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BOR - Papers in Press, published online ahead of print June 29, 2005.
Biol Reprod 2005, 10.1095/biolreprod.105.043703
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BIOLOGY OF REPRODUCTION 73, 1025–1031 (2005)
DOI: 10.1095/biolreprod.105.043703
© 2005 by the Society for the Study of Reproduction, Inc.

Cumulus Cells Accelerate Aging of Mouse Oocytes1

Yi-Liang Miao , Xin-Yong Liu , Tian-Wu Qiao , De-Qiang Miao , Ming-Jiu Luo , and Jing-He Tan 2 

College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Tai-an City 271018, People's Republic of China


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The role of cumulus cells (CCs) that surround oocytes in maturation, ovulation, and fertilization has been extensively studied, yet little is known about their role in oocyte aging. Although early studies have shown that when ovulated oocytes are aged in vitro displayed similar morphological alterations as those aged in vivo, a recent study found that vitro culture of mouse oocytes retarded oocyte aging. The objective of this study was to test the hypothesis that CCs would accelerate oocyte aging. During in vitro aging with CCs of both in vivo-matured and in vitro-matured mouse oocytes, activation rates increased, whereas the maturation-promoting factor (MPF) activity decreased significantly as during in vivo aging of the ovulated oocytes. During aging after denudation of CCs, however, activation rates of both in vivo-matured and in vitro-matured oocytes remained low and the MPF activity decreased much more slowly compared to that of oocytes aged with CCs. Although many oocytes aged in vivo and in vitro with CCs showed a partial cortical granule (CG) release, very few cumulus-free oocytes released their CGs during in vitro aging. When denuded oocytes were cultured with cumulus-oocyte-complexes at a 1:2 ratio or on a CC monolayer, activation rates increased, while MPF activity decreased significantly. The results strongly suggested that CCs accelerated the aging progression of both in vivo-matured and in vitro-matured mouse oocytes.

activation, aging, cumulus cells, gamete biology, mouse oocyte, MPF, oocyte development, ovum


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mammalian oocytes are normally fertilized soon after ovulation. If fertilization does not occur in time, unfertilized oocytes that remain in the oviduct (aging in vivo) or culture of these oocytes in a suitable medium (aging in vitro) can lead to a time-dependent process of oocyte aging [1, 2]. The most prominent manifestations of oocyte aging include an increased susceptibility to activating stimuli [3– 9], a decrease in maturation-promoting factor (MPF) activity [10–13], the onset of anaphase II [12, 14–16], and partial exocytosis of cortical granules (CGs) [1, 17–22]. It is well known that fertilization or artificial activation of aged oocytes resulted in abnormal development [23–26]. However, culture of metaphase II (MII) eggs derived from both in vivo and in vitro maturation is common in many research and clinic applications. For instance, many experimental designs involve culture of oocytes before micromanipulation or insemination. This in vitro culture of eggs could compromise their subsequent competence of development. Therefore, control of oocyte aging in vitro should have advantages for recently developed embryo technologies.

Oocytes that mature both in vivo and in vitro are enclosed with cumulus cells (CCs). The CCs stay with in vivo-matured oocytes for different periods after ovulation, depending upon the species [1, 14, 20, 27, 28], but they will stay with in vitro-matured oocytes until artificially removed. The role of the surrounding CCs in maturation, ovulation, and fertilization of oocytes has been extensively studied [29–32], yet little is known about their role in oocyte aging. Longo [28] found that mouse oocytes aged in vitro showed similar morphological alterations as those aged in vivo after ovulation. Webb et al. [33] showed that the cytoskeletal organization of the mouse egg changes similarly during aging in vivo and in vitro. Tan [14] found no marked difference in morphological changes or the rate of degeneration between in vivo-aged goat oocytes and those aged in vitro. The spontaneous reduction in MPF activity with in vitro aging of in vitro-matured oocytes has been documented in cattle [13] and pigs [10, 11]. However, Abbott et al. [16] reported that in vitro culture of mouse oocytes retarded the spontaneous activation of cell cycle progression that normally occurred in in vivo unfertilized eggs [12]. The difference between studies by Abbott et al. [16] and by others is that Abbott et al. cultured ovulated oocytes that were free of CCs. Because the CCs of mouse oocytes did not show signs of degeneration in vivo until 12 h after ovulation [28], eggs used by Xu et al. [12] should have always been with CCs during the entire period of their in vivo aging (13–22 h after hCG injection). In addition, aging of in vitro-matured mouse oocytes has not been studied. We therefore proposed a hypothesis that CCs would accelerate the progression of in vitro aging of mouse oocytes. To test this hypothesis, we assessed the effects of CCs on the susceptibility to activating stimuli, MPF activity, exocytosis of cortical granules (CGs), and anaphase onset during in vitro aging of both in vivo-matured and in vitro-matured mouse oocytes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Mice of the Kun-ming breed (Shandong Agricultural University) were kept in a room with 14L:10D cycles, with darkness starting at 20:00 h. The animals were handled according to rules stipulated by the Animal Care and Use Committee of Shandong Agricultural University.

Recovery of Oocytes

Ovulated oocytes Female mice were induced to superovulate ~6–8 wk after birth with eCG (10 IU, i.p.) followed 48 h later by hCG (10 IU, i.p.). Both eCG and hCG used in this study were from Ningbo Hormone Product Co., Ltd., P.R. China. The superovulated mice were killed at different times after hCG injection and the oviductal ampullae were broken to release the cumulus-oocyte complexes (COCs) at different stages of in vivo aging.

In vitro-matured oocytes Procedures for oocyte maturation in vitro were the same as those reported by Miao et al. [34] from this laboratory. Briefly, female mice 3 wk after birth were killed at 46 h after eCG administration, and the large follicles on the ovary were aspirated for COCs. The recovered COCs were cultured in groups of around 40 for 14 h in 100-µl microdrops of TCM-199 (Gibco, Grand Island, NY) supplemented with 10% fetal calf serum (Gibco) and 10 IU/ml eCG. At the end of maturation culture, well-expanded COCs were selected and allocated to different treatments.

Preparation of denuded oocytes After being dispersed and washed three times in M2 medium, the COCs from the two sources described above were denuded of CCs by pipetting with a thin pipette in M2 containing 0.1% hyaluronidase (Sigma).

In Vitro Aging

The COCs and DOs were cultured in wells (25–35 oocytes per well) of a 96-well culture plate (made in China) containing 200 µl of Chatot-Ziomek-Bavister (CZB) medium [35] and covered with mineral oil at 37.5°C under 5% CO2 in humidified air. At different time intervals of in vitro aging, the COCs and DOs were allocated to parthenogenetic activation or for examination of MPF activity.

Activation and Assessment of Activated Oocytes

Oocytes were activated with ethanol and 6-DMAP in combination. Before activation treatment, COCs were denuded of CCs by pipetting in M2 containing 0.1% hyaluronidase (Sigma). In this study, only oocytes with first polar bodies were treated for activation or histone H1 kinase assay. The denuded oocytes were first treated with 5% (v/v) ethanol in M2 medium for 5 min at room temperature, then washed three times and cultured in CZB containing 2 mM 6-DMAP for 6 h. At the end of culture, oocytes were observed under a microscope for activation. Only those oocytes with one pronucleus or two pronuclei, or two cells each having a nucleus, were considered activated. Oocytes for controls were cultured for 6 h in CZB containing no 6-DMAP without prior ethanol treatment. Controls were set for each experiment and data were used only when no control oocytes were activated in the experiment.

Assay of Histone H1 Kinase Activity

Procedures for assay of histone H1 kinase activity were those reported by Bement and Capco [36] and Gallicano et al. [37] with modifications. Briefly, a sample of 15 oocytes was washed twice in the collecting medium (80 mM glycero-2-phosphate, 20 mM EGTA, 15 mM MgCl2, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 g/ml pepstatin, 10 µg/ml aprotinin, and 50 nM cAMP-dependent protein kinase inhibitor, pH 7.3), frozen in 10 µl of the same medium, and stored at –70°C. The cAMP-dependent protein kinase inhibitor (IP-20) from rabbit was purchased from Sigma (production number P0300). At assay, oocytes were disrupted by three cycles of freeze-thawing, and the sample was added to 25 µl of a mixture containing histone H1 at 2.5 mg/ml, 0.7 mM ATP, and {gamma}-[32P]ATP at 50 µCi/ml. The reaction was allowed to run for 15 min at 20°C, and was terminated by pipetting 25 µl of the reaction mixture onto a 1 x 2 cm piece of P81 phosphocellulose (Whatman). Filters were then washed three times (2 min per wash) in 10 ml of phosphoric acid per liter of H2O. Filters were then transferred wet into scintillation vials, 10 ml scintillation fluid was added, and samples were assayed with a Beckman scintillation counter for radioactivity (cpm).

Staining and Observation of CGs and Chromosomes

The zonae pellucidae were removed by treating the oocytes with 0.5% pronase (Roche) in M2. After being washed three times in a washing solution (M2 supplemented with 0.3% BSA [Sigma] and 0.01% Triton X-100), oocytes were fixed with 3.7% paraformaldehyde in M2 for 30 min at room temperature. The oocytes were then blocked three times for 5 min each in a blocking solution (M2 containing 0.3% BSA and 100 mM glycine). After permeabilization for 5 min in M2 containing 0.1% Triton X-100 (Sigma), oocytes were washed twice again for 5 min each in blocking solution. They were then cultured in 100 µg/ml of fluorescein isothiocyanate-labeled lens culinaris agglutinin (Sigma) in M2 for 30 min in the dark. Finally, the oocytes were washed three times in the washing solution. The DNA was stained in the final incubation for at least 10 min with M2 containing 10 µg/ml propidium iodide. After washing, oocytes were mounted on glass slides and observed with a laser scanning confocal microscope.

Assay for Zona Pellucida Hardening

The assay for zona pellucida (ZP) hardening was performed as described by Gulyas and Yuan [38] and Xu et al. [12] with minor modifications. Briefly, 20 cumulus-free oocytes were treated with 1 µg/ml {alpha}-chymotrypsin (type II, 40–60 U/mg protein; Sigma C-4129) contained in a 100-µl drop of PBS covered with mineral oil at 30°C. Oocytes were monitored every 2 min during the first 30 min of the treatment and then every 5 min until the end of the treatment (3 h). The time at which 50% of the ZP underwent a complete dissolution (with denuded oocytes stuck on the bottom of the dish) was assessed as t50 for ZP dissolution.

Data Analysis

For each treatment, three replicates were run. Statistical analyses were carried out by analysis of variance. Differences between treatment groups were evaluated with the Duncan multiple comparison test. Data are expressed as mean ± SEM and P < 0.05 is considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of CCs on Activation and MPF Activity of Ovulated Oocytes

To study the effect of in vivo aging, some oocytes collected from oviducts 12–36 h after hCG administration were activated with ethanol and 6-DMAP, whereas others were examined for MPF activity. While activation rates increased, the MPF activity of oocytes decreased significantly from 12 to 24 h after hCG administration. After that, however, while the MPF activity continued to decline, few oocytes were activated (Table 1). To study the effect of in vitro aging, freshly ovulated oocytes collected 12 h after hCG administration were cultured in vitro, and activated or examined for MPF activity at different times of in vitro aging (from 18 to 36 h after hCG administration). Activation rates and MPF activity of oocytes aged in vitro with CCs (i.e., COCs) changed in a similar manner as those of in vivo-aged oocytes, although their activation rates were lower and their MPF activity was higher at 18 and 24 h after hCG administration (Table 1). However, activation rates and MPF activity of oocytes cultured denuded (DO) changed in a manner different from those of oocytes cultured with cumulus. Thus, activation rates of the denuded oocytes remained below 30% from 12 to 24 h after hCG administration and their MPF activity decreased much more slowly compared to that of oocytes aged with CCs (Table 1).


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  		TABLE 1. Activation and MPF activity of in vivo mature oocytes after different hours of aging in vivo or in vitro

Effects of CCs on Activation and MPF Activity of In Vitro-Matured Oocytes

Mouse oocytes at the germinal vesicle stage were in vitro-matured in TCM-199 for 14 h. At the end of maturation culture, some of the oocytes were denuded of CCs. The in vitro-matured COCs and DOs were then in vitro-aged in the CZB medium. At different times of in vitro aging, some oocytes were activated and others were examined for MPF activity. The activation rate of COCs increased significantly from 14 to 32 h of culture (0 to 18 h of aging) but began to decrease afterward (Table 2). The MPF activity of these oocytes decreased steadily during the entire aging period (Table 2). The activation rate of DOs, however, remained constant in most parts of the aging period (from 20 to 38 h of culture), although it increased mildly from 14 to 20 h of culture (Table 2). Accordingly, the MPF activity of these oocytes did not change significantly from 20 to 38 h of culture (Table 2).


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  		TABLE 2. Activation and MPF activity of in vitro matured oocytes after different hours of aging in vitro

Changes of CGs and Chromosomes of Ovulated Oocytes During In Vivo or In Vitro Aging

The COCs collected 12 h after hCG administration were in vitro-aged either with the cumulus intact (COC) or after they had been freed of CCs (DO) for up to 24 h after hCG administration. The in vivo-aged oocytes were recovered from oviducts 24 h after hCG injection. Both the in vitro-aged and in vivo-aged oocytes were stained and observed under a confocal microscope for CGs and chromosomes, or tested for ZP hardening. In all the freshly ovulated oocytes collected 12 h after hCG administration (Fig. 1A) and most (87.7%) of the oocytes aged in vitro without CCs (Fig. 1B), CGs were densely populated in a line just beneath the oolemma, with a typical normal CG-free domain. Metaphase chromosomes of these oocytes were viewed on edge (Fig. 1A) or in a polar orientation (Fig. 1B). However, many oocytes aged in vitro with CCs (63%) and those aged in vivo (57%) showed a partial CG release (i.e., a reduced density of CGs beneath the oolemma and a reduction or loss of the CG-free domain; Fig. 1, C and D; Ducibella et al. [21]). While the chromosomes of oocytes aged in vitro were still tightly aligned at the metaphase plate (Fig. 1C), a fraction (23%) of oocytes aged in oviducts showed an anaphase configuration with a separation of chromosomes and their CGs disappeared from most parts of the cortex (Fig. 1D). In summary, while many oocytes with a cumulus showed partial CG releases, very few cumulus-free oocytes released their CGs during in vitro aging, and while a proportion of oocytes aged in vivo for 12 h displayed an anaphase appearance, those aged in vitro did not (Table 3).



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  		FIG. 1. Confocal micrographs (equatorial sections through the spindle) of ovulated mouse oocytes showing changes of cortical granules (green) and chromosomes (red) during in vivo or in vitro aging. In freshly ovulated (12 h after hCG injection) oocytes (A) and most of the oocytes that aged in vitro for 12 h without CCs (B), CGs were densely populated in a line just beneath the oolemma, with typical normal CG-free domains, and metaphase chromosomes were viewed on edge (A) or in a polar orientation (B). Many oocytes aged in vitro with CCs for 12 h (C) showed a partial CG release (i.e., a reduced density of CGs beneath the oolemma and a loss of the CG-free domain, but their chromosomes were still tightly aligned at the metaphase plate). A fraction of oocytes aged in the oviducts for up to 24 h after hCG administration (D) showed an anaphase configuration with a separation of chromosomes and their CGs disappeared from most of the cortex. Bar = 20 µm


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  		TABLE 3. Changes of cortical granules (CGs) and chromosomes of ovu lated oocytes during in vivo or in vitro aging with (COC) or without cu mulus cells (DO) for up to 24 h post hCG

The result that many oocytes aged in vivo and in vitro with cumulus showed a partial CG release was further substantiated by the ZP hardening assay, which showed that the half-time (t50) for chymotrypsin-mediated dissolution of the ZP increased significantly in the ZP from oocytes aged in vivo or in vitro with cumulus for up to 24 h after hCG administration compared to the ZP from oocytes isolated at 12 h after hCG administration (Fig. 2). However, although oocytes aged in vitro without CCs showed little CG exocytosis, they displayed a transnormally long t50 of the ZP dissolution.



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  		FIG. 2. Changes in chymotrypsin digestion time of ZP (t50 is the time at which 50% of the ZPs per group were completely digested) of ovulated oocytes during in vivo or in vitro aging with (COC) or without CCs (DO) for up to 24 h after hCG administration. vvCOCvvA, in vivo-matured oocytes aged in vivo with cumulus; vvCOCvtA, in vivo-matured oocytes aged in vitro with cumulus; vvDOvtA, in vivo-matured oocytes aged in vitro without cumulus. Oocytes in the control group were recovered from oviducts 12 h after hCG injection. Means with different letters indicate statistically significant differences (P < 0.05)

Co-aging with COCs or Culture on CC Monolayers of Denuded In Vivo-Matured and In Vitro-Matured Oocytes

In this experiment, all the oocytes were collected from oviducts 12 h after hCG injection. Some of the oocytes were denuded of cumulus cells immediately after collection (i.e., DOs) and cultured in the same drop with oocytes with cumulus intact (i.e., COCs) or on a CC monolayer. At 6 h of culture (18 h after hCG injection), DOs (designated as DOs aged with COCs) and COCs (designated as COCs aged with DOs) were processed separately for activation treatment or H1 kinase assay. Oocytes for controls were freed of cumulus cells and activated or frozen for the H1 kinase assay immediately after collection from oviducts. While activation rates increased and MPF activity decreased significantly in the companion COCs, in comparison with controls, rates of DOs aged alone or with COCs at a 1:1 ratio did not change significantly (Table 4). However, activation rates increased while MPF activity decreased significantly in DOs that were aged on a CC monolayer or with COCs at a 1:2 ratio (Table 4).


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  		TABLE 4. Effects of co-culture with COCs or cumulus cell monolayers (CCM) on activation and MPF activity of denuded in vivo matured oocytes

The same experiment was then conducted on the in vitro-matured oocytes. In this experiment, all the oocytes were collected at 14 h of in vitro maturation. Some of the oocytes were denuded of cumulus cells immediately after collection (i.e., DOs) and aged in the same drop with oocytes with cumulus intact (i.e., COCs) or on a CC monolayer. At 12 h of aging (26 h after the onset of in vitro maturation), DOs (designated as DOs aged with COCs) and COCs (designated as COCs aged with DOs) were processed separately for activation treatment or H1 kinase assay. Oocytes for controls were freed of cumulus cells and activated or frozen for the H1 kinase assay immediately after collection. It was found that activation rates increased, whereas MPF activity decreased significantly in the companion COCs. However, activation rates of DOs co-aged with COCs or on a CC monolayer did not change significantly, although their MPF activity declined markedly, in comparison with those of the newly matured oocytes and the DOs aged alone (Table 5). That MPF activity of the in vitro-matured DOs co-aged with either COCs or the CC monolayer was significantly lower than that of the DOs aged alone suggested that CCs had caused a reduction in the MPF activity of these oocytes, although they failed to enable these oocytes to be activated.


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  		TABLE 5. Effects of co-culture with COCs or cumulus cell monolayer (CCM) on activation and MPF activity of the denuded in vitro matured oocytes


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Although early studies have shown that ovulated oocytes aged in vitro displayed similar morphological alterations as those aged in vivo [14, 28, 33], and that a spontaneous reduction in MPF activity [10, 11, 13] or cytoskeletal alteration [39] has been reported with in vitro aging of in vitro-matured oocytes, Abbott et al. [16] found that in vitro culture of mouse oocytes retarded spontaneous activation of cell cycle progression that normally occurred in in vivo unfertilized eggs [12]. The difference was that Abbott et al. [16] cultured ovulated mouse oocytes after denudation of CCs. We therefore proposed a hypothesis that CCs would accelerate the progression of in vitro aging of mouse oocytes. The work in this paper showed that CCs accelerated the spontaneous activation of both in vivo-matured and in vitro-matured mouse oocytes.

Our results showing that many oocytes aged in vivo and in vitro with a cumulus showed a partial CG release was further substantiated by the observation that the t50 for chymotrypsin-mediated ZP dissolution increased significantly in the ZP from oocytes aged in vivo or in vitro with cumulus for up to 24 h after hCG administration compared to the ZP from freshly ovulated oocytes. However, although oocytes aged in vitro without CCs showed little CG exocytosis, they displayed an unusually long t50 of ZP dissolution. Downs et al. [40] found that serum maintained the ability to fertilize mouse oocytes matured in vitro by preventing the hardening of the ZP. Ducibella et al. [41] observed that a reduction in the number of CGs was accompanied by a conversion of ZP2 to ZP2f when mouse oocytes were matured in vitro in serum-free medium; but in contrast, the loss of CGs of oocytes matured in vitro in the presence of serum is not accompanied by a modification of ZP2. Schroeder et al. [42] reported that fetuin, a component of fetal bovine serum, inhibited ZP hardening and conversion of ZP2 to ZP2f during mouse oocyte maturation in vitro in the absence of serum. Vanderhyden and Armstrong [43] showed that the presence of CCs and serum significantly increased the penetrability of rat oocytes by spermatozoa. Therefore, the present result suggested an inhibitory effect of both the serum and the CCs on the conversion of ZP2 to ZP2f during oocyte aging, because our oocytes were aged in the CZB medium that contained no serum.

In this study, when denuded ovulated oocytes were cocultured with COCs at a 1:2 ratio or especially on a CC monolayer, activation rates increased, while MPF activity decreased significantly. This indicated that the CCs accelerated oocyte aging not through their connections with the oocytes but rather by releasing one or more aging-promoting factors (APFs) into the culture medium. When denuded ovulated oocytes were aged in CZB medium conditioned for 6 h with ovulated COCs, they showed an increased activation rate (54.7%) that was not significantly different from that of oocytes aged with an intact cumulus (59.8%) after activation (our unpublished data), indicating that a soluble APF is responsible. There is evidence that factors secreted by CCs are very important in promoting oocyte maturation and acquisition of developmental competence. For example, maturation in media conditioned with CCs or COCs was found to improve oocyte maturation and developmental competence [44–47], and denuded oocytes have been observed to take up amino acids, sugars, and ribonucleosides [48–50]. However, the COC-derived factors that promote oocyte maturation are probably different from the APFs, because structural alterations of CCs, including nuclear pycnosis and cytoplasmic vacuolization, were apparent immediately after ovulation, and the number of degenerated CCs increased thereafter [28].

Besides, at 18 and 24 h after hCG administration, activation rates were markedly higher and MPF activity was lower in ovulated oocytes aged in vivo than in oocytes aged in vitro with CCs (Table 1). And while a proportion of oocytes aged in vivo for 12 h displayed an anaphase appearance, those aged in vitro for the same period did not (Table 3). Webb et al. [33] also noticed that the maximum percentages of activated oocytes were higher in mouse eggs aged in vivo than in eggs aged in vitro. This might suggest a role of the oviduct in the acceleration of oocyte aging. According to Longo [28], although about 30% of the cumulus masses examined contained cells that plated out when cultured and remained viable for up to 3 days in vitro, almost all CCs of COCs aged in vivo showed signs of degeneration 12–24 h postovulation.

Aging of in vitro-matured oocytes has been studied in pigs [10, 11, 39] and cattle [13], but not in mice. In this study, we showed that activation rates and MPF activity of in vitro-matured mouse oocytes changed during aging with or without CCs, similar to those of ovulated oocytes aged under the same conditions (compare Tables 1 and 2). However, as a whole, the activation rates of in vitro-matured oocytes were lower than those of in vivo-matured oocytes, whereas the MPF activity of the former was higher than that of the latter during in vitro aging. In other words, the lower activation rates of in vitro-matured oocytes were a result of their higher MPF activity, in comparison with those of ovulated oocytes. This further provided evidence for the hypothesis that maintenance of arrest in MII requires high levels of MPF activity, and entry into interphase requires a decrease in this activity below a critical threshold [12]. Besides, unlike the activation rates of denuded ovulated oocytes, the activation rates of denuded, in vitro-matured oocytes did not change significantly during co-aging with COCs or on CCM, although their MPF activity declined markedly (Tables 4 and 5). The inefficient aging-promoting effect of coculture on in vitro-matured oocytes could have resulted from a poor responsiveness of the denuded oocyte, or from a low metabolic activity of the CCs or a reduced metabolic coupling between the CCs and the oocyte in the companion COCs. Our recent unpublished work showed that the activation rate of in vitro-matured DOs was significantly higher when co-aged with ovulated COCs (47.7%), than with in vitro-matured COCs (5.1%). This suggested that it was not the in vitro-matured oocyte itself, but rather the physiological state of the CCs or the extent of metabolic coupling between the CCs and the oocyte in the companion in vitro-matured COCs, that affected the activation responsiveness.

During aging in vivo or in vitro, with or without CCs, activation rates of ovulated oocytes increased at first but stopped increasing, and began to decrease at 24 h after hCG administration, although the MPF activity continued to decline after this time (Table 1). Similar changes in activation and MPF activity were observed during the in vitro aging process of in vitro-matured oocytes with CCs (Table 2). In one study [33], activation rates of mouse oocytes declined markedly after extended periods of in vivo and in vitro aging. These observations might suggest that: 1) the decrease in MPF activity below a critical threshold is not the only requirement for oocyte activation, and 2) extended aging causes damage to the oocyte's component parts, such as deterioration of components of cytoskeleton [39, 51, 52] and initiation of apoptosis [26, 53], leading to its inability to respond to activating stimuli.


   FOOTNOTES
 
1 Supported by grant 30430530 from the China National Natural Science Foundation, and by the "973" Project of the China Science and Technology Ministry through grant G200016108. Back

2 Correspondence: Jinghe Tan, Laboratory for Animal Reproduction and Embryology, College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Tai-an City, Shandong Province, People's Republic of China 271018. FAX: 0538 8241419; tanjh{at}sdau.edu.cn Back

Received: 10 May 2005.

First decision: 23 May 2005.

Accepted: 23 June 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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