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Regular Article |
a Department of Animal Biology, University of Valencia, Burjassot, Valencia, Spain 46100
b Department of Pediatrics, Obstetrics and Gynecology, University of Valencia, Valencia, Spain 46010
ABSTRACT
The present study aims to shed light on the origin of abnormal oocytes ovulated by aged females. In order to reach this goal, cellular and morphological traits of ovulated oocytes from hybrid (C57Bl/6JIco female x CBA/JIco male) female mice retrieved after exogenous ovarian stimulation at the age of 12, 40–42, 50–52, or 57–62 wk were analyzed. Aging of female mice was associated with 1) decreased number of ovulated oocytes; 2) increased percentage of cumulus-free oocytes; 3) raised percentage of oocytes with intracellular mitochondrial aggregates; 4) reduced percentage of oocytes displaying a normal distribution of chromosomes in the metaphase-II plate; 5) increased percentage of normal oocytes exhibiting a DNA-containing polar body (PB); 6) higher percentage of oocytes with chromosome scattering; 7) increased percentage of chromosome-scattered oocytes without a DNA-containing PB and with intracytoplasmic mitochondrial aggregates; 8) raised percentage of oocytes exhibiting chromosome decondensation; 9) lower percentage of chromosome-decondensed oocytes lacking both a DNA-containing PB and intracytoplasmic mitochondrial aggregates; 10) increased percentage of abnormal/degenerated oocytes; 11) reduced percentage of abnormal/degenerated oocytes displaying cellular fragmentation; and 12) higher percentage of abnormal/degenerated oocytes with mitochondrial aggregates exhibiting no nuclear/chromosomal DNA fluorescence, cellular fragmentation, milky or dark cytoplasm, or cellular remains enclosed by the zona pellucida. Although several studies suggest aging females may ovulate aged or overripened oocytes, these data support the hypothesis that old females ovulate an increased percentage of atretic/apoptotic oocytes coming from rescued follicles that would have become atretic earlier in life.
aging, apoptosis, gamete biology, meiosis, ovulation
INTRODUCTION
It is well-known that female aging is associated with decreased viability of oocytes and preimplantation embryos. The negative effect of female aging can go even further not only raising the probability of obstetrics complications and/or fetal and perinatal problems but also increasing the risks of mortality and morbidity in newborns and later in life [1]. Although many hypotheses have been proposed [2, 3], little it is known about the real causes of the decreased viability of oocytes from reproductively aged females. It has been proposed that one potential cause of the high incidence of abnormal oocytes in reproductively old females is the production of aged or over-ripened oocytes due to an uncoupling of the normal developmental events leading to ovulation in aging females [2]. This hypothesis is supported by the fact that in vitro aging of mouse oocytes induces apoptotic cell death as evidenced by morphological traits such as cellular shrinkage and cellular fragmentation as well as the presence of caspase activity and the occurrence of DNA cleavage [4]. However, the increased percentage of nonviable oocytes shed by aged mice may also be explained by other mechanisms such as the ovulation of a higher percentage of atretic/apoptotic oocytes coming from rescued follicles that would typically have become atretic earlier in life [2].
In the present study, we aim to shed light on the origin of abnormal oocytes ovulated by aged females. In order to reach this goal, we have analyzed several cellular and morphological traits of oocytes from aging mice retrieved from oviducts after exogenous ovarian stimulation.
MATERIALS AND METHODS
Housing of Mice
F1 hybrid female mice (C57Bl/6JIco female x CBA/JIco male) (Criffa, Santa Perpetua de la Mogoda, Barcelona, Spain) were maintained on a 14L (0800–2200 h):10D photoperiod in a temperature-controlled room at 21–23°C. Females were fed a standard diet supplemented or not supplemented with vitamin C (Rovimix C-F; Roche, Neiully Sur Seine, France; 10 g/kg diet) and vitamin E (Rovimix E-505D; Roche; 0.6 g/kg diet) either from weaning or from the age of 32 wk until they were killed at the age of 12, 40–42, 50–52, or 57–62 wk of age. Female mice were marked by ear punching at weaning and housed in groups of 10 in 35.5 x 23.5 x 18.5-cm plastic cages with wood shavings and a wire mesh top. During the last 3 wk prior to exogenous ovarian stimulation, the phase of the estrous cycle was determined by examining the color, moistness, and degree of swelling of the vagina [5].
Oocyte Collection
Females were superovulated by i.p. injection of 10 IU eCG (Folligon; Intervet-International, Boxmeer, Holland) followed 48 h later by 10 IU of hCG (Chorulon; Intervet-International). In each experiment, a control and an experimental female were killed consecutively. The timing of eCG and hCG administration was scheduled at 1730 and 1800 h so as to control the time of ovulation precisely and prevent spontaneous release of endogenous LH [6]. Females were killed at 14 h after hCG injection. Oviducts were excised and placed into 2 ml of medium M2 [7] supplemented with 4 mg BSA/ml (BSA fraction V; Sigma Chemical Co., St. Louis, MO). Ovulated cumulus-enclosed and cumulus-free oocytes were released from the ampullae. Mucous cumulus cells were removed by gentle agitation in M2 supplemented with 80 IU hyaluronidase (Sigma). After removing cumulus cells, oocytes were washed several times and left for 30 min in 20-µl droplets of medium M16 [8] supplemented with 4 mg BSA/ml overlaid with mineral oil (light white oil; Sigma) at 37°C in a humidified atmosphere of 5% CO2 in air. After the 30-min culture period, oocytes were fixed for 30 min in 3.7% formaldehyde in microtubule stabilization buffer (80 mM potassium PIPES, 5 mM EGTA, 1 mM MgCl2, 0.2% Triton X-100, and 0.5 µM taxol, pH 6.8) [9]. Thereafter, oocytes were washed and stored at 4°C in medium M2. All the animal experiments performed were conducted in accordance with the National Research Council (NRC) publication Guide for the Care and Use of Laboratory Animals (1996).
Analysis of Chromosomal Distribution, DNA Organization, and Cellular Morphology
The quality of oocytes was determined by examining both 1) the morphological appearance of oocytes immediately after retrieval under a stereomicroscope and 2) the distribution of chromosomes in the metaphase-II (MII) spindle and degree of chromosomal/DNA decondensation by fluorescence microscopy after a 5-min exposure of fixed oocytes to the DNA probe 150 µM 4’-6-diamino-2-phenylindole (DAPI; Sigma) in medium M2. Morphological anomalies were classified into several groups including cellular fragmentation, milky or dark appearance of the cytoplasm, presence of cellular remains enclosed by the zona pellucida, and completely empty zonae pellucidae. Anomalies in chromosomal distribution and DNA decondensation were classified as follows: 1) scattering of chromosomes if one or more individual chromosomes were separated from the chromosomal plate by a distance greater than or equal to the chromosomal length (Fig. 1B); 2) chromosomes showing varying degrees of DNA decondensation ranging from the presence of a disorganized or irregular chromosomal plate with a slight or negligible decondensation of chromosomes (Fig. 1C) to the formation of one or more chromatin masses (restitution nuclei) in which individual chromosomes were not discernible (Fig. 1E) [10–12]. Intermediate stages exhibited decondensed chromosomes scattered throughout the cytoplasm (Fig. 1D); and 3) immature oocytes with chromosomes in anaphase-I (Fig. 1F) or telophase-I (Fig. 1G) configuration. In addition to this chromosomal/DNA analysis, DAPI staining of oocytes allowed us to 1) identify degenerated oocytes displaying no intracytoplasmic nuclear/chromosomal DNA fluorescence (Fig. 1H); 2) ascertain whether mitochondria were homogeneously (Fig. 1, A and G) or heterogeneously (presence of intracellular mitochondrial aggregates) (Fig. 1, B–F and H) distributed throughout the cytoplasm of morphologically normal oocytes as well as throughout the cytoplasm of morphologically abnormal oocytes exhibiting cellular fragmentation (Fig. 1I), milky (Fig. 1J) or dark (Fig. 1K) appearance of the cytoplasm or cellular remains enclosed by the zona pellucida (Fig. 1L); and 3) determine whether oocytes were carrying a DNA-containing polar body (PB). The absence of a DNA-containing PB was coincident with the absence of any PB-like cellular mass within the perivitelline space. In order to check and confirm that the intracytoplasmic organelle aggregates evidenced by DAPI staining corresponded to mitochondrial complexes, ovulated oocytes coming from 20 different females were stained for 30 min with the mitochondrial probe 500 nM MitoTracker Red CMXRos (Molecular Probes, Inc., Eugene, OR) in medium M2. Oocytes were, thereafter, fixed for 30 min in 3.7% formaldehyde in microtubule stabilization buffer and stained with DAPI before being observed under the fluorescence microscope. As shown in Figure 2, both probes stained the same intracellular organelle aggregates. Oocytes analyzed in this study were used in another two studies aimed to ascertain whether 1) oral antioxidants counteract the negative effects of female aging on oocyte quantity and quality and 2) the stage of the estrous cycle at the time of eCG injection affects the quality of ovulated oocytes (unpublished data).
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Statistical Analysis
Univariate and multivariate ANOVA and repeated-measures design of multivariate ANOVA were applied for comparisons of means. A Kolmogorov-Smirnov one-sample test was used to check whether variables were normally distributed. If the normality assumption was violated, square root (if data were counts) or logit (if data were percentages) transformation of the variable was applied to induce normality. When the multivariate ANOVA test was applied, Box M-test of the homogeneity of the covariance matrices was used to check whether the dependent variables had the same variance-covariance matrix in each level of the between-subjects factors. Because there is no reason to use the multivariate ANOVA procedure if the dependent variables are not correlated, Bartlett test of sphericity was applied to test the hypothesis that the population correlation matrix was an identity matrix. A Bonferroni test (when the variances were assumed to be equal) and Dunnett T3 test (when the variances were assumed to be unequal) were applied to perform posthoc pairwise multiple comparisons within the four age groups. A Levene test was used to test the homogeneity of variance for each dependent variable across all level combinations of the between-subjects factors. Simple linear regression models were used to detect linear relationships between variables. Values shown in the text and tables are means ± SEM. Significance was defined as P 
0.05. The entire statistical analysis was carried out using the Statistical Package for Social Sciences (SPSS Inc., Chicago, IL).
RESULTS
Diet, interaction between diet and female age, and interaction between diet and onset of oral antioxidant administration did not have any significant effect on length or regularity of the estrous cycle, cellular morphology, chromosomal distribution, and presence or absence of both/either intracytoplasmic mitochondrial aggregates and/or a DNA-containing PB. For this reason, data from females fed the control and the antioxidant diet were grouped and analyzed together.
Estrous Cycle
Both the number of 4- or 5-day estrous cycles and the number of cycles longer than 5 days during the last 3 wk prior to eCG injection were not affected by female age (number of 4- or 5-day estrous cycles: 2.5 ± 0.3, 2.6 ± 2.5, 2.4 ± 0.2, and 2.7 ± 0.2; number of cycles longer than 5 days: 0.5 ± 0.13, 0.4 ± 0.06, 0.4 ± 0.06, and 0.3 ± 0.06 in females of 12, 40–42, 50–52, and 57–62 wk of age, respectively). Very few animals were acyclical (1/26, 3.8% in females 12 wk of age and 2/62, 3.2% in females 57–62 wk of age). However, the number of 2- or 3-day estrous cycles was significantly (P 0.012) lower in 57–60-wk-old females (0.14 ± 0.05) when compared to females of 40 wk of age (0.57 ± 0.12).
Types of Oocytes Recovered from Oviducts
Table 1 shows the effect of female aging on the presence or the absence of either a DNA-containing PB and/or intracytoplasmic mitochondrial aggregates in oocytes surrounded by or free of cumulus cells (number of empty zona pellucidae was not taken into account to calculate percentages). Absolute number of ovulated oocytes surrounded by (P 0.0005) or free of cumulus cells (P 0.0005) decreased as the age of females increased from 12 to 40–42, 50–52, and 57–62 wk of age. Moreover, the percentage of cumulus-free oocytes increased with female age (P 0.0005). The number of 4- or 5-day estrous cycles during the last 3 wk prior to eCG injection (covariate) affected (P 0.001) both the absolute number (slope: -0.438 ± 0.132; P 0.001) and percentage (slope: -0.635 ± 0.243; P 0.010) of cumulus-free oocytes.
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The subpopulation of cumulus-free oocytes exhibited a higher percentage of oocytes with mitochondrial aggregates (86.4 ± 1.9% vs. 8.6 ± 1.3%) and percentage of oocytes without a DNA-containing PB (81.8 ± 17% vs. 44.8 ± 2.0%), when compared to the subpopulation of cumulus-enclosed oocytes. No intracytoplasmic mitochondrial aggregates were observed in any of the three telophase-I oocytes retrieved from oviducts. In contrast, the single anaphase-I oocyte observed in this study exhibited clusters of mitochondria throughout the cytoplasm (Fig. 1F).
Female age had no effect on percentage of ovulated oocytes without a DNA-containing PB (56.0 ± 2.5%, 60.7 ± 2.6%, 56.1 ± 2.7%, and 64.3 ± 2.9% in females of 12, 40–42, 50–52, and 57–62 wk of age, respectively). However, the percentage of oocytes (with or without a DNA-containing PB) with intracellular mitochondrial aggregates increased with female age (oocytes with a PB: 1.7 ± 0.5%, 4.7 ± 1.0%, 13.0 ± 2.5%, and 12.7 ± 2.5%, P 0.0005; oocytes without a PB: 5.5 ± 1.4%, 32.3 ± 3.6%, 37.7 ± 2.8%, and 44.9 ± 3.1%, P 0.0005, in females of 12, 40–42, 50–52, and 57–62 wk of age, respectively). By contrast, the percentage of oocytes (with or without a DNA-containing PB) without intracytoplasmic mitochondrial aggregates decreased with female age (oocytes with a PB: 42.3 ± 2.6%, 34.7 ± 2.8%, 30.9 ± 2.8%, and 22.9 ± 2.6%, P 0.001; oocytes without a PB: 50.5 ± 2.4%, 28.3 ± 2.5%, 18.4 ± 2.1, and 19.4 ± 2.6%, P 0.0005, in females of 12, 40–42, 50–52, and 57–62 wk of age, respectively). The covariate number of 4- or 5-day estrous cycles during the last 3 wk prior to eCG injection was also significantly (P 0.001) associated with three of the four types of oocytes recovered from oviducts (oocytes without mitochondrial aggregates and with a PB: slope 0.353 ± 0.138, P 0.011; oocytes without mitochondrial aggregates and without a PB: slope 0.416 ± 0.152, P 0.007; and oocytes with mitochondrial aggregates and without a PB: slope -0.553 ± 0.209, P 0.009).
Oocytes Displaying a Normal Distribution of Chromosomes in the MII Plate
Table 2 shows the effect of female aging and the presence or the absence of a DNA-containing PB on oocytes exhibiting a normal distribution of chromosomes in the MII plate. The majority of normal oocytes came from the subpopulation of ovulated oocytes enclosed by cumulus cells (1956 out of 1974 normal oocytes). Normal oocytes were characterized by the fact that no mitochondrial aggregates were observed inside their cytoplasm. Moreover, total (cumulus-enclosed plus cumulus-free oocytes) percentage of normal oocytes with a DNA-containing PB was significantly higher than total percentage of normal oocytes lacking in a DNA-containing PB (60.3 ± 1.9% vs. 39.7 ± 1.9%, P 0.0005).
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Aging of females was associated with decreased total (cumulus-enclosed plus cumulus-free oocytes with/without a DNA-containing PB) percentage of normal oocytes (87.6 ± 2.1%, 55.7 ± 4.0%, 47.0 ± 3.6%, and 33.1 ± 3.1% in females of 12, 40–42, 50–52, and 57–62 wk of age, respectively, P 0.0005). In contrast, the total percentage of normal oocytes exhibiting a DNA-containing PB increased with female age, at least until the age of 50–52 wk (47.3 ± 2.6%, 58.6 ± 2.8%, 65.5 ± 3.4%, and 62.6 ± 4.5% in females of 12, 40–42, 50–52, and 57–62 wk of age, respectively, P 0.019). The number of 4- or 5-day estrous cycles during the last 3 wk prior to eCG injection was a significant (P 0.001) covariate only when used to correct the effect of female aging on total (cumulus-enclosed plus cumulus-free oocytes with/without a DNA-containing PB) percentage of normal oocytes (slope: 0.580 ± 0.233; P 0.014).
Oocytes with Chromosome Scattering
Table 3 shows the effect of female aging and the presence or the absence of either a DNA-containing PB and/or intracytoplasmic mitochondrial aggregates on percentage of oocytes displaying chromosome scattering. The majority of oocytes with chromosome scattering came from the subpopulation of ovulated oocytes free of cumulus cells (261 out of 305 oocytes displaying this anomaly). In addition, this anomaly was significantly (P 0.0005) associated with the absence of a DNA-containing PB and, in particular, with the absence of a DNA-containing PB and the presence of intracytoplasmic mitochondrial aggregates (240 out of the 305 oocytes displaying this anomaly). This trend was observed in both the subpopulation of cumulus-enclosed oocytes and the subpopulation of cumulus-free oocytes.
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Aging of females from 12 to 40–42 wk of age was associated with a significant increase in total (cumulus-enclosed plus cumulus-free oocytes) percentage of oocytes with chromosome scattering, although no further increase was observed from 40 to 42 wk to 57 to 62 wk of age (4.4 ± 1.4%, 14.8 ± 2.3%, 10.3 ± 1.8%, and 12.1 ± 2.0% in females of 12, 40–42, 50–52, and 57–62 wk of age, respectively, P 0.0005). Taking into account the four types of oocytes with chromosome scattering established according to the presence or the absence of both/either a DNA-containing PB and/or intracytoplasmic mitochondrial aggregates, 12-wk-old females showed decreased percentage of PB-less oocytes with intracytoplasmic mitochondrial aggregates when compared to females from the other three groups of age (44.9 ± 12.1%, 78.0 ± 6.1%, 93.3 ± 4.6%, and 81.0 ± 6.1%, P 0.001, in females of 12, 40–42, 50–52, and 57–62 wk of age, respectively). In contrast, 12-wk-old females exhibited a higher percentage of PB-less oocytes lacking in intracytoplasmic mitochondrial aggregates than females from the other three groups of age (52.6 ± 11.6%, 19.1 ± 5.6%, 0.0 ± 0.0%, and 10.6 ± 4.8%, P 0.0005, in females of 12, 40–42, 50–52, and 57–62 wk of age, respectively). Number of 4- or 5-day estrous cycles during the last 3 wk prior to eCG injection had no significant effect on total percentage of oocytes with chromosome scattering and percentage of chromosome-scattered oocytes with/without a DNA-containing PB and with/without intracytoplasmic mitochondrial aggregates.
Oocytes with Chromosome Decondensation
Table 4 shows the effect of female aging and the presence or the absence of both/either a DNA-containing PB and/or intracytoplasmic mitochondrial aggregates on percentage of oocytes exhibiting chromosome decondensation. The majority of oocytes with chromosome decondensation came from the subpopulation of ovulated oocytes free of cumulus cells (447 out of 519 oocytes displaying this anomaly). Oocytes with chromosome decondensation were characterized by the fact that most of them showed intracytoplasmic mitochondrial aggregates irrespective of the presence or the absence of a DNA-containing PB (466 out of 519 oocytes displaying this anomaly), although the vast majority of them were oocytes lacking in a DNA-containing PB (344 PB-less oocytes vs. 122 oocytes with a DNA-containing PB). Both the subpopulation of cumulus-enclosed oocytes and the subpopulation of cumulus-free oocytes showed the same pattern.
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Total percentage of oocytes (cumulus-enclosed oocytes plus cumulus-free oocytes) exhibiting chromosome decondensation increased as the age of females increased from 12 to 40–42, 50–52, and 57–62 wk (5.0 ± 1.0%, 18.4 ± 2.2%, 20.7 ± 2.6%, and 25.9 ± 2.8%, respectively, P 0.0005). Furthermore, the covariate number of 4- or 5-day estrous cycles during the last 3 wk prior to eCG injection had a significant (P 0.008) effect on total percentage of oocytes with chromosome decondensation (slope: -0.400 ± 0.184; P 0.031). The presence or the absence of a DNA-containing PB and/or intracytoplasmic mitochondrial aggregates in oocytes with chromosome decondensation was also affected by female aging. In particular, 12-wk-old females ovulated a higher percentage of chromosome-decondensed oocytes lacking in both a DNA-containing PB and intracytoplasmic mitochondrial aggregates when compared to the other three groups of age (25.9 ± 9.6%, 8.3 ± 3.5%, 0.2 ± 0.2%, and 7.1 ± 2.5% in females of 12, 40–42, 50–52, and 57–62 wk of age, respectively, P 0.002).
Morphologically Abnormal Oocytes and/or Degenerated Oocytes
Table 5 shows the effect of female aging on distribution of different types of morphologically abnormal oocytes and/or degenerated oocytes retrieved from oviducts after exogenous ovarian stimulation. Like oocytes displaying chromosome scattering or chromosome decondensation, the majority of abnormal/degenerated oocytes came from the subpopulation of ovulated oocytes free of cumulus cells (347 out of 447 oocytes displaying this anomaly). Furthermore, 80.8 ± 4.8% (68 out of 91) of oocytes without nuclear/chromosomal DNA fluorescence, 89.3 ± 3.1% (134 out of 146) of oocytes with cellular fragmentation, 86.4 ± 4.5% (100 out of 110) of oocytes with a milky or dark cytoplasm, and 85.9 ± 5.1% (49 out of 58) of oocytes with cellular remains enclosed by the zona pellucida exhibited intracytoplasmic mitochondrial aggregates.
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Total (cumulus-enclosed plus cumulus-free oocytes) percentage of abnormal/degenerated oocytes exhibited a concomitant increase with female age (2.7 ± 0.8%, 11.1 ± 1.8%, 21.9 ± 2.6%, and 28.9 ± 3.0% in females of 12, 40–42, 50–52, and 57–62 wk of age, respectively, P 0.0005). However, percentage of abnormal/degenerated oocytes displaying cellular fragmentation decreased as the age of females increased from 12 to 57–62 wk (69.4 ± 11.8%, 50.6 ± 6.2%, 29.8 ± 4.9%, and 17.0 ± 3.5% in females of 12, 40–42, 50–52, and 57–62 wk of age, respectively, P 0.0005). Percentage of abnormal/degenerated oocytes with mitochondrial aggregates exhibiting no nuclear/chromosomal DNA fluorescence, cellular fragmentation, milky or dark cytoplasm, or cellular remains enclosed by the zona pellucida increased as females aged from 12 wk to 40–42 wk of age (oocytes without nuclear/chromosomal DNA fluorescence: 30.0 ± 12.3%, 59.1 ± 14.8%, 92.5 ± 5.5%, and 91.7 ± 5.8%, P 0.001; oocytes with cellular fragmentation: 55.0 ± 15.7%, 90.3 ± 4.9%, 100.0 ± 0.0, and 91.3 ± 6.0%, P 0.001; cellular remains enclosed by the zona pellucida: 0.0 ± 0.0%, 72.7 ± 14.1%, 94.7 ± 5.3%, and 86.2 ± 6.5%, P 0.023; and oocytes with a milky or dark cytoplasm: 0.0 ± 0.0%, 90.0 ± 10.0%, 83.3 ± 11.2%, and 85.4 ± 7.0%, P 0.273, in females of 12, 40–42, 50–52, and 57–62 wk of age, respectively). Number of 4- or 5-day estrous cycles during the last 3 wk prior to eCG injection was not a significant covariate in any of the analyses performed with abnormal/degenerated oocytes.
DISCUSSION
The present study shows that aging of female mice is associated with 1) decreased number of ovulated oocytes retrieved from oviducts after exogenous ovarian stimulation; 2) increased percentage of ovulated cumulus-free oocytes; 3) raised percentage of oocytes with intracellular mitochondrial aggregates; 4) reduced percentage of oocytes displaying a normal distribution of chromosomes in the MII plate; 5) increased percentage of normal oocytes exhibiting a DNA-containing PB; 6) higher percentage of oocytes with chromosome scattering; 7) increased percentage of chromosome-scattered oocytes without a DNA-containing PB and with intracytoplasmic mitochondrial aggregates; 8) raised percentage of oocytes exhibiting chromosome decondensation; 9) lower percentage of chromosome-decondensed oocytes lacking in both a DNA-containing PB and intracytoplasmic mitochondrial aggregates; 10) increased percentage of abnormal/degenerated oocytes; 11) reduced percentage of abnormal/degenerated oocytes displaying cellular fragmentation; and 12) higher percentage of abnormal/degenerated oocytes with mitochondrial aggregates exhibiting no nuclear/chromosomal DNA fluorescence, cellular fragmentation, milky or dark cytoplasm, or cellular remains enclosed by the zona pellucida.
These results are in agreement with previous studies showing an age-associated decrease in number of oocytes shed in response to eCG and hCG injections as well as an age-associated increase in incidence of oocytes displaying c-meiosis (colchicine-meiosis: anomalies in chromosomal distribution similar to those induced by treatment with colchicine) and morphological abnormalities [2, 3]. It is important to note that the negative effect of female aging on absolute number and percentage of ovulated oocytes free of cumulus cells, percentage of oocytes with intracellular mitochondrial aggregates, percentage of oocytes with a normal distribution of chromosomes in the MII plate, and percentage of oocytes with chromosome decondensation was significant after correcting for the covariate number of 4- or 5-day estrous cycles during the last 3 wk prior to eCG injection. In other words, female aging was an independent factor that inflicted by itself a negative effect on oocyte viability.
As mentioned in the Introduction, one potential cause of the high incidence of abnormal oocytes ovulated by reproductively old females is the production of aged or over-ripened oocytes due to an uncoupling of the normal developmental events leading to ovulation [2]. According to vom Saal et al. [2], oocytes may age in the ovary as a consequence of a prolonged preovulatory phase during the cycle or a prolonged interval between the LH surge and ovulation. The present study shows that percentage of oocytes with intracellular mitochondrial aggregates increased with female age. Clustering of mitochondria was, in turn, associated with several cellular and morphological abnormalities of oocytes including chromosome scattering, chromosome decondensation, cellular fragmentation, milky or dark cytoplasm, absence of nuclear/chromosomal DNA fluorescence, and presence of cellular remains enclosed by the zona pellucida. The aggregation of mitochondria in clusters of varying sizes appears to be a trait related to oocyte aging. In fact, the percentage of ovulated oocytes displaying intracytoplasmic Mito-Tracker-red-CMXRos-positive patches increased from 0% to 77% after an in vitro ageing period of 1 to 13–48 h, respectively (unpublished observation). But given that in the present study we did not find significant differences between females of 12, 40–42, 50–52, and 57–60 wk of age in number of estrous cycles longer than 5 days during the last 3 wk prior to eCG and females were induced to ovulate by injection of exogenous hCG rather than by the endogenous LH-surge, neither a prolonged preovulatory phase during the cycle nor a prolonged interval between the LH surge and ovulation can explain the present data.
Aging females may, however, ovulate aged or over-ripened oocytes by other mechanisms including age-associated increases in levels of LH during the follicular phase that may induce a premature resumption of meiosis [13] and age-induced alterations in cell-cycle progression that may cause an asynchrony in ovulation relative to maturation kinetics, i.e., the occurrence of accelerated maturation associated with normal ovulation timing [14]. Although these mechanisms may explain the ovulation of over-ripened oocytes, the fact that we did not observe an age-associated increase in percentage of ovulated MII oocytes lacking in a DNA-containing PB (indirect evidence of the occurrence of a premature resumption of meiosis or accelerated maturation) would provide evidence against these possibilities.
An alternative hypothesis is based on the ovulation of an increased percentage of oocytes coming from rescued follicles that would typically have become atretic earlier in life [2]. These rescued follicles may ovulate nonviable oocytes showing clear signs of atresia/apoptosis related with events occurring when residing within follicles including organelle aggregation, loss of contact of cumulus cells with the oocyte, degeneration of the entire granulosa wall leaving the oocyte completely denuded of cumulus cells, shrinkage of the oocyte accompanied or followed by pseudomaturation that includes germinal vesicle breakdown, and likely expulsion of the first PB [15]. Data from the present study are compatible with this hypothesis. First, percentage of cumulus-free oocytes increased with female age. Second, most of the cumulus-free oocytes exhibited mitochondrial aggregates. Third, all cumulus-free oocytes showed cytoplasmic condensation, i.e., retraction of the plasma membrane from the zona pellucida. Finally, most of cumulus-free oocytes were lacking in a DNA-containing PB that may be explained by the occurrence of the process of pseudomaturation before the injection of exogenous hCG. Furthermore, we should bear in mind that the majority of abnormal/degenerated oocytes and oocytes with chromosome scattering and chromosome decondensation came from the subpopulation of ovulated oocytes free of cumulus cells.
In conclusion, the present study shows that female aging is associated with the presence of an increased percentage of ovulated oocytes denuded of cumulus cells and a higher percentage of oocytes displaying intracellular mitochondrial aggregates. Although several studies suggest that aging females may ovulate aged or over-ripened oocytes due to an uncoupling of the normal developmental events leading to ovulation, data from this study support the hypothesis that old females ovulate an increased percentage of atretic/apoptotic oocytes coming from rescued follicles that would have become atretic and therefore would not have been ovulated earlier in life.
FOOTNOTES
First decision: 25 January 2001.
1 This work was supported by grant GV99-138-1-04 from Conselleria de Cultura, Educació i Ciència, Generalitat Valenciana, grant 1FD97-1035-C02-01 from CICYT (Ministry of Education and Culture, Madrid, Spain) and European Union; and grant FIS 00/0668 from Instituto de Salud Carlos III, Fondo de Investigación Sanitaria, Ministerio de Sanidad y Consumo. 
2 Correspondence: Juan J. Tarín, Department of Pediatrics, Obstetrics and Gynecology, Faculty of Medicine, University of Valencia, Avda. Blasco Ibañez 17, 46010 Valencia, Spain. FAX: 34 96 386 4815; tarinjj{at}uv.es
Accepted: February 16, 2001.
Received: December 13, 2000.
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W. Ma, D. Zhang, Y. Hou, Y.-H. Li, Q.-Y. Sun, X.-F. Sun, and W.-H. Wang Reduced Expression of MAD2, BCL2, and MAP Kinase Activity in Pig Oocytes after In Vitro Aging Are Associated with Defects in Sister Chromatid Segregation During Meiosis II and Embryo Fragmentation After Activation Biol Reprod, February 1, 2005; 72(2): 373 - 383. [Abstract] [Full Text] [PDF]
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 T. Hamatani, G. Falco, M. G. Carter, H. Akutsu, C. A. Stagg, A. A. Sharov, D. B. Dudekula, V. VanBuren, and M. S.H. Ko Age-associated alteration of gene expression patterns in mouse oocytes Hum. Mol. Genet., October 1, 2004; 13(19): 2263 - 2278. [Abstract] [Full Text] [PDF]
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 J. J. Tarin, V. Gomez-Piquer, C. Manzanedo, J. Minarro, C. Hermenegildo, and A. Cano Long-term effects of delayed motherhood in mice on postnatal development and behavioural traits of offspring Hum. Reprod., August 1, 2003; 18(8): 1580 - 1587. [Abstract] [Full Text] [PDF]
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