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Department of Anatomy of Domestic Animals, Faculty of Veterinary Medicine, University of Milan, 10-20133 Milano, Italy
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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ATP, early development, gamete biology, meiosis, microtubule, mitochondria, ovum, parthenogenesis, pig oocyte
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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While the only parameter that can reliably measure the efficiency of an in vitro system is the ability to produce a viable fetus from a cultured embryo, for practical and economical reasons, the most commonly used measure of efficiency remains the proportion of immature oocytes developing to the blastocyst stage.
Several studies have clearly demonstrated that oocyte quality is the most crucial factor affecting blastocyst rate [2]; however, the essence of oocyte quality remains elusive and it certainly includes many aspects [3]. We have previously identified post-transcriptional modification of the maternal mRNA molecules stored in the ooplasm as one of these aspects [4, 5]. In the present work we took into consideration the role of cytoplasmic compartmentalization, which has been suggested to play an important role in the completion of coordinate nuclear and cytoplasmic oocyte maturation [6]. In particular, the activity and cytoplasmic distribution of mitochondria was chosen as an easily detectable marker of cytoplasm compartmentalization; microtubules were also studied as the recognized responsible partner for the establishment and maintenance of cellular asymmetry during oocyte maturation [7].
The activity and organization of mitochondria are necessary features among the diverse events involved in cytoplasmic maturation [8]. The primary function of mitochondria is to generate ATP, which is necessary for several functions, including motility, maintenance of cellular homeostasis, and regulation of cell survival [9]. The pattern of mitochondria distribution and their metabolic activity undergo changes during oocyte maturation in many species, including mouse [8, 10], cow [11], human [12], and pig [13].
The transfer of mitochondria within different areas of the cell is mediated by a cytoskeletal network of microtubules [14]. Microtubules, homologous polymers of 
- and ß-tubulin, are dynamic components of the cell cytoskeleton; they are ubiquitously present in mammalian cells and are involved in diverse functions such as determining cell shape and movement, transportation of molecules and organelles, meiosis, and mitosis. In the mouse oocyte, two populations of microtubules can be identified: one linked to the DNA, and another located in the cytoplasm [15]. Despite the role that microtubules play during meiotic maturation and competence acquisition, relatively little information is available for species other than the mouse. In particular, no clear data are available on microtubule formation and distribution during pig oocyte maturation. In fact, some studies described only a microtubule population linked to the DNA, while a cytoplasmic microtubule network has not been identified at all [16, 17]; in others, a fine mesh of cytoplasmic microtubules has been reported only around the time of germinal vesicle breakdown [18, 19].
To identify the cellular mechanisms that are associated with a reduced viability of pig oocytes we used an IVM system in which pig oocytes can complete the meiotic process at a high rate, but they predictably show either a low or a high developmental competence to the blastocyst stage in vitro, following parthenogenetic activation. This system, therefore, uncouples nuclear and cytoplasmic maturation, making it possible to study the role of ATP content, migration of active mitochondria, and microtubule dynamics throughout the entire process of IVM.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Porcine Follicular Fluid Preparation
Ovaries were collected at the slaughterhouse. Porcine follicular fluid (pff) was aspirated from follicles of prepuberal gilts with a diameter of 2–7 mm in 15-ml tubes. Cellular debris was allowed to settle for 20 min, and tubes were centrifuged for 15 min at 400 x g at 4°C. The supernatant was aspirated and filtered through a 0.22-µm membrane (Sterilin, Milan, Italy). Aliquots of 2.5 ml of pff were stored at –20°C until use.
Recovery and Maturation of Cumulus-Oocyte Complexes
Cumulus-oocyte complexes (COCs) were obtained from ovaries of slaughtered gilts weighing 160 ± 15 kg and aged 240 ± 15 days. Follicles with a diameter of 2–7 mm were aspirated with an 18-gauge needle and vacuum pressure of 50 ml/min. The follicles aspirate was collected in 15-ml tubes, and COCs were washed twice in prewarmed (37°C) aspiration medium. The medium was composed of TCM-199 supplemented with 6.5 mg/ml Hepes, 1.1 mg/ml sodium bicarbonate, 4 mg/ml BSA, 75 µg/ml penicillin, and 50 µg/ml streptomycin (199D). Only COCs with large, compact cumulus and homogeneous oocyte cytoplasm were selected for IVM. IVM was performed as previously described [20] with minor modifications. Briefly, COCs were cultured in TCM-199 supplemented with 1.1 mg/ml sodium bicarbonate, 0.1 mg/ml sodium pyruvate, 0.5 mM cysteamin, 8.2 µg/ml insulin, 10 ng/ml epidermal growth factor, 1 mM dibutryl cAMP, 0.5 IU/ml porcine FSH:LH (Pluset; Serono, Rome, Italy), 1.0 µg/ml 17 ß-estradiol, 75 µg/ml penicillin, and 50 µg/ml streptomycin (199-IVM). Based on previous preliminary data [21], 25% or no pff was added to 199-IVM medium. COCs were matured in this medium for 22 h at 38.5°C in an atmosphere of 5% CO2 in air. COCs were washed in fresh medium and matured for an additional 24 h in 199-IVM without dibutryl cAMP.
Meiotic Assessment
At the end of each IVM experiment, a representative sample of oocytes (approximately 10% of the total 552 oocytes used for the developmental studies) was denuded by gently pipetting in medium 199D containing 0.1% hyaluronidase at 38.5°C, washed in medium 199D, and mounted on microscope slides. Samples were fixed for 48 h in acetic acid:ethanol (1: 3) and stained with 0.1% (v/v) acetic orcein for 30 min. They were destained in glycerol:acetic acid:water (1:1:3) and meiotic stage was evaluated using a Nikon Eclipse E600 microscope (Nikon Instruments, Sesto Fiorentino, Italy) at 200x and 400x magnification.
Other oocytes of each group were used for the measurement of ATP content or for the staining of active mitochondria and microtubules. All the remaining oocytes were parthenogenetically activated and cultured in vitro. The relative procedures are described below.
Parthenogenetic Activation and In Vitro Culture
After IVM, oocytes were denuded as described above, washed for 10 min in TCM-199 supplemented with 20% (v/v) fetal calf serum (FCS), and then incubated in TALP medium for 30 min at 38.5°C. Parthenogenetic activation was performed according to the method described by Boquest et al. [22] by sequentially exposing the oocytes to 5 µM ionomycin in TALP for 5 min at 38.5°C in the dark; and to 2 mM 6-DMAP in medium NCSU-23 [23] for 3 h at 38.5°C in an atmosphere of 5% CO2, 5% O2, and 90% N2. In a separate experiment, 16 h from the end of the incubation in 6-DMAP, the incidence of second polar body retention was assessed by acetic orcein staining as described above and compared between the two maturation treatments in a total of 90 oocytes.
Presumptive parthenotes were washed thoroughly in NCSU-23 and cultured in groups of 25–35 in 50-µl NCSU-23 drops under mineral oil at 38.5°C in the same atmosphere as above. Embryonic cleavage was recorded 48 h postactivation. On Day 5 postactivation, half of the medium was replaced with fresh NCSU-23 containing 20% (v/v) FCS to reach a final FCS concentration of 10% (v/v) in the in vitro culture drop. At Day 7 postactivation, in vitro culture was interrupted and embryonic development was evaluated. The experiment was replicated five times.
Measurement of the ATP Content of Oocytes
ATP content of denuded, nonmatured and matured oocytes (n = 282) was assessed using a commercial assay based on a luciferin-luciferase reaction (Bioluminescent Somatic Cell Assay Kit; Sigma) as previously described [11]. Briefly, samples were rinsed twice in PBS and then transferred individually in 50 µl of PBS (sample buffer) into plastic tubes on melting ice. Then, 100 µl of somatic cell reagent (FL-SAR) was added to each tube. Samples were incubated on melting ice for exactly 5 min in the dark, and then 100 µl of assay buffer (dilution 1:25 with ATP assay mix dilution buffer, FL-AAB) was added, and the tubes were kept for exactly 5 min at room temperature in the dark. ATP content was measured with a luminometer (Stratec Biomedical Systems AG, Birkenfeld, Germany). An eight-point standard curve (0–7 pmol/tube) was included in each experiment (n = 3), and the content of ATP was determined from the formula for the standard curve.
Staining of Active Mitochondria and Microtubules
Oocytes at different stages of maturation were stained with MitoTracker Orange CMTM-Ros (Molecular Probes Europe, Leiden, The Netherlands). A stock solution of the dye was prepared according to manufacturer's specifications. MitoTracker Orange CMTM-Ros stains selectively active mitochondria and is well retained after fixation; the dye was used at a concentration of 250 nM in medium 199D for 30 min at 38.5°C. After staining, oocytes were briefly rinsed in PBS and fixed in 3.7% paraformaldehyde in PBS for 30 min at 37°C. DNA was stained with 4',6'-diamidino-2-phenylidole (DAPI; 0.2 µg/ml), and samples were mounted on glass slides in a glycerol-based mounting medium. Oocytes were stored below 0°C until confocal microscopy was performed.
Immunolocalization of microtubules was carried out as previously described [17] with minor modifications. Briefly, oocytes were fixed in 3.7% paraformaldehyde in PBS for 30 min at 37°C. They were permeabilized in PBS containing 0.5% Triton X-100 for 20 h at 37°C and then incubated in PBS containing 115 mM glycine and 1% Triton X-100 for 30 min. After washing for 15 min in PBS, oocytes were incubated with a fluorescein isothiocyanate-conjugated anti-alpha-tubulin antibody diluted 1:50 for 90 min at room temperature. After two washes in PBS, DNA was stained with DAPI (0.2 µg/ml) and oocytes were mounted as described above. When mitochondria and microtubules were stained in the same oocytes, the two protocols described here were performed in sequential order.
A total of 86 oocytes were analyzed, divided into three replicates.
Confocal Microscopy
Stained samples were examined using a TCS-NT laser scanning confocal microscope (Leica Microsystems, Heidelberg, Germany) equipped with Ar/Kr and He/Ne lasers. Blocking filters used were band path, 530 ± 30 (microtubules); long path, 590 (mitochondria); and long path, 450 (DNA). Active mitochondria distribution was assessed through one equatorial optical section of 6.7 µm thickness. Laser intensity was 1.5 mV. Objective (10x and 40x Leica Floutar; Leica Microsystems, Heidelberg, Germany), pinhole (1 Airy unit), filters, offset, gain, and Photon Multiply Tube settings were kept constant throughout the experiments. Microtubule distribution was assessed by sequential scanning of at least 40 µm of sample with 2.6 µm step size and controlled oversampling.
Blastomere Count of Parthenogenetic Blastocysts
Blastocysts at Day 7 after activation were fixed in 60% methanol in PBS overnight at 4°C. After a wash in PBS, nuclei were stained with propidium iodide and the samples were mounted on microscope slides. Nuclei were counted with the use of a Nikon Eclipse E600 microscope at 200x and 400x magnification.
Statistical Analysis
Developmental competence of oocytes matured in the presence of different amounts of pff and the relative distribution of active mitochondria at different times of IVM were evaluated by chi-square analysis. The number of blastomeres forming blastocysts from different treatments was evaluated by Kruskal Wallis analysis of variance (ANOVA). Oocyte ATP content was evaluated by ANOVA followed by a least significant difference test. When appropriate, results are presented as mean ± SEM.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The oocyte maturation rate did not change significantly when IVM was performed in the presence or absence of 25% pff (Table 1). No difference in the second polar body retention rate was observed between treatments (Table 1). However, parthenogenetic development to the blastocyst stage was significantly higher in the 25% pff group (41.5%) compared with that of the 0% pff treatment (19.9%). Moreover, blastocysts in the 25% pff group had a significantly higher number of cells than the 0% pff group (76.1 ± 6.3 and 47.2 ± 6.5, respectively; Table 1).
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ATP Content of Oocytes
At collection, oocyte ATP content was 1.21 ± 0.1 pmol/ oocyte (Table 2). During IVM, this value increased significantly in both the 0% pff and 25% pff groups. However, there was no statistically significant difference in the ATP content at the end of IVM between the two treatments (3.08 ± 0.42 and 3.60 ± 0.51 pmol/oocyte, respectively).
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Active Mitochondria and Microtubule DistributionDuring IVM
Three mitochondrial distributions were identified: peripheral, semiperipheral, and diffused (Fig. 1). At collection, 71% (22/31) of the oocytes presented a peripheral distribution of active mitochondria, while 13% had a semiperipheral distribution, and 16% exhibited a diffused pattern (Table 3). After 46 h of IVM in the 0% pff treatment, 57% (12/21) of the oocytes had maintained the peripheral distribution, 24% presented a semiperipheral pattern, and 19% reached the diffused state. Conversely, in the 25% pff group, 59% (20/34) of the oocytes presented a diffused distribution of active mitochondria, 21% displayed a semiperipheral pattern, and 20% remained in a peripheral type of distribution.
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No microtubules were detected at collection in any of the oocytes analyzed (10/10). While maturation proceeded, in all oocytes, disregarding treatment, small asters of microtubules were detected in conjunction with DNA starting at 20 h of IVM, which in our system, corresponds to the meiotic stage of germinal vesicle breakdown. Association of microtubules to DNA continued throughout meiosis until the end of IVM (Fig. 2). Independently from the distribution of this DNA-linked microtubule population, 87% (36/ 42) of the oocytes in the high-competence group (25% pff) presented a cytoplasmic localization of microtubules during IVM (Fig. 2A). The cytoplasmic mesh of microtubules started to appear around 12 h of IVM at the periphery of the oocyte, extended to the inner parts of the cytoplasm over the following 12 h, reached a peak at 24–28 h, and then tended to fade. At 36 h of IVM, microtubules were no longer detectable in the cytoplasm. Conversely, in the low-competence group (0% pff), no oocytes presented a diffused pattern of distribution of cytoplasmic microtubules, and only 10% (2/22) presented a weak and partial peripheral distribution of microtubules, appearing at around 24 h of IVM and fading by 42 h of IVM.
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The contemporary staining of the samples for active mitochondria, tubulin, and DNA allowed us to time the relative relocation of mitochondria with respect to cytoskeletal formation. Mitochondria relocation toward the center of the cytoplasm started at around 16 h of IVM, before germinal vesicle breakdown, and was complete by 36 h of IVM. No further mitochondria relocation was recorded after this time until the end of IVM (46 h). No specific connections between mitochondria and nuclear material were observed.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The role played by ATP in the acquisition of meiotic competence has been studied in many species, but it remains difficult to define in detail [9]. Our results indicate a statistically significant increase during IVM of ATP content in pig oocytes similar to what has been reported in the cow [11]. Recently, an increased oxidative activity of mitochondria has also been reported during IVM of pig oocytes [30]. To the contrary, no ATP increase was observed during mouse oocyte maturation [31]. In cattle, COCs presenting a progressively worsening morphology displayed a decreasing average content of ATP, both at the beginning (germinal vesicle) and at the end of IVM (MII). Because oocyte morphology was linked to blastocyst rate, a relation between ATP content and developmental competence was evident in this species [11]. This relation was not observed in our experiments because the increase in ATP during maturation was not influenced by the amount of pff employed during IVM and its concentration was not related to oocyte developmental competence. Our observations agree with a recent study performed in pigs [32] in which it was observed that no significant differences in ATP content were present between oocytes matured in vivo or in vitro. Therefore, pigs may be different from other species in that a higher rate of embryonic development had been associated with higher concentrations of ATP not only in cattle [11], but also in humans and mice [31].
Changes in mitochondrial organization are believed to be a faithful indicator of oocyte capacity to sustain embryonic development [33] and to occur during IVM in many species including mouse [8, 10], cow [11], and human [12]. Pig mitochondria migrate during maturation from the peripheral part of the oocyte to the inner region of the cell both in vitro [13] and in vivo [30], even though information is scarce on the correlation between this phenomenon and subsequent oocyte development. Our results indicate that relocation of active mitochondria is significantly more frequent in the oocyte group with high developmental competence. Conversely, little or no relocation was observed after IVM in the low-competence group, suggesting that the lack of mitochondrial distribution through the ooplasm is a marker of cytoplasm immaturity and is strongly linked to low developmental ability.
The transfer of mitochondria within different areas of the cytoplasm is mediated by the cytoskeletal network of microtubules in the oocytes of mice [14] as well as pigs [13]. While there is general agreement on the involvement of microtubules in forming the meiotic spindle, the dynamics of cytoplasm-associated microtubules in pig oocytes remains to be elucidated. Some studies failed to identify a cytoplasmic microtubule network altogether [16, 34], while other reports describe only a finely distributed mesh of cytoplasmic microtubules surrounding the germinal vesicle nucleus [18, 19, 35]. Therefore, the question remains open whether the missed distribution of mitochondria during the maturation of defective oocytes was due to the lack of formation of an appropriate microtubule network or was caused by an uncoupling between mitochondria and microtubule, possibly related to the lack of specific motor molecules such as dynein and kinesin [36, 37]. Here we described the formation of a microtubule network in the ooplasm of the high-competence group that began to form before germinal vesicle breakdown at 12 h of IVM from the periphery of the oocyte, extended to the inner parts of the cytoplasm over the following 12 h, reached a peak at 24–28 h, and then tended to fade. At 36 h of IVM, microtubules were no longer detectable in the cytoplasm, but persisted until the end of maturation in correspondence with the DNA. The contemporary staining of active mitochondria, DNA, and microtubules allowed us to demonstrate that the lack of relocation of active mitochondria to the inner part of the oocyte is related to the absence of an appropriate and timely formation of the microtubule network in the cytoplasm. In the oocytes belonging to the low-competence group we did not observe a diffused pattern of distribution of cytoplasmic microtubules, and only 10% of them presented a weak and partial peripheral distribution of microtubules, appearing at around 24 h of IVM and fading by 42 h of IVM. This suggests that the lack of mitochondria relocation observed in the defective oocyte group may be due to their inability to form a cytoplasmic microtubule network rather than to the inability of mitochondria to migrate along the tubules. The formation of a normal meiotic spindle, observed in both groups of oocytes, explains why the rate of meiotic progression to the second metaphase was not altered in the low-competence group. This observation clearly illustrates the hypothesis that defective oocyte developmental competence is due to the uncoupling of nuclear and cytoplasmic maturation. While the normal formation of nucleus-associated microtubules allows the correct segregation of chromosomes during the reductive meiotic divisions, the lack of a microtubule cytoplasmic network prevents a correct relocation of mitochondria, which is likely to reflect a more generally altered compartmentalization of the ooplasm. It is not clear, however, whether the missing relocation of mitochondria also has a direct effect because it was not linked to differences in the total ATP content of the oocytes. However mitochondria are also involved in Ca2+ signaling during egg activation [38], and the recent observation that estrogen receptor beta is localized to mitochondria [39] suggests that a specific distribution of these organelles may be necessary for a correct oocyte response to estradiol-17ß, which is present at high levels in preovulatory follicles.
We conclude that low developmental competence is associated with the lack of a microtubule cytoplasmic network, which prevents the correct relocation of mitochondria, and is likely to reflect a more generally altered compartmentalization of the ooplasm. This can occur independently from the formation of the microtubule machinery required for the completion of chromosome disjunctions and does not affect the overall ATP content.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Fulvio Gandolfi, Istituto di Anatomia degli Animali Domestici, via Celoria, 10-20133 Milano, Italy. FAX: 39 025 031 7980; fulvio.gandolfi{at}unimi.it
Received: 16 November 2004.
First decision: 16 December 2004.
Accepted: 17 January 2005.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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