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Regular Article |
a Section of Pathology and Veterinary Clinics, University of Trás-os-Montes and Alto-Douro, 5000 Vila Real, Portugal
b Laboratory of Histology and Embryology and Center of Marine and Environmental Research and
c Laboratory of Cell Biology, Institute of Biomedical Sciences Abel Salazar, University of Porto, 4099-003 Porto, Portugal
ABSTRACT
Bovine ovarian cumulus-oocyte complexes (COCs) are used for in vitro maturation and fertilization after selection by size and morphology, but their developmental potential remains low. Stereology could provide more objective criteria for selecting the most competent complexes, but its application is lacking in cattle. COCs from small (1–4 mm) antral follicles were aspirated from diestrous ovaries of Holstein-Friesian cows, fixed in glutaraldehyde, randomly embedded in glycol-methacrylate, and sectioned at 20 µm. The unbiased nucleator principle was used for estimating the mean volumes of complexes, oocytes, cumulus cells, and nuclei of oocytes and cumulus cells. The thickness of the zona pellucida and the relative numerical percentages of the several morphologic types of cumulus cells were also evaluated. The optical disector procedure was used for cumulus cell sampling. Volume estimation based on a real physical unique point did not differ from those based on a particular point among many or on a virtual central point, and the mean cumulus cell volume was estimated by using the single section bearing the unique reference point. Quantitative data showed that COCs appear heterogeneous for all studied parameters and that the cumulus mass contains three different cell populations.
cumulus cells, follicle, follicular development, gametogenesis, oocyte development, ovary, ovulatory cycle, ovum
INTRODUCTION
Bovine follicles aspirated from slaughterhouse ovaries are used for different research lines, including regulation of granulosa cell proliferation and steroidogenesis, evaluation of factors influencing in vitro maturation and fertilization of oocytes, and the production of calves from cows of major genetic value. The in vitro production of bovine embryos by means of maturation and fertilization generally plateaus at a blastocyst rate of about 30% [1]. This rather limited success may be at least partially attributed to the methods of selection of the cumulus-oocyte complexes (COCs). These methods are usually based on parameters such as the morphology of the cumulus, the combined morphology of the cumulus and of the ooplasm, the size of the follicle and of the oocyte, and the level of follicle atresia.
Few studies have so far evaluated the impact of the cumulus morphology on subsequent development [2–12]. The thickness of the cumulus varies in accordance to follicle size, and cumulus integrity has been related to the health status of the follicles [2]; complexes with a thicker cumulus also have higher developmental rates [6, 8–10]. However, some oocytes from large follicles failed to produce embryos, whereas some oocytes from medium-size follicles succeeded [11]. In this respect, COCs with a compact multilayered cumulus and a homogeneous ooplasm have a higher developmental ability than do dark COCs with an expanded cumulus and irregular ooplasm [4, 12]. Blondin and Sirard [7] observed a correlation between the atretic aspect of the cumulus and the atresia of the follicle of origin, with very compact cumulus originating from healthier follicles and cumulus with visible degenerative changes coming from atretic follicles. However, competent oocytes may originate from early atretic follicles, and many healthy follicles contain incompetent oocytes [7]. In addition, there seems to be a relationship between oocyte diameter and developmental competence; bovine oocytes are considered to be in growth phase up to 110 µm in diameter, to acquire full meiotic competence at 115 µm, and to have full developmental competence at 120 µm [13, 14].
Because of these difficulties in the classification of bovine COCs, a quantitative morphologic analysis could provide a more accurate approach. Stereologic methods are tools for obtaining quantitative information about three-dimensional structures, mainly based on two-dimensional data contained in sections. In the present context, stereology also can be considered a gold standard against which both the often used qualitative and the nonstereologic semiquantitative or quantitative approaches for COC selection could be compared and even validated. One major breakthrough in stereology was the publication of the disector principle [15]. Other subsequent major theoretical advances allowed the replacement of assumption-based (biased) methods with design-based (unbiased) approaches. Whereas the previous techniques for estimating particle size and number relied on assumptions based on the shape and orientation of particles, the more recent methods are not based on those parameters and allow a priori unbiased estimates without the need of validation studies [16–18]. However, the application of unbiased stereologic tools has been limited to a few studies of nonbovine mammalian follicles [19–23].
The aim of the present work was to use the unbiased stereologic approach to evaluate Bos taurus COCs with a compact and complete cumulus mass and noniform ooplasm that were collected during the diestrous phase of the cycle. The study was based on two unbiased stereologic principles, the nucleator [24] and the disector [15], and allowed quantitative characterization of all COC substructures. The volumes of the complexes, oocytes, cumulus cells, and their nuclei and the thickness of the zona pellucida were estimated, and the local stereologic estimators were used to make line plots of size distributions. Cumulus homogeneity was further evaluated in terms of the relative number of cumulus cell populations around the oocyte.
MATERIALS AND METHODS
Collection of COCs
Ovaries that were showing signs of diestrous were collected from eight Holstein-Friesian cyclic cows at a local slaughterhouse. The identification of the stage of the estrous cycle was based on the appearance of the corpus luteum (color, size, and vasculature) [25]. In the selected ovaries, 1) a point of the follicle was covered over and the apex of the bisected corpus luteum was brown, orange, or yellow; 2) the corpus luteum had a diameter of 1.6–2 cm; and 3) the vasculature was visible or even prominent on the surface of the corpus luteum.
Ovaries were transported to the laboratory within 1.5–3 h at ambient temperature in sterile PBS with 1% (v/v) penicillin/streptomycin (Sigma, Barcelone, Spain). After washing in PBS, small antral follicles (1–4 mm at the ovary surface) were aspirated with a 5-ml syringe using a 19-gauge needle. Follicular contents were transferred to Petri dishes and observed under a stereomicroscope at 38°C. Twenty-four COCs, with a compact and complete cumulus mass and with a uniform or a nonvisible ooplasm, were selected. These COCs corresponded to category 1 as defined by several authors [2, 7, 12, 26–29] and to types I and II of Hasler et al. [30]. COCs were then washed twice in PBS containing 0.5–1% (v/v) penicillin/streptomycin and 2.5% (v/v) heat-treated newborn calf serum (Sigma).
Processing of COCs
COCs were fixed with 3% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, for 2 h at 4°C [31]. After washing in buffer for 3 h, COCs were dehydrated (2 h each in 70°C and 96°C ethanol, 12 h in 100°C ethanol) and infiltrated for 12 h in a 1:1 mixture of ethanol:glycol methacrylate (GMA; Historesin, Leica, Germany) followed by infiltration in pure GMA for 4 days at 4°C. For embedment, each COC was placed in a GMA-filled plastic mold (Leica) and continuously rotated with dissection needles until it finally landed in a random position at the bottom. This procedure allowed the generation of randomly oriented sections that can be regarded, for practical purposes, as isotropic uniform random planes, with no significant chance of bias [32, 33]. Considering the nearly spherical shape of cumulus cells, oocytes, and COCs, is it unlikely that the isotropy requirement of the nucleator was compromised.
COCs were completely sliced with a rotary microtome (RM 2155; Leica) set at the nominal thickness of 20 µm and equipped with a tungsten carbide type-D knife (Leica). Sections were collected into distilled water at 48°C, spread with needles, transferred to slides coated with poly-L-lysine (Sigma) by immersion contact, and air dried. On average, a total number of 12 sections were obtained per COC. These sections were stained for 5 min with a 2:1 mixture of aqueous 1% methylene blue:azure II (Sigma).
Depending on the target parameter, sections were analyzed under a 100x (N.A. 1.35), 20x, or a 10x objective, calibrated with a stage micrometer. The light microscope (BX50; Olympus, Tokyo, Japan) was coupled to a Sony CCD camera, and z-axis movement was monitored with a 0.5-µm-resolution microcator (Heidenhain, Traunreut, Germany). Both peripherals were connected to a computer running the Olympus-Denmark stereologica analysis software Grid (version 1.09).
Morphometric Determination of the Thickness of the Zona Pellucida
The thickness of the zona pellucida (ZP) was estimated by direct linear measurements of a section judged perpendicular to a tangent plane at the surface of the ZP. In this section, two sets of measurements were made in diametrically opposite zones (Fig. 1). Although this procedure is not strictly unbiased, it is sufficiently accurate for practical purposes. Three measures were performed, with one intercepting the whole ZP thickness and the other two restricted to the inner (denser) and outer (lighter) ZP portions.
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Target Parameters
The five primary estimated parameters were the mean volumes of 1) COCs, 2) oocytes with or without the ZP, 3) nuclei of oocytes, 4) cumulus cells, and 5) nuclei of cumulus cells. We also derived the relative numerical percentages of the several morphologic types of cumulus cells (Fig. 1).
Stereologic Techniques
Volume estimation
The mean volumes (
N) of COCs, oocytes, and cumulus cells were estimated with the unbiased stereologic technique of the nucleator [24]. This procedure is a very efficient estimator of the mean particle volume, requiring an arbitrary fixed point within a uniquely definable subspace of the particle, in practical terms for example, the center of the nucleolus of a cell, the nucleolus having been selected by uniform random probability using the disector principle [18]. For an isolated cell with a single nucleolus, there is no need of such sampling for performing the nucleator. For each nucleolus thus sampled, an isotropic direction is generated from a random point within the nucleolus (for practical purposes the nucleolus center), and the distances in each direction out from the point to the boundary of either the nucleus (to estimate the nuclear volume) or the cell limit (to estimate the cell volume) are recorded. From a series of these measurements, the mean particle volume in the so-called number-weighted distribution is estimated from
where the ln refers to the distances from the sampling point to the edge of the particle (such as the nucleus or the cell). The nucleator is generally implemented in thick physical sections and by using optical sections generated within it. In this case, the optical disector is used as the particle selection method (as described in Sampling of Cumulus Cells). To increase the efficiency of this estimator, distance measurements are usually performed in two or eventually more systematic random directions from the unique reference point. In our study, to increase the precision of the volume estimates the four-way nucleator technique was used [20], with measurements made every 90° (Fig. 1). All the estimating procedures were carried out with the referred software Grid.
The single point condition of the nucleator procedure can be met in mononucleated cells and even more efficiently in cells with just one nucleolus [24]. However, we found oocytes with a single recognizable point (one nucleolus) and oocytes with multiple nucleoli and even oocytes without any nucleolus or nucleus. Thus, some adaptations were made: 1) when several nucleoli were present, the bigger one was chosen as the reference point; 2) when two or more nucleoli of similar size were present, the first nucleolus to appear in focus when moving downward from the top of the section was chosen; 3) in the absence of nucleoli, the unique (virtual) point was the center of the nucleus in the optical plane where it was largest (the section with the largest nuclear profile was used); and 4) in the absence of a nucleus, the unique (virtual) point was the oocyte center in the section bearing the largest (and sharpest) profile. To validate empirically this approach, we also measured the distances from the virtual center of the nucleus and from the virtual center of the oocyte in those COCs to which the nucleator technique could be directly applied because a definable unique physical point was available.
For cumulus cells, because nucleoli were hardly distinguishable from chromatin clumps the virtual center of the nucleus was considered the unique fixed point for the nucleator procedure, for both nuclear and cell volume estimates. The referred virtual point was always defined in the optical section where the nuclear profile of the measured cell was largest. In this case, an empirical validation of the procedure could obviously not be performed.
Sampling of cumulus cells A systematic uniform random strategy [34] was used from section to field sampling. Whereas in a pilot trial half of the sections resulting from each COC were used, in studies encompassing all the sampled oocytes only one section was used. Within each sampled field, the cells were finally selected for volume estimation by using optical disectors [16]. Under the 100x lens, the focal plane was first brought to 2 µm below the section surface and then moved 10 µm further down the section. Every cumulus cell was sampled when its maximum nuclear profile came into focus as the focal level moved from 2 to 12 µm below the surface of the section. A counting grid bearing boundary lines (Fig. 2) was used to avoid edge effects [35]. A mean of 116 cumulus cells were sampled per COC.
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For data recording purposes, the cumulus mass was divided into two portions: zone I (the first three cumulus cell layers counting from the oocyte) and zone II (from the fourth layer up to the COC periphery). During data recording, the morphology of the cumulus cells was also considered (see Results). To assess whether the relative numerical estimates of cumulus cells could be restricted to the section used with the nucleator for the COC volume estimation, a complete set of estimates for cumulus cells was also made in that particular section. In this procedure, a mean number of 140 cumulus cells were sampled per COC.
Percentual distribution of cumulus cell types
According to its particular morphology, each disector-sampled cell was first assigned into one of the defined cell types, and its nuclear and cell volumes were then estimated. The relative numbers of the three cumulus cell types (see Results) were directly derived from the sampling with optical disectors, because in this technique all particles have the same chance of being sampled regardless of their size [24]. Numerical percentages (NN) of each cell type (C) were estimated as follows:
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Statistical Analysis
The statistical analysis was performed with the software Statistica (5.1) and Excel (5.0) for Windows. Results are presented as means accompanied by the SEM (SEM = SD/n1/2) and by the coefficients of variation (CV = SD/mean) computed for each group of COCs. Correlation analyses were performed between volume estimates based on the physical points and parallel estimates based on the virtual points. Paired t-tests were additionally performed for the volumes obtained by the two approaches. Unpaired t-tests were used to compare the subsets of COCs and oocytes derived from the primary groups. Unpaired t-tests were also used for comparing the stereologic parameters of cumulus cells, estimated from a sample of all the sections through a COC or just from the section used for estimating the volume of the corresponding COC. After checking assumptions of normality and homogeneity of variances, data from cumulus cells grouped by each zone of the cumulus were analyzed by two-way ANOVA to test the isolated and interactive effects of the cumulus zone and of the cell type upon the volumes of cumulus cells. For disclosing eventual specific differences in the volumes of the different cumulus cell types, the ANOVA was followed by Duncan multiple range test.
RESULTS
Qualitative Findings
Under the stereomicroscope, the majority of the selected COCs presented a roundish cumulus mass, with the oocyte at the center. Some complexes, however, had a very irregular cumulus shape with the oocyte located either in a central position or in a more eccentric position. In most cases, the ooplasm was homogeneous, but fine dark granules could be observed occasionally under the oolema.
Under the light microscope, oocytes had a round regular surface with the ooplasm homogeneously filled with dark and light clumps. When present, the nucleus was eccentric, round or oval, with regular contour and small, dispersed chromatin clumps. The nucleolus or nucleoli were generally eccentric. The ZP was well delimited and showed a darker inner region and an outer lighter portion.
Three distinct cumulus cell types were identified based on cell morphology: C1 cells with a small and very dense nucleus, C2 cells with an intermediate large and dense nucleus, and C3 cells with a large and light nucleus (Fig. 1).
Quantitative Findings
From the 24 COCs collected for this study, 10 had oocytes with only one nucleolus (thus allowing the direct use of the nucleator). Ten other COCs had oocytes with more than one nucleolus (which further showed a variable degree of fragmentation), one COC had an oocyte without a nucleolus, and three COCs had oocytes without a nucleus. In all these cases, the nucleator was used after the operator defined the fixed point, according to the rules outlined in Materials and Methods. Hereinafter, we refer to the implementation of the nucleator based on a virtual point as the virtual point nucleator.
The mean volumes obtained with the nucleator and with the virtual point nucleator for COCs, oocytes, and oocyte nuclei suggest that results are essentially the same regardless of the reference point used (Table 1). This finding was confirmed by the statistical analysis, which showed that correlations between the standard nucleator and the virtual point nucleator are quite high, with r-values ranging from 0.96 to 0.99. Although the results of the paired t-tests largely agree with those of the correlation analysis, thus negating differences between the virtual and the golden standard approaches, there is one apparent exception regarding the volume of the complex when the virtual center of the nucleus was used. In this case, the significant difference between volumes was due to the fact that, although the mean values obtained with the two approaches were identical, the value was slightly higher whenever the nucleus center was used (Table 1).
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Based on these results, we computed the mean values from all COCs as analyzed as a single group (Table 2). For this approach, the volume adopted for a particular COC was in accordance with the priority rules for choosing the nucleator reference point as described in Materials and Methods. The reported values should thus be considered as representative of this type of COC and those to be used in comparisons with data from other studies. Table 2 also includes the thickness estimation of the ZP, assuming that the outer zone is the thickest. For all estimates, the variability among complexes was quite high, especially for the oocyte nucleus, but there was no linear correlation between any of the reported parameters or values.
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If we consider the size distribution of the volumes of both COCs and oocytes, it is possible to build more homogeneous groups than those presented in Table 2. Such exercise was based on apparent cutoff points (Fig. 3). Regarding COCs, about 75% had volumes below 30 x 106 µm3, thus making a subset with a lower but still high CV (42%). Only six COCs had values greater than 60 x 106 µm3, but under the referred criteria five of those values were outliers. In relation to oocytes, about 87.5% had volumes below 2 x 106 µm3, lowering the observed CV of the subset to 23% (Fig. 3). Only three oocytes displayed higher values, all greater than 3.5 x 106 µm3. These values were considered outlier values under the default criteria implemented in the software Statistica. In both instances (Fig. 3), the newly formed subsets (containing the majority of the COCs and of the oocytes) were significantly different, based on the Student t-tests (P < 0.001). However, the more homogeneous subsets that could be created a posteriori from our data were not formed with the same basic elements (they did not hold the same group of COCs and corresponding oocytes), as is reflected in the different percentages.
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Mean cell and nuclear volumes for cumulus cells were first estimated using five COCs in a comparative study between estimates obtained with the five sections used for the nucleator approach (with the nucleolus as the fixed point) and with a sample made of half of the total sections obtained from the same COCs (Table 3). Because unpaired t-tests showed no differences between the two strategies, the analysis was further extended to 17 COCs (Table 3). For the other COCs parameters, the observed variability of cumulus cells was quite high as indicated by the CVs.
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Table 4 contains the volume estimates for the different cumulus cell types (C1 to C3) within the several regions defined for the cumulus (see Materials and Methods). This analysis was made on the single sections used for estimating the volumes of 17 COCs and of their components as displayed in Table 3, i.e., the sections that had either a defined physical reference point or a virtual reference point defined by the nucleus or oocyte center. Considering each cumulus cell type separately, Duncan's analysis revealed no significant differences in nuclear volume within the different cumulus zones. In the same way, the overall mean size of each cell type was the same regardless of the location in the cumulus. On the contrary, several significant differences were found among the different cell types within the same cumulus zone, in relation to either the nuclear or cell volumes, with differences being more numerous and significant when comparing C1 with C3; C2 is clearly an intermediate type of cell (Table 4). Because cumulus zones I and II produced similar results, for comparative purposes the overall mean volumes were computed for the cumulus as an unique global entity.
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The distribution frequency of cell (Fig. 4A) and nuclear (Fig. 4B) volumes of cumulus cells were also plotted. In accordance with the statistical analysis of the mean values, the overlapping of distributions was more evident in relation to cell types C1 and C2, with cell type C3 being clearly on the more voluminous right side. There was a two-peak distribution of cell volume for C2 cells, with the lower peak (smaller cells) being clearly under the main distribution peak of C1 cells. The distinction between the different cell types was greater when analyzing the nucleus (Fig. 4B).
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For estimating the numerical distribution of the different cumulus cell types within the cumulus, a pilot study using five COCs was carried out to compare the results obtained from the single sections used for estimating the volumes of the COCs with those from half of the sections from the corresponding COCs. The results supported the idea that similar estimates were obtained with the two sampling approaches (Table 5). Even the high variability among complexes was the same, regardless of the sections used. As expected, variance tended to decrease from cell type C1 (the least numerous) to cell type C3 (the more numerous cells in the COCs).
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The final estimation of the numerical distribution of cumulus cell types within the cumulus was made in a total of 17 COCs used for estimating the respective volumes of the COCs (Table 6). The results demonstrated that the same numerical frequency can be found both in the closest and farthest oocyte zones, which explains why results from the two cumulus zones do not differ from data computed for the cumulus as a whole. Additionally, the largest cell type (C3) was clearly confirmed as the predominant cumulus cell in the aspirated COCs, whereas the smallest cell type (C1) was the least abundant.
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Still regarding cumulus cells, the two-way ANOVA revealed that both the mean volumes and the numerical distribution of each cell type within the cumulus mass was dependent solely upon the cell type and not on the cumulus zone nor on any interaction between cell type and cumulus zone.
DISCUSSION
In this study, a stereologic approach was used to evaluate COCs aspirated from ovarian small (1–4 mm) antral follicles of diestrous cows. We primarily estimated the mean volumes of COCs, oocytes, nuclei of oocytes, cumulus cells, and nuclei of cumulus cells. COCs contained three distinct populations of cumulus cells, and their relative numerical distribution around the oocyte was the same regardless of the distance from the oocyte. Results also showed that the aspirated COCs were heterogeneous with respect to all quantitative parameters studied, both in relation to the oocytes and cumulus cells. This finding may be a relevant basis for understanding the rather erratic nature of the maturation rate of in vitro COCs and the subsequent embryo developmental potential [6, 8, 10, 11].
The methodology used was based on some keystones of modern stereology: 1) using grids bearing boundary lines [35] for sampling based on the disector principle [15], 2) estimating volumes with the nucleator [24], and 3) choosing sections or microscopic fields in a systematic random fashion [34]. Despite this approach, some technical and theoretical factors could have undermined the attempt to obtain unbiased estimates. Although we used aspirated follicles because they are the most common entities for in vitro processing in veterinary sciences, the same stereologic principles and techniques (and thus potential biases) could be applied to intact follicles gathered from microdissection.
When estimating the volume of embedded biologic material, the possibility of shrinkage must be taken into account. To minimize this potential bias, COCs were embedded in GMA, according to recent recommendations [18] and studies with murine ovarian follicles [20, 22, 23].
Isotropy is a requirement for performing the nucleator principle [24], and if the particle being evaluated is not isotropic in itself, either isotropic or vertical uniform random sections should be used [18]. In addition to the basically isotropic nature of both COCs and their components, the embedding strategy used was sufficiently accurate to ensure that sections could be considered isotropic uniform random planes. Because each COC was randomly rolled in the resin before being set down, this method somewhat mimicked the isector, an unbiased procedure that ensures a priori isotropic uniform random sections [36].
Although the theoretical background for the use of virtual points instead of physical fixed points for the nucleator can be derived from the study describing the nucleator [24], no formal validation has been produced for substituting a virtual central point for a missing actual physical point. However, such a substitution was recently discussed and advanced for another volume estimator, the spatial rotator [37]. The use of a virtual central point was also recently implemented using both the nucleator [38] and the rotator [39] for estimating the nuclear volume of neurones deprived of well-defined nucleoli and thus of identifiable physical points. However, in these studies no attempt was made to evaluate the potential bias caused by choosing a virtual point.
We attempted to vaildate the use of virtual points with our material, but only when it was possible to compare the estimates made from the physical (but seldom central) point with those from the virtual (and always central) reference, which was not possible with cumulus cells. Data on COCs (Table 1) demonstrated the validity of this approach for estimating the volume, regardles of whether the virtual center of the nucleus or that of the oocyte was chosen. Thus, the volume estimates when using mixed reference points (Table 2) did not differ significantly from those based in only one kind of point. For future studies, the mixed criteria could be adopted for estimating the volumes of COCs, oocytes, and nuclei of oocytes. Based on our results and on the previous use of virtual points by other authors [37–39], our strategy of using only a virtual reference for estimating cumulus cell volume seems adequate for providing unbiased data.
The way cumulus cells should be sampled is also important; should those cells be disector-sampled for measurements using a systematic sample of the sections from a COC, or would it be adequate to use the unique section used for measuring the COC volume by the nucleator? The nucleator principle has already been used for estimating the total number of cumulus cells in integral ovarian follicles of mice [23] and sheep [22]. In both studies, the sampling strategy was based on the fact that on one isotropical section through the unique point of a nucleated bag, one can sample in a uniform, systematic, random way either a few particles or a few points on the wall of the bag [16].
We thus postulated that this principle could be used not only for sampling cumulus cells for counting but also for producing local volume estimates. For the set of five COCs, we obtained nearly the same mean cell and nuclear volumes regardless of whether the sample was from all the sections through a COC or just the COC section having a physically well-defined and unique point (Table 3). Accordingly, when a larger pool of 17 COCs was analyzed, including some COCs with no single physical reference or no physical reference at all, both nuclear and cell volumes did not differ from the estimate based on cumulus cell sampling in some sections from a COC.
From the biologic point of view, the present quantitative approach unveiled new relevant data on this type of aspirated COC, with two main findings: 1) the high variability shown by most of the studied parameters, despite careful selection and handling of follicles of similar size, and 2) the presence of three distinct populations of cumulus cells and their relative abundance in COCs.
The between-COC variability in these estimates does not contradict data from other studies. For instance, in a study of mouse ovarian follicles that were collected by microdissection, Bagger et al. [20, 23] achieved very high CVs (up to 123%) for nucleator estimates of the volumes of the oocyte, antrum, and follicle and of the total number of granulosa cells per follicle.
The three populations of cumulus cells were distinguishable by their qualitative aspects (Fig. 1) and by their specific cell and nuclear sizes (Table 3). Most of the cumulus cells in COCs were of the largest cell type, C3, whereas the least numerous were of the smaller cell type, C1, with cell type C2 clearly an intermediate kind of cell in both size and relative number (Fig. 4 and Tables 4–6). However, despite the fact that the three populations appear distinguishable qualitatively and quantitatively, they nevertheless overlap somewhat in a continuum, thus suggesting a structural/functional gradient within the cumulus itself.
Neither structural nor functional populations of cumulus cells have been identified previously in aspirated intact follicles, although it is well known that in mammals a functional heterogeneity may exist among mural, antral, and cumulus granulosa cells [40]. The only study estimating follicular cell volume in an unbiased way was performed on pelleted granulosa cells from aspirated human COCs, in which cell location relative to the oocyte was lost [21]. Our data thus support the need for extending the present quantitative approach to follicles with different sizes and from different ovarian cycle stages to examine any possible relationship between structure and function that could enhance developmental success.
ACKNOWLEDGMENTS
We thank João Carvalheiro (Laboratory of Cell Biology) for the iconographical work and Elsa Oliveira (Laboratory of Cell Biology) and Maria Wanda Silva and Maria Helena Oliveira (Laboratory of Histology and Embryology) for technical support.
FOOTNOTES
First decision: 8 November 2000.
1 Partially supported by grants from PRODEP (1/PRODEP/96) to A.M.C. and FCT (Praxis XXI-PCNA/C/BIA/100/96; UMIB; Sapiens-36363/99, 35231/99) to M.S. 
2 Correspondence: Mário Sousa, Laboratory of Cell Biology, Institute of Biomedical Sciences Abel Salazar, University of Porto, Lg. Prof. Abel Salazar, 2, 4099-003 Porto, Portugal. FAX: 351 22 206 22 32;msousa{at}icbas.up.pt.
Accepted: June 4, 2001.
Received: September 12, 2000.
REFERENCES
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) of bovine COCs estimated by the nucleator, using different kinds of unique fixed points
