1 (also known as cyclin B) [18, 19]. In the meiotic process, a first threshold in MPF activity is required to organize the microtubules into a bipolar structure [20, 21]. A second threshold in MPF activity at the end of meiosis I. enables the formation of stable connections between microtubules and kinetochores and makes chromosomes align on the spindle's equator [22]. MPF activity drops and anaphase sets in, with homologous chromosomes segregating approximately 12 min after final alignment on the metaphase plate [23]. Subsequently, MPF activity increases and the MII spindle is formed. Until oocyte activation, high MPF activity is maintained and meiosis is arrested [24].
In vivo, mouse oocytes are ovulated in the MII stage around 12 h after the LH surge and are expected to be fertilized within 6 h after ovulation [25]. Outside this window, postovulatory oocyte aging sets in. Postovulatory aging effects are biochemical changes caused by mitochondrial dysfunction, like diminished intracellular ATP levels [26, 27]. The changed biochemical parameters will be reflected in morphology: cytoskeletal abnormalities, which are a prelude to aneuploidies [16, 28] and membrane and cortical changes [29]. Aged oocytes often display zona hardening, which keeps sperm from entering, or diminished cortical reaction, leading to polyspermy [25, 30]. Activation of aged oocytes frequently leads to inaccurate Ca2+ signals [27], which, together with a reduction in the anti-apoptotic BCL2 protein, induces apoptosis in the zygote or embryo [31].
The aim of the present study was to compare the timing of the meiotic progression of oocytes grown in a follicle culture system, initially developed as a microdroplet system under oil overlay [32], with oocytes grown in an adapted oil-free system [33]. The frequently hypothesized influence of progesterone on the meiotic progression in mouse cultured oocytes was also studied using a specific progesterone receptor (PGR) antagonist Org 31170. To learn the time window for fertilization in oil-free cultured oocytes, early postovulatory aging effects on the spindle structure were investigated.
This study was performed with F1 mice (C57BL/6J x CBA/Ca; Harlan, Horst, The Netherlands and Charles River, Brussel, Belgium, respectively) housed and bred according to the national standards for animal care and approved by the ethical committee for animal experiments of the Free University Brussels (Project 01-395-1).
Preantral follicles were mechanically isolated from ovaries from 13- to 14-day-old F1 mice in L15 Leibovitz-glutamaxI medium supplemented with 10% heat-inactivated fetal bovine serum (HIA FBS), 100 IU/ml penicillin, and 100 µg/ml streptomycin (all Gibco; Invitrogen, Merelbeke, Belgium) and cultured in 
-minimal essential medium with glutamaxI (-MEM; Gibco; Invitrogen) containing 5% HIA FBS, 5 µg/ml insulin, 5 µg/ml transferrin, 5 ng/ml selenium (all from Sigma, Bornem, Belgium), 10 mIU/ml r-FSH throughout, and 10 mIU/ml r-LH only added at the first day of culture. At the end of culture (Day 12), meiotic resumption was induced by replacing the sampled medium with culture medium supplemented with r-hCG (hormones r-FSH [Gonal-F], r-LH [LHadi], r-hCG [Ovidrel] kindly donated by Ares Serono, Geneva, Switzerland) and r-EGF (Roche, Mannheim, Germany) [34] with final concentrations of 1.2 or 1.5 mIU/ml r-hCG and 4 or 5 ng/ml r-EGF in oil-free and oil-containing culture, respectively.
Two types of culture systems were used: with or without mineral oil (Sigma) overlay, in which the essential differences were the use of oil, the incubation volume, and the frequency of medium refreshments. In the oil-free system, 12–14 preantral follicles were put as single units in 75 µl culture medium in a 96-well plate (Costar; Elscolab, Kruibeke, Belgium), and 30 µl conditioned medium was refreshed with preincubated culture medium every 4 days [35]. In each culture, one time point consisted of two plates. In the oil-overlay culture, 18–20 follicles were cultured in 20-µl microdroplets in a 60-mm petri dish (Falcon, Becton Dickinson, Belgium) covered with 5 ml mineral oil and refreshed every 2 days with 10 µl medium [32]. In each culture, one time point consisted of one plate. Spent medium was pooled per plate and frozen at –20°C for estrogen and progesterone content measurement.
The oil-free system was used to detect the effects of PGR antagonist Org 31170 (kindly donated by Organon, Oss, The Netherlands) in a continuous exposure throughout follicle growth and meiotic maturation of 5 µM Org 31170 supplemented in culture medium as described above. This concentration is comparable to concentrations used in literature describing preovulatory follicle culture [9]. Dimethyl sulfoxide (DMSO) was used as a solvent, resulting in a 0.002% DMSO concentration in Org 31170 containing culture medium. This concentration is 100x less than the maximal dose of DMSO continuously used in the oil-free follicle culture system without effects on follicle growth, oocyte maturation, spindle integrity and steroid output (our unpublished data).
All manipulations were done on a heated stage (37°C), and cultured follicles were grown at 37°C, 100% humidity, and 5% CO2 in air.
Follicle Morphology Assessment
At the first day of culture, the quality of the follicles was ascertained. Follicle diameter (100–130 µm), oocyte-granulosa cell connection, and theca cell presence were scored under an inverted microscope with a Hoffman contrast-modulation system at magnification x400 (inverted microscope IX90; Olympus, Aartselaar, Belgium). On each refreshment day, all follicles were individually evaluated and classified for their growth profile under a stereomicroscope (MZ8; Leica, Brussels, Belgium). In vitro-grown follicles kept their follicular structure for the first 4 days of culture. Diffuse follicles displayed granulosa cell growth through the basal membrane overgrowing the theca cell monolayer that had formed (Days 5–8). The final growth stage was characterized by the formation of antral-like cavities, which separated the granulosa cells into mural and cumulus granulosa cells (Days 8–12). The different stages of in vitro follicle growth are depicted in Figure 1. After the hCG stimulus, mucification was scored as the degree of expansion of the cumulus cells surrounding the oocyte [32].
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Effect of oil overlay in follicle culture on meiotic progression. In both culture systems, oocytes were harvested to assess nuclear maturation before hCG stimulation (0 h) and at regular time points after hCG stimulation (4, 6, 8, 10, 12, 14, 16, and 18 h; Fig. 1). Cumulus expansion was scored. After cumulus cell removal, the nuclear maturation stage of the oocytes was recorded and the oocytes were immediately fixed in a microtubule-stabilizing buffer.
Effect of PGR antagonist on meiotic progression in oil-free cultured oocytes. Oocytes were cultured simultaneously in the absence or presence of 5 µM Org 31170. At 6, 8, 10, 12, 14, 16, and 18 h after maturation induction, oocyte maturation was scored, and immediately hereafter oocytes were fixed. Solvent control plates were processed in the same way at 12 h and 18 h after hCG.
Postovulatory spindle aging in oil-free cultured oocytes. To study postovulatory oocyte aging, oocyte maturation was scored after cumulus cell removal at 18, 21, 24, and 27 h after hCG (Fig. 1). Oocytes were immediately fixed for chromosome and spindle analysis.
Nuclear Maturation Assessment: Light Microscopy
Oocytes, mechanically freed from surrounding cumulus cells, were assessed under the stereomicroscope for the presence of an intact nucleus (GV) or an extruded PB. Absence of both structures indicated an intermediate stage, the germinal vesicle breakdown (GVBD).
Oocyte Fixation and Immunofluorescent Spindle Staining
Denuded oocytes were fixed per culture per time point in a microtubule-stabilizing buffer for 45 min on a heated stage (37°C) [36]. For immunofluorescent staining, the oocytes were incubated subsequently with mouse monoclonal anti--tubulin antibody (1:100; Sigma), secondary polyclonal anti-mouse IgG labeled with Alexa Fluor (1:200; Molecular Probes, Leiden, The Netherlands), and subsequently Ethidium Homodimer-2 (1:2000; Molecular Probes) each for 45 min at 37°C. Oocytes were mounted in 90% glycerol with 2 mg/ml diaminobicyclooctane (Sigma) between a cover slip and a microscope slide [36]. Slides were stored at 4°C until analysis on a two-channel fluorescence laser scanning confocal microscope (Olympus Fluoview; Omnilab NV, Aartselaar, Belgium) excitation with Argon laser (wavelength 488 nm), filter BA 510–540 for Alexa Fluor (emission at 519 nm), and filter BA 610 IF for Ethidium homodimer-2 (emission at 624 nm) [35].
The successive meiotic maturation stages were assessed after microtubule and chromatin staining (for a detailed description, see Fig. 2). Oocytes were considered as containing abnormal spindles when MII spindle structure or chromosomal alignment was aberrant.
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Assessment of Steroid Production of Cultured Follicles
During refreshment of the follicle culture, spent medium was collected, pooled per plate, and stored at –20°C upon steroid measurement. During the last phase of follicle growth (Days 8 and 12), 17β-estradiol (E2) production was measured with a radioimmunoassay from Clinical Assays (DiaSorin; Sorin Fueter, Brussels, Belgium) having a functional sensitivity of 20 ng/L and a total imprecision profile of <10% coefficient of variation (CV) for concentrations between 50 and 750 ng/L. Progesterone secretion was determined both before and after the maturation stimulus with Prog-CTRIA (Cis bio international, Gif-sur-Yvette cedex, France), with a functional sensitivity of 0.5 µg/L and a total imprecision profile of <10% coefficient of variation (CV) for concentrations between 3 and 50 µg/L.
Oocytes were considered per plate, and data for visual nuclear maturation and spindle analysis were analyzed with a generalized linear mixed model, with condition and time as fixed factors and test as a random variable. Coefficient estimates and their respective variance-covariance matrix were used to build all-group multiple comparisons for time (per condition) and condition (per time). When the interaction time was not significant, responses of condition were averaged over time and subjected to multiple comparisons. All multiple comparisons were corrected for simultaneous hypothesis, with a global significance level of 95% for each response variable. Analysis was performed in R (R Development Core Team; R Foundation for Statistical Computing, Vienna, Austria). Steroid measurement resulted in one data point per culture plate, representing a pool of conditioned medium from the surviving follicles. Data were log-transformed to obtain a normal distribution and processed with a general linear mixed model. Differential results were considered significant if P < 0.05.
Effect of Oil Overlay on Follicle Culture and Development
In the oil-containing system, four culture experiments were performed. Each time point contained two to four plates ranging from 31–59 oocytes for light microscope evalution and 27–43 oocytes for spindle and chromosome analysis. In the oil-free condition, six culture experiments were performed to obtain 6–13 plates per time point. This resulted in 60–139 oocytes for light microscopy evalution per time point, out of which 16–93 oocytes were analyzed for spindle and chromosome configuration. To investigate the aging effects in oil-free cultured oocytes, three culture experiments were performed, resulting in six plates per time point and a total oocyte number ranging from 70–77, out of which 56–57 oocytes were analyzed for spindle configuration. Discrepancy between oocyte numbers for light microscopy evaluation and spindle analysis is due to losses during fixation and staining procedures.
At the end of follicle culture (Day 12), a follicle survival rate of at least 90% was reached, and at least 80% of the follicles had formed an antral cavity in both culture conditions.
Effect of Oil Overlay on Cumulus Cell Mucification
When evaluating the effects of the maturation stimulus upon the follicle, the first visible parameter is the expansion and mucification of the cumulus cell mass around the oocyte. No signs of mucification were observed before 6 h after stimulation (Fig. 3). At 6 h the outer layers of cumulus cells displayed some expansion. At 8 and 10 h the cumulus was mucified, exept for two to three layers lining the oocyte. Complete expansion in the majority of the COC was obtained 12 h after hCG in follicles cultured without oil and 14 h after hCG in follicles in the oil-containing culture. Mucification clearly progressed centripetally in time. While performing cumulus cell removal, an increase in interconnectivity between the cumulus cells, caused by increased extracellular matrix deposition, was noticed in time. At the time full expansion was achieved (12 h), single cumulus cells were dispersed throughout the medium after the mechanical cumulus cell removal. Performing this same manipulation at 14 h and 16 h after the hCG stimulus, the cumulus cells appeared adhering together in large strings presumably caused by the presence of a more extensive hyaluronic acid network. Though these observations apply to the oil-containing cultures, these features were far more prominent in the oil-free culture.
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Effect of Oil Overlay on Nuclear Maturation: Light Microscopy
The first GVBD oocyte was detected at 4 h after the maturation stimulus (Fig. 4A). By 8 h after hCG, more than 80% of the oocytes underwent GVBD both in the oil-free and the oil-containing culture system. The first PB oocyte was detected 8 h after the maturation stimulus in oil-free cultures; 2 h later, first PB extrusion was seen in the oil-containing culture. At 12 h after hCG, a PB rate of 80% was reached in the oil-free system, while under oil it took 2 h more to obtain an equal amount of mature oocytes.
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Effect of Oil Overlay on Nuclear Maturation: Spindle and Chromosome Conformation
Since light microscopical assessment of GVBD and PB includes several successive stages during meiosis (prometaphase I [PMI], metaphase I [MI], anaphase I [AI], telophase [TI], and MII), we analyzed the meiotic process at the subcellular level by fluorescent staining of the microtubules and chromatin (Fig. 2). AI and TI are meiotic stages of short duration and are only sporadically found. For the sake of clarity they were included in the MI category, resulting in the following categories: 1) GV-stage oocytes, 2) PMI-stage oocytes (forming the MI spindle), 3) MI-stage oocytes (MI combined with few oocytes forming the MII spindle), and 4) MII-stage oocytes. Data are represented in cumulative groups.
Before the maturation stimulus, all cultured follicles contained a GV oocyte, which displayed a surrounded nucleolus conformation (SN) and only very few polymerized microtubules in the cytoplasm, predominately at the oocyte's cortex and around the nuclear membrane. At 4 h after the hCG stimulus, most of the oocytes still reside in SN GV conformation, with more pronounced polymerization of microtubules from microtubule organizing centers (MTOC) appearing as asters.
The first PMI oocytes were seen at 4 h (Fig. 4B). By 8 h after hCG nearly all oocytes (>80%) had initiated meiosis. The first fully formed MI spindle with well-aligned, condensed chromosomes is seen around 4 h after hCG in the oil-containing culture and at 6 h in the oil-free system. The percentage of MI-stage oocytes and beyond steadily increases to a plateau of more than 80% at 12 h after hCG for the oil-free system and at 14 h for the oil-containing culture. This means that the time needed from first chromosome condensation and GVBD to completion of the MI spindle in almost all oocytes is 8 h in the oil-free culture (4–12 h), while it takes 10 h in oocytes cultured in the oil-containing system (4–14 h). The longer period of MI formation suggests more heterogeneous progression to full MI spindle formation in oil-containing culture. Secondly, significantly fewer oocytes reached the MI stage at 12 h after stimulation if cultured in the oil-containing culture, indicating a slower progression of meiosis from meiosis induction to MI-spindle formation.
The first meiotic division was completed by the migration of homologous chromosomes to adjacent spindle poles (AI) and the extrusion of the first PB (TI). After extrusion of the first PB, a new aligned MII spindle is formed, with few asters throughout the cytoplasm. The first MII-stage oocytes were observed at 10 h after the hCG stimulus, and a maximal MII rate of 80% is reached at 16 h for the oil-free system and at 18 h for the oil-containing culture.
Abnormally formed MII spindles were found sporadically (2%–6%) in both culture systems.
Effect of Oil Overlay on Steroidogenesis
Oil overlay during culture of the follicle had a pronounced impact on steroidogenic environment. E2 was measured in conditioned medium during the antral follicle growth phase (Days 8 and 12 of culture). Overall, the oil-containing culture had an E2 concentration that was 7 times lower at the beginning of the antral phase (Day 8; P < 0.001) and 14 times lower in the preovulatory stage (Day 12; P < 0.001; Table 1).
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Progesterone concentration was determined both before and after the hCG stimulus. In oil-containing and oil-free culture, there was a significantly higher progesterone concentration in the post-hCG conditions compared to the basal pre-hCG progesterone concentration (Table 1). Basal progesterone concentration in conditioned medium before the maturation stimulus was on average eight times lower in oil-containing culture (Day 12; P < 0.001). After the maturation stimulus, the overall progesterone concentration in conditioned medium was 41 times lower (Day 13; P < 0.001) in the oil-containing culture. However, progesterone production is induced by the hCG stimulus and should be considered in terms of time after hCG. In oil-free culture there is a significant rise in progesterone after the stimulus up to 8 h after hCG (P < 0.05), after which the progesterone concentration remains at a plateau up to 27 h.
Effect of PGR Blocker Org 31170 on Meiotic Progression in Oil-Free Cultured Oocytes
In total, four replicate experiments were performed, each containing one plate per time point in the absence or presence of 5 µM Org 31170. Per time point and condition, 32–44 oocytes were analyzed for nuclear maturation under the light microscope and by fluorescent spindle and chromosome staining. The use of Org 31170 or an equivalent solvent concentration did not alter follicle growth parameters. A follicle survival rate of at least 90% was reached, and at least 80% of the follicles had formed an antral cavity in all culture conditions. Mucification was not affected by the presence of a PGR antagonist in time after hCG. After visual inspection of the cumulus-free oocytes, no differences were detected in GVBD or PB rate in time after hCG between both conditions. In-depth spindle analysis of these oocytes did not reveal a delay or an acceleration caused by Org 31170 exposure in successive time points after the maturation stimulus (Fig. 5).
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Effect of Postovulatory Oocyte Aging in Oil-Free Cultured Oocytes
We studied the effect of prolonged culture after the maturation stimulus to determine the optimal time window in which in vitro-grown oocytes could be fertilized. Postovulatory aging effects were analyzed in oocytes kept within the cultured follicle environment for 21, 24, and 27 h. Throughout these time points, more than 90% PB oocytes were obtained. However, subcellular spindle and chromosome analysis showed an increase in MII oocytes with malformations at 24 h and 27 h after hCG compared to 18 h after hCG (P < 0.05). An interphase-like organization of the cytoskeleton, with microtubules emanating from the spindle and spreading throughout the oocyte's cytoplasm, was frequently observed (Fig. 2 and Table 2). This cytoskeletal organization increased with postovulatory time. An enlargement of the metaphase plate, probably due to a slight decondensation of the chromatin material, was seen progressively more in oocytes aged in vitro. Elongation of the spindle structure was also found. A round spindle shape or unaligned chromosomes were found in equal amounts in control (18 h after hCG) and aged oocytes (24–27 h after hCG) and are hence not related to postovulatory aging in this setting. As different abnormalities coexisted in the same oocyte, a total amount of oocytes with integrated abnormalities at each time point is given in Table 2. Control oocytes (18 h after hCG) had few integrated abnormalities (6%). Abnormalities significantly increased to 32% and 47% in MII oocytes at 24 and 27 h after the maturation stimulus.
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In this study, the timing of meiotic maturation of in vitro-grown mouse oocytes was studied in the absence or presence of an oil overlay. Secondly, we determined the time window after the maturation stimulus in which normal spindle morphology is maintained.
Mucification is a process induced by the LH surge and comprises the deposition of the glycosaminoglycan hyaluronic acid (HA), produced by hyaluronic acid synthase 2 in the cumulus cells and granulosa cells lining the antrum. In our culture systems, expansion and mucification start centripetally in the outer cumulus cell layers from 6 h after hCG onward, and complete expansion of the COC is obtained around 12–14 h after hCG. Salustri et al. [37] demonstrated that during IVM in mouse the synthesis of HA reaches maximal values between 3 and 6 h after stimulation and is considerably decreasing again after 12 h of stimulation. The extracellular matrix between the cumulus cells demonstrated a progression towards a very sticky mass at 16 h after the hCG stimulation, due to the intensified covalent coupling of matrix proteins like inter alpha-trypsine inhibitor heavy chain proteins, tumor necrosis factor alpha induced protein 6, and versican, which stablize the cumulus extracellular matrix [38].
Nuclear progress was studied on cumulus-free oocytes at precise time intervals after the hCG stimulus. Before the hCG stimulus, all preovulatory follicles contained a GV oocyte with an SN configuration, corresponding to the type IV GV in the classification of Mattson and Albertini [36]. We used Ethidium Homodimer 2, which is a DNA and RNA stain; however, identical results were also obtained after evaluation of D12 oocytes with Hoechst, a DNA-specific stain (Hoechst 33258; data not shown). Transition from a nonsurrounded to a surrounded nucleolus is described to coincide with the timing of antrum formation and the acquisition of meiotic competence. SN configuration is also temporally linked to transcriptional repression and requires the tight association with companion cumulus cells [36, 39–41]. This illustrates that oocytes from in vitro-grown follicles with or without oil are fully grown and have acquired the competence to undergo meiosis.
LH action induces the onset of meiosis. In vivo, this process progresses rapidly (2 h after the hCG stimulus), with nearly all triggered oocytes from the cohort in GVBD by 4–5 h post-LH. Most oocytes (>70%) complete the first meiotic spindle at 8 h after stimulation, and MII oocytes will be ovulated at 12 h [11–13, 42]. As described in in vitro-matured oocytes, oocytes cultured from the preantral follicle stage onward also demonstrate a delay in the meiotic process, regardless of the use of oil overlay. Only 8 h after the hCG stimulus, most oocytes (>80%) display PMI morphology. Formation of the MI spindle in the majority of the oocytes (>80%) is observed only after 12 h in the oil-free culture system and after 14 h in the oil-containing culture. MII spindle formation was only obtained at 16 h or 18 h post-hCG after oocyte oil-free culture or oil-containing culture, respectively, whereas in vivo MII spindles are already present at 12 h post-hCG upon ovulation. Differences in meiotic timing between in vivo- and in vitro-matured oocytes are described to be caused by aberrant spindle formation. With the fixation method used in this study, in vitro-grown oocytes display barrel-shaped spindles with flattened poles, unlike the tapered spindle poles and smaller spindle volume in in vivo-matured oocytes. This might be due to restricted access of MTOC to the forming spindle by remaining anchored to the oocyte cortex, and it coincides with focused localization of 
-tubulin, a pivotal regulator of microtubule nucleation, at the spindle poles in in vivo-matured oocytes [13, 43]. Presumably, these morphologic observations are a consequence of metabolic and transcriptional deficiencies in IVM oocytes [13, 42, 44]. The occurence of barrel-shape spindles in the present study indicates that the meiotic cytoskeleton reorganization during IVM of oocytes is comparable after in vitro and in vivo growth. Major anomalies like chromosomal aberrations or severe spindle deformations were only observed in maximally 6% of the oocytes at the time points up to 18 h after hCG, indicating that the presently used culture systems generate genetically normal oocytes.
Since the occurance of GVBD morphology is gradually increasing over a time period of 4 h (between 4 and 8 h), there seems to be both a delay and an asynchrony in onset of meiosis in in vitro-grown compared to in vivo-grown oocytes. However, previous work in microdroplet culture under oil showed GVBD morphology between 2 and 3 h after mechanical oocyte removal and transfer to IVM medium containing hCG and EGF [45]. The quick onset of meiosis could be due to the mechanical disruption of the contacts that vehiculate the inhibitory signals from the granulosa cells, in contrast to the hormone-induced maturation in the intact follicle as practiced in this study. In an intact follicle, the signal transduction pathways need to be activated first in the granulosa cell compartment [46]. These findings suggest that the delay and heterogeneity of meiotic resumption in cultured oocytes is not caused by an intrinsic deficiency of in vitro-grown oocytes, but is rather an effect of a different reaction time after the stimulus of the granulosa cells.
The in vitro-cultured oocytes display a time difference between meiotic entry (>80% PMI at 8 h after hCG in culture with and without oil) and progression to MII (>80% MII at 16 h after hCG in oil-free culture and at 18 h after hCG in oil-containing culture) of 8–10 h in the oocytes grown without or with oil overlay, respectively. This time is needed to complete spindle formation and initiate AI, which is under CCNB1/MPF control [21]. It was described in IVM mouse oocytes that it takes 4 h after GVBD to form the first metaphase spindle with semi-aligned chromosomes. Four hours later, upon maturation of the kinetochores, final alignment induces AI only taking ±12 min to separate the sister chromatids [23, 24]. The duration of MI spindle formation and kinetochore maturation in oil-free culture (8 h) is therefore comparable to in vivo grown-oocytes; however, in oocytes grown in an oil-containing system the formation of the MI spindle is significantly delayed.
This finding in mice is consistent with an IVM study in pig, where the absence of oil overlay accelerated meiosis. These authors hypothesized that it was due to the high concentrations of endogenously produced progesterone [6]. Progesterone is described in pig to reduce connexin-43 via the PGR in LH- or FSH-stimulated cumulus cells. This will reduce gap junctional communication and hereby stimulate GVBD [47]. The high progesterone concentration in oil-free pig IVM also correlated with an earlier activation of mitogen-activated protein kinase (MAPK) and CDC2A after GVBD, which accelerates further the meiotic processes [6, 47]. In rodents, Pgr mRNA in granulosa cells is highly upregulated in the first few hours after maturation induction [46], and in our setting, progesterone production increased rapidly from 4 h of hCG, with maximal levels at 8 h. In this respect, the idea of progesterone being a mediator of the meiotic process was likely, but not conclusive in current literature [7–9, 48]. Our results using the specific PGR antagonist Org 31170 indicate that progesterone was no prerequisite for meiotic maturation induction, and the absence of any effect on cumulus expansion and meiotic timing strongly suggest that reduced progesterone-mediated action through this PGR is not responsible for a meiotic delay in mouse.
Both oil-free and oil-containing culture systems produce physiologic steroid profiles over time, with increasing E2 production during follicle growth (Days 8 and 12) and a switch to progesterone output upon the maturation stimulus. The rise in progesterone after hCG plateaus at 8 h after hCG, which could be in concordance with the rise in Pgr mRNA expression until around 5–7 h after hCG in the superovulated rat [46, 49]. Although the concentration of steroids in which follicles are residing is different due to the sequestration effect of oil, follicle survival (>90%) and follicle differentiation (>80%) are equal in both systems, suggesting only moderate local effects of steroids on folliculogenesis and oogenesis. This is also seen in the aromatase knockout mouse, where limited effects were seen on oocyte quality [50].
The use of oil overlay and a smaller culture volume (20–75 µl) will induce several differences in follicular environment, like chemical changes (e.g., ammonium), protein levels (e.g., growth factors), and metabolism (e.g., nutrients, metabolites). However, recent literature indicates that another lipophilic substance, meiosis-activating sterol (FF-MAS), influences maturation. Although in general FF-MAS is presumed not to be indispensable for meiotic induction [7], it did enhance the MI to MII transition of in vitro-maturing mouse oocytes by mechanisms not yet resolved [51]. FF-MAS also positively affected developmental capacity and genetic stability of the oocyte, were it appears to protect oocytes from precocious segregation of chromatids [52], but positive effects on meiosis were only observed in mouse models with a genetic background leading to meiotic defects [51]. IVM without oil overlay in pig had a beneficial effect on the preimplantation embryonic development, probably attributable to higher steroid and FF-MAS, which could enhance oocytes' cytoplasmic maturation [6]. In primate models the abolishment of progesterone did lower oocyte development competence [53, 54]. These data would suggest a potential dose-effect of progesterone on distal endpoints in development. However, historical lab data show neither statistical difference in cleavage rate nor blastocyst formation capacity (oil system, 73 ± 9 and 72 ± 16; oil-free system, 74 ± 9 and 54 ± 10, for 2-cell rate (%) and blastocyst/2-cell rate (%), respectively; [33]). These findings may have many origins (the different species used, meiotic induction by FSH vs. hCG, oil quality, etc.), but are also likely to be an effect of concentration. In our follicle culture system, entire follicles consisting of a functional steroid-producing theca/mural cell compartment are cultured for 12 days with a steroid-rich medium carry-over from basal steroid production during growth after meiotic maturation induction. This ascertains the presence of a basic level of steroids during meiotic maturation.
Since the differential hormonal environments in which oocytes mature induce subtle alterations in meiosis kinetics in different species, this could also have implications for the human ART-clinic, where different ovarian hyperstimulation protocols were shown to significantly influence endocrine parameters in serum and follicular fluid [55] and where the upcoming technique of IVM of immature oocytes used mineral oil overlay [56, 57].
In the oil-free follicle culture system, full MII spindle formation is reached 16 h after the stimulus in >80% of the cultured oocytes. Prolongation of culture after hCG, thereby inducing oocyte aging within the follicular structure, did not further increase the yield of MII oocytes, and normal MII spindle rate decreased considerably 21 h after hCG. This indicates an optimal fertilization window of 5 h, comparable with in vivo [25], to obtain genetically balanced embryos. Classification of the spindle and chromosome abnormalities showed a large proportion of abnormal oocytes with astral microtubules emanating from the spindle structure. The spindle dissolution observed in this study was described earlier by others in in vivo postovulatory aged oocytes from 15 h after ovulation onward in mouse (comparable to our 27 h after hCG) [16] and in human [28, 58, 59]. It was mentioned that the aged spindle resembled the interphase-like appearance of microtubuli seen after activation of the oocyte and in the early embryo. This indicates that the time course and indications of postovulatory aged oocytes are comparable after in vivo aging in the fallopian tube or in vitro aging within the in vitro follicle structure. Sometimes this interphase-like appearance of the spindle is accompanied by a broadening of the metaphase plate of chromosomes. An intracellular environment inducing interphase-like spindle structure could also be a trigger for chromosome decondensation. The maintenance of a stable meiotic spindle structure with perfectly aligned chromosomes during MII arrest requires a high MPF activity, high MAPK activity, the presence of the cytostatic factor, and MAPK-associated proteins like MAPK1IP1 (also known as MISS) and CDK2AP2 (also known as DOC1R) [60, 61]. Depletion of CDK2AP2 or MAPK1IP1 in the oocyte induces disorganized spindles, like microtubules emanating from the poles to the cytoplasm and cytoplasmic aster formation [62, 63]. A possible explanation for the microtubule defects seen in postovulatory aged oocytes could hence be a gradual decrease in spindle stabilizing proteins. A gradual decrease in MPF activity with aging has been described in in vitro-matured, aged COCs, which contained significantly less MPF activity at 26 h after maturation induction compared to 14 h [64]. However, exact interrelations between the different components of MII spindle stabilization during MII arrest remain to be determined. The process of aging is correlated with a gradual weakening of spindle stability and, as recently described, a gradual loss of transcripts for checkpoint components, a prelude for aneuploidies [17]. These findings demonstrate the importance of a well-characterized time window for fertilization in order to obtain genetically balanced embryos.
In conclusion, we can state that the processes maintaining the spindle structure are present and active during intrafollicular oocyte aging after 12-day in vitro follicle culture, since oocytes keep an organized spindle for at least 5 h after MII formation (21 h after hCG), but are gradually weakening in time after hCG like their in vivo-grown counterparts.
Oil overlay during follicle growth does not impair the capability of cultured oocytes to proceed to a normal MII stage, but tends to delay the time course of maturation events, mainly in MI spindle formation. Meiotically mature oocytes were obtained 16 and 18 h after stimulation in oil-free and oil-containing culture, respectively. Despite the highly differential progesterone concentrations between oil-free and oil-containing culture systems, progesterone receptor signaling does not appear to be the etiological factor for the delay in meiotic progression.
Up to 21 h after the hCG stimulus, spindle structure remains intact in oil-free culture, i.e., 5 h after they have reached MII spindle morphology. This interval represents the time window for cultured oocytes to maintain normal spindle morphology and consequently genetic stability in view of performing IVF. The most prominent postovulatory aging effect was an interphase-like appearance of the cytoskeleton.
ACKNOWLEDGMENTS
The authors would like to thank Ms. Anne Gerard for technical assistance with the radioimmunoassay and Ms. Sandra De Schaepdryver for editorial support.
FOOTNOTES
1Supported by FWO G.0479.06 and VUB-OZR 1227. 
Correspondence: 2FAX: 32 2 477 50 60; e-mail: ingrid.segers{at}vub.ac.be
Received: 3 May 2007.
First decision: 28 May 2007.
Accepted: 16 December 2007.
REFERENCES
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  Y.-L. Miao, K. Kikuchi, Q.-Y. Sun, and H. Schatten Oocyte aging: cellular and molecular changes, developmental potential and reversal possibility Hum. Reprod. Update, May 8, 2009; (2009) dmp014v1. [Abstract] [Full Text] [PDF]
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