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a Tufts University School of Veterinary Medicine, North Grafton, Massachusetts 01536
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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20-mm diameter, was higher for oocytes initially having expanded cumuli than for those having compact cumuli. The maturation rate was strongly correlated (r2 = 0.92) with the proportion of oocytes having condensed chromatin at 0 h. Oocytes with diffuse chromatin were found essentially only in follicles 20-mm diameter that yielded compact granulosa, indicating follicle viability. Presence of diffuse chromatin was inversely related to maturation rate. We conclude that the major signal for chromatin condensation, and thus acquisition of meiotic competence, occurs in viable follicles after 20-mm diameter in the horse. Condensation of chromatin in oocytes in smaller apparently viable follicles, while associated with acquisition of meiotic competence, may represent a pre-atretic change.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In studies comparing meiotic competence of horse oocytes with differing cumulus morphology, oocytes with expanded cumuli matured in higher proportions than did oocytes with compact cumuli [8, 14, 15]. In the horse as in other species, cumulus expansion is strongly correlated with follicle atresia [7, 8], again indicating that horse oocytes from atretic follicles have significantly higher rates of maturation in vitro than do oocytes from viable follicles. Meiotic competence of horse oocytes appears to be associated with the chromatin configuration within the germinal vesicle. There are two major chromatin configurations found in germinal vesicle-stage horse oocytes [16–18]: chromatin condensed into one dense mass within the germinal vesicle (condensed chromatin, CC) and chromatin spread diffusely throughout the germinal vesicle (fluorescent nucleus, FN; [17, 18]). Oocytes in the FN configuration do not appear to contribute to the population reaching metaphase II in vitro [15]. However, the FN configuration was found most commonly in oocytes from viable follicles [8]. The association of the FN configuration with follicle viability suggests that oocytes with the FN configuration represent viable but juvenile oocytes that lack meiotic competence, but that at some point in follicle development they would progress to a competent stage [8]. If this theory is valid, a progression of chromatin toward the CC configuration would be observed as viable follicles grow toward preovulatory size. However, the relationship between follicle size and oocyte chromatin configuration in the horse has not been examined previously.
Because the horse is a seasonal breeder, ovulating only in the spring, summer, and early fall, most laboratories studying horse oocytes restrict work to the breeding season. While many reports on horse oocytes do not specify the season in which the study was conducted, of 10 papers in which the season is specified, only 1 was conducted during the nonbreeding season [19]. However, other than in the study of Brück et al. [6], in which no difference in maturation rates was seen between the early and late breeding season, the effect of season on equine oocyte meiotic competence has not been determined. Few data are available on the effect of season on meiotic competence in any mammalian species. Work in a seasonal primate (squirrel monkey) showed that oocytes recovered in the nonbreeding season had negligible maturation rates (6%) and that this was overcome by treatment with FSH [20].
The aim of this study was to evaluate the interactions among season, cumulus expansion, chromatin configuration, follicle size, and meiotic competence in horse oocytes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Follicles were classified by diameter into 4 size categories: T, 1–5 mm; S, 6–10 mm; M, 11–20 mm; and L, > 20 mm. The follicles were opened with a scalpel blade, the granulosa layer of each follicle was scraped using a 0.5-cm bone curette, and the cells were washed from the curette into individual Petri dishes using holding medium (M199 with Hanks' salts and 25 mM Hepes [Gibco Life Technologies, Grand Island, NY]) and ticarcillin (0.1 mg/ml; SmithKline Beecham Pharmaceuticals, Philadelphia, PA). The Petri dishes were labeled with the follicle size category.
Oocyte Classification
The contents of the Petri dishes were examined using a dissection microscope at x10–20. Cumulus morphology was classified as being compact (Cp) or expanded (Ex), as described previously [8]. Briefly, a Cp cumulus was defined as being densely cellular, having a smooth surface over the cumulus hillock, and having even or granular coloration; Ex as having individual cells visible protruding from the surface of the cumulus hillock to having full expansion with copious matrix visible between cumulus cells.
After classification, cumulus-oocyte complexes (COC) were grouped according to their size and cumulus categories and were kept at room temperature in holding medium until all oocytes had been collected (1–4 h). One half of each group of COC were assigned to be fixed directly for staining (0-h group), and one half were cultured for 24 h before fixation (24-h group). For evaluation, oocytes were denuded of cumulus by pipetting in a solution of 0.25% trypsin and 1 mM EDTA in Hanks' salts without CaCl2, MgCl2, or MgSO4 (Gibco). The oocytes were fixed in buffered formol saline for a minimum of 24 h at room temperature. Fixed oocytes were labeled for chromatin evaluation by being placed on a glass slide with 6 µl of mounting medium (3:1 glycerol:PBS containing 2.5 g/ml Hoechst 33258). The oocytes were evaluated using a fluorescence microscope with a 365-nm exciter filter. Chromatin configuration was classified as previously described [8, 17]. Germinal vesicle-stage oocytes were classified as having a diffusely fluorescent nucleus with or without aggregates of chromatin (FN) or as having chromatin condensed entirely into one mass within the germinal vesicle (CC; Fig. 1). CC was further broken down into tightly condensed, having a smooth, oval outline; and loosely condensed, having a larger area and an irregular outline (Fig. 1, [17]). Oocytes with a hairlike, faint, or jagged distribution of chromatin throughout the germinal vesicle ("crinkled FN") were considered to be degenerating as previously discussed [15]. Other classifications were diakinesis, having individual strands of dense chromatin visible [17]; metaphase I; metaphase II; and degenerating (having abnormal chromatin configurations or no chromatin visible).
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Oocyte Maturation
Oocytes intended for maturation were incubated in groups in 200-µl droplets of M199 with Earle's salts and 10% neonatal calf serum (Gibco), 5 g/ml FSH (Schering, Kenilworth, NJ), 25 g/ml gentamycin, and 1.25 g/ml Fungizone (Gibco) under light white mineral oil (Sigma, St. Louis, MO) at 38.2°C in a humidified atmosphere of 5% CO2 in air. After 24-h incubation, the cumulus was removed and the oocytes were fixed, stained, and evaluated as above.
Follicle Populations
Additional data were recorded on the follicle and luteal population of the ovaries from which the oocytes described above were collected, as well as from additional ovaries obtained during the same time period for other projects. These data were total number of T, S, M, and L follicles present; presence of luteal structures; and classification of mural granulosa cells in those follicles from which no oocyte was recovered. For this last category, the degree of mural granulosa expansion was based on the system used for cumulus granulosa expansion. Mural and cumulus granulosa morphology are strongly correlated with each other and with follicle viability [8].
Statistical Analysis
The proportion of ovaries containing luteal structures was evaluated first, to determine the relationship between season and ovulation in this population of mares. Based on the number of luteal structures per ovary for each month of the study (Fig. 2), five seasons were defined: anestrus (ANS): December, January, and February; spring transition (SPR): March and April; early ovulatory (EOV): May, June, and July; late ovulatory (LOV): August and September; fall transition (FAL): October and November. These groupings were used to determine the effect of season on oocyte and follicle parameters.
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Difference in number of follicles per ovary among seasons was examined by ANOVA, using the log10 of the mean follicles per ovary on each collection day. Effects of follicle size, cumulus morphology, season, and day on initial chromatin configuration and on maturation rate were analyzed using the Generalized Linear Mixed Model (GLIMMIX) software (SAS 6.12, Cary, NC). Because there was no overdispersion (no effect of day), comparisons among strata were performed using chi-square analysis, with Fisher's exact test used for comparisons in which a value of less than 5 was expected for any cell. Correlations between percentage of oocytes in diakinesis at 0 h and those in metaphase I or II (MI or MII) at 0 h were performed by Spearman's rank correlation. For analysis of the percentage of oocytes actively maturing in culture (meiotically competent oocytes), the percentage of oocytes found in MI or MII after culture was adjusted by subtracting the percentage of oocytes already at MI or MII at 0 h for that oocyte class. Maturation to MI or MII was used as an endpoint rather than MII alone to ensure that competent Cp oocytes, which mature more slowly than do Ex oocytes [18], were included in the analysis. Correlation between the percentage of oocytes in the CC configuration at 0 h and those maturing at 24 h was performed by linear regression analysis after arcsin transformation.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Proportion of luteal structures per ovary ranged from less than 0.1 in December through March to over 0.5 in May and July (Fig. 2).
Follicle Characteristics
For the determination of follicle size and average number of follicles per ovary by season, 1397 ovaries from 80 separate collection days (January of Year 1 to April of Year 2; average of 17.5 ovaries per collection day) were evaluated. There were 10 753 follicles recorded, for an average of 7.7 follicles per ovary. The average number of follicles recorded per ovary for the various seasons were 7.3, 8.5, 7.6, 7.1, and 7.9 for ANS, SPR, EOV, LOV, and FAL, respectively. The difference in the number of follicles per ovary among the different seasons was not significant (P > 0.1).
The overall proportion of follicles that were T, S, M, and L was 43%, 35%, 19%, and 3%, respectively. The proportions of follicles that were T, S, M, and L for the different seasons are presented in Table 1. The distribution among follicle sizes differed significantly among seasons (P < 0.001), with the exception that the distribution was similar between EOV and LOV and between FAL and ANS (P > 0.1). The proportion of T follicles decreased during the ovulatory seasons, whereas the proportions of M and L follicles increased during this time.
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The expansion status of cumulus granulosa cells, or of mural granulosa cells in follicles from which no oocyte was recovered, was examined in 8016 follicles (April of Year 1 to April of Year 2). The proportion of Cp follicles increased significantly with increasing follicle size (P < 0.001), being 30%, 46%, 53%, and 63% for T, S, M, and L follicles, respectively. Within each follicle size group, the proportion of Ex and Cp follicles varied significantly among seasons (P < 0.01; Fig. 3), with the highest proportion of Cp follicles occurring in SPR and EOV and the lowest proportion in LOV.
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The rate of oocyte recovery from Cp follicles was significantly higher than that from Ex follicles (1741 of 3103, 56% vs. 2183 of 4913, 44%; P < 0.001). The oocyte recovery rate from Cp follicles decreased as follicle size increased (P < 0.001), being 55%, 60%, 52%, and 49% for T, S, M, and L follicles, respectively. In contrast, the recovery rate from Ex follicles was significantly lower for T (38%) than for all other follicle sizes (51%, 51%, and 52% for S, M, and L follicles, respectively; P < 0.001).
Chromatin Configuration
Chromatin configuration was determined in 1711 oocytes fixed at 0 h (795 Cp and 916 Ex) and for 1668 oocytes fixed after 24-h culture (788 Cp and 880 Ex). The chromatin configurations of the various categories of oocytes at 0 h are presented in Table 2. There were significant effects of cumulus morphology (P < 0.001), follicle size (P < 0.001), and season (P < 0.05) on the proportion of oocytes in the CC configuration, but no significant interactions among these factors. There was no independent effect of day on proportion of CC oocytes (extra-dispersion scale = 1.01), indicating that the variation in maturation rate from day to day was explained entirely by the mean CC% for that group and the number of oocytes evaluated. The proportion of oocytes in the CC configuration increased with increasing follicle size for both Cp and Ex oocytes (P < 0.001). For each follicle size except L, the proportion in the CC configuration was significantly higher in Ex than in Cp oocytes (P < 0.001).
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In T, S, and M Cp oocytes, the proportion in the FN configuration at 0 h was constant at 22–23%, whereas the proportion in the FN configuration was 6% in L Cp oocytes and was less than 5% in all size classes of Ex oocytes (Table 2; Fig. 4). Maturing chromatin configurations (MI or MII) were found only in L Cp oocytes (3%) and in Ex oocytes. Diakinesis was seen mainly in T Cp oocytes (10%) and was present in less than 4% of oocytes in all other groups. There was a negative correlation (r = -0.97) between the proportion of oocytes in diakinesis and the proportion of oocytes in further stages of maturation (MI or MII) at 0 h.
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Within the CC configuration, there was a significant difference among follicle sizes in the proportion of tightly condensed and loosely condensed CC subgroups. The proportion of the CC oocytes that had tightly condensed chromatin was 36%, 35%, 41%, and 70% for T, S, M, and L Cp groups, respectively (P < 0.01); this proportion was 62%, 57%, 71%, and 73% for T, S, M, and L Ex groups, respectively (P < 0.05). The proportion of CC oocytes having tightly condensed chromatin was significantly higher for Ex than for Cp oocytes at each follicle size except L (P < 0.001).
There were significant effects of cumulus morphology and follicle size on oocyte maturation rate (P < 0.001), but no interactions between these factors. There was no significant effect of season on oocyte maturation rate. There was no independent effect of day on maturation rate (extra-dispersion scale = 0.99).
The proportions of oocytes in MI or MII after culture for total Cp and total Ex oocytes were 278 of 788 (35%) and 564 of 880 (64%), respectively. There was a significant effect of follicle size on maturation rate; the proportion of oocytes in MI or MII after 24-h culture increased with increasing follicle size for both Cp oocytes (P < 0.01) and Ex oocytes (P = 0.010), being 77 of 258 (30%), 109 of 325 (34%), 76 of 179 (43%), and 16 of 26 (62%) for T, S, M, and L Cp, respectively, and 214 of 365 (59%), 219 of 332 (66%), 112 of 159 (71%), and 19 of 24 (79%) for T, S, M, and L Ex, respectively. For each follicle size except L, the proportion of mature oocytes after culture was significantly higher in Ex than in Cp oocytes (P < 0.001).
The relationship of the proportion of oocytes actively maturing in culture (meiotically competent oocytes) to the proportion of oocytes in the CC and FN configurations at 0 h in the same cumulus/follicle size classes is presented in Figure 4. The proportion of oocytes maturing in culture was strongly correlated to the proportion of oocytes in the CC configuration at 0 h (adjusted r2 = 0.92; P < 0.001).
Season
The proportion of CC oocytes at 0 h differed significantly among seasons in T Ex, S Ex, and M Cp oocytes (P < 0.05; Table 3). In these classes, the proportion in the CC configuration was lowest during the breeding season (EOV for M Cp, LOV for T Ex and S Ex). In those oocytes having a substantial proportion in the FN configuration (T, S, and M Cp oocytes), the proportion of FN oocytes differed significantly among seasons, being highest in EOV (36%, 40% and 44% for T, S, and M Cp oocytes, respectively) and low in ANS (16%, 10%, and 10%, respectively; Table 3).
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There was no significant effect of season on maturation rate, nor were there interactions among follicle size, cumulus, and season. However, the proportion of oocytes maturing per CC oocyte present in the 0-h group did differ among seasons: for total Cp and total Ex oocytes, the proportion of oocytes maturing after culture was significantly lower than the CC% at 0 h for both Cp and Ex oocytes in ANS, and for Ex oocytes in SPR (P < 0.01). The proportion of oocytes maturing in culture was not significantly different from the proportion of oocytes in the CC configuration at 0 h in all other seasons.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The strong correlation (r2 = 0.92) between the proportion of oocytes in the CC configuration at 0 h and the proportion of oocytes maturing in culture supports our earlier hypothesis that the CC configuration represents the meiotically competent horse oocyte [8, 15]. The proportion of oocytes in the FN configuration was inversely related to maturation rate, supporting previous findings that FN oocytes are not meiotically competent [15].
Condensation of chromatin is associated with meiotic competence in other species and has been most thoroughly described in the mouse [21]. However, in the mouse, by the time the follicular antrum is formed, > 90% of oocytes have CC and are meiotically competent. The findings of our study indicate that in the horse, there is a gradual increase in meiotic competence with increasing follicle size, suggesting that the signal for acquisition of meiotic competence is less strongly related to follicle size in the horse than it is in other species. However, there is another possible interpretation of these data. FN oocytes were found essentially only in Cp follicles of 20-mm diameter (T, S, and M groups; Table 2). If oocytes progress gradually from FN to CC configurations as the follicle grows, one would expect an inverse relationship between these two configurations. Instead, the increase in CC% with increasing follicle size was associated with a loss of degenerating oocytes, while the FN% remained constant, until follicles reached over 20-mm diameter. During the peak ovulatory season there was even a greater proportion of FN oocytes in 10- to 20-mm CP follicles than in smaller follicles (36%, 40%, and 44% for T, S, and M, respectively; Table 3), suggesting that FN oocytes were maintained during follicle growth while other configurations were lost. However, after 20-mm diameter, the proportion of FN oocytes dramatically decreased, and the proportion of CC oocytes—and the maturation rate—increased (Fig. 4).
One possible explanation for these findings would be that follicles containing FN oocytes are "failures" and are eliminated at 20-mm diameter. However, the strong association of the FN configuration with follicle viability [8], and the increase in proportion of FN oocytes during the breeding season—when follicle growth leading to ovulation is at its most efficient—argue against this theory. A preferable explanation would be that oocytes destined to be preovulatory are maintained in the FN configuration until around 20 mm, when they gain physiological meiotic competence and progress to the CC configuration.
Progression to CC in smaller follicles may only occur if the follicle is destined for atresia. In a previous study [8], we found that histologically viable follicles containing CC oocytes were more likely to show signs of luteinization than those with FN oocytes, even at small follicle diameters. Luteinization has been associated with "incipient atresia" of horse follicles [22]. The relationship of the CC configuration to atresia in small follicles is supported by data from Pedersen et al. [23], who reported that 6 of 11 follicles of < 20-mm diameter containing a CC oocyte were apoptotic, whereas there was no apoptosis in follicles > 30 mm in diameter containing CC oocytes. Apoptosis was also not found in follicles containing FN oocytes. These data suggest that progression of oocytes to the CC configuration in small follicles having Cp granulosa—those likely to be classified as viable histologically—may be a sign that the follicle can no longer suppress oocyte development, and thus is entering atresia.
The differences between the subdivisions within the CC category (tightly condensed chromatin and loosely condensed chromatin) were surprising, as these two configurations are morphologically similar to one another and may be difficult to differentiate. However, the increased proportion of tightly condensed chromatin with increasing follicle size, and with atresia, suggests that this is the configuration that is furthest progressed. This is supported by the finding that the variation in maturation rate was entirely explained by the variation in tightly condensed chromatin, and that addition of loosely condensed chromatin did not tighten the regression analysis. The proportion of oocytes with tightly condensed chromatin is less in all groups than is the proportion of oocytes maturing, suggesting that some oocytes with loosely condensed chromatin are also maturing. Loosely condensed chromatin appears to be the most likely other configuration to be competent because of the morphological continuum between it and tightly condensed chromatin, and because the other configurations (FN, diakinesis, degenerating) were inversely related to maturation rate.
The "diakinesis" chromatin configuration (individual loops of chromatin visible throughout the germinal vesicle, with no fluorescent background [17]) was inversely related not only to meiotic competence of oocytes after culture, but also to presence of further stages of maturation within the follicle. This suggests that this configuration does not represent progress toward MI, as proposed earlier [17]. Since the diakinesis chromatin configuration was found in appreciable proportions only in the same oocyte classes that had a high FN% (T, S, and M Cp), this configuration may represent a variation in FN morphology or an intermediate step between FN and CC. Configurations similar to that we describe as diakinesis have been reported by other laboratories as "germinal vesicle with diplotene chromatin" but were seen only in a few oocytes [4, 24]. Evaluation of stained oocytes using time-lapse, low-level fluorescence is needed to definitively determine the progression of horse oocytes through the various chromatin configurations during maturation.
Oocyte Recovery and Classification
The total number of visible antral follicles in our study (average 7.7 per ovary) agrees with the findings of Wesson and Ginther [25] (approximately 15 follicles per mare) and with previous studies on oocyte recovery [5, 7, 18] but is lower than that reported by Driancourt et al. [26]. These workers found an average of 16 follicles per horse ovary that were between 1.5 and 10 mm, using serial histological sections [26], whereas in our study, an average of 6 follicles per ovary were in this size category. This suggests that follicles 1- to 5-mm diameter are undercounted by gross ovary dissection.
On the basis of the findings of this study, to select horse oocytes for maximum meiotic competence, Ex oocytes, and Cp oocytes from follicles of > 20-mm diameter, should be selected. However, the proportion of follicles > 20 mm is low (3%), and almost half of the follicles present on the horse ovary are in the smallest size category (1- to 5-mm diameter). Thus, when oocytes are recovered from all follicles present on excised horse ovaries, the majority of oocytes recovered are from the lowest meiotic competence groups. These findings are of major importance both for selection of oocytes for in vitro work and for management of transvaginal ultrasound-guided ovum pickup (OPU) programs. In cattle, OPU may be performed as often as every 3 days with high oocyte recovery and meiotic competence. In horses, frequent OPU sessions in a given mare would increase the proportion of small, growing follicles and would thus decrease the meiotic competence of the oocytes recovered. A low maturation rate (20–45%) in oocytes collected after repeated OPU in mares has been reported [4]. Frequent OPU sessions were also associated with an increase in the proportion of recovered oocytes having diffuse chromatin [27].
Seasonality
The proportion of ovaries bearing luteal structures throughout the year, as seen in this study, was similar to that previously described by others [28] and reflects the seasonal nature of ovulatory activity in our population of mares. While the numbers of large follicles decreased significantly during the nonbreeding season, surprisingly there was no effect of season on the total number of visible antral follicles present. This result is similar to the findings of Wesson and Ginther [25] on dissection of pony mare ovaries, but it contradicts the report of Driancourt et al. [29]. These latter authors performed histological sections on ovaries removed from 2-yr-old pony fillies by hemicastration. However, they used young animals, had small numbers (5 ovaries per season), and removed only ovaries bearing a preovulatory follicle; and these limitations may have affected their results.
The constant number of visible antral follicles throughout the year indicates that growth of follicles to the antral stage is independent of season in the mare. The increase in proportion of follicles of > 10-mm diameter during the ovulatory seasons (Table 1) suggests that follicle growth in this group occurs in response to gonadotropin stimulation. The maximum follicle size found in mares with artificially suppressed FSH concentrations was approximately 15 mm [30–32]. Our finding that the proportion of viable (Cp) follicles increased during the early breeding season (SPR and EOV) contradicts that of Driancourt et al. [29]; however, as noted above, this latter study may have had confounding factors. We found that the lowest proportion of Cp follicles in every size group occurred not during anestrus but during the late ovulatory season (Fig. 2).
The seasonal variation in the percentage of Cp oocytes having the FN configuration, in follicles 1–5 mm, 6–10 mm, and 10–20 mm (from 10% to 15% in ANS to 36–44% in EOV), suggests that oocyte germinal vesicle chromatin status is responsive to gonadotropin stimulus, even in follicles less than 10 mm. It is possible that during anestrus (a period of low gonadotropin stimulation), follicles may grow to 10–15 mm but are functionally gonadotropin-starved, and thus pre-atretic changes, including condensation of chromatin in the enclosed oocyte, are commonly observed. In the early breeding season, increasing gonadotropin concentrations simulate growth of a greater proportion of Cp follicles to larger sizes, and a higher proportion of FN oocytes are maintained. In apparent contradiction to this hypothesis, the proportion of Cp follicles (Fig. 3) and of FN oocytes within the Cp groups (Table 3) is reduced in the late breeding season, even though gonadotropin concentrations remain high at this time. One possible explanation for this is down-regulation of gonadotropin receptors causing functional loss of gonadotropin stimulation.
The overall meiotic competence of each oocyte cumulus/size category, of total Cp oocytes and total Ex oocytes, and of all oocytes combined, did not change with season. There appeared to be a decreased meiotic competence of both Cp and Ex CC oocytes during ANS, which was compensated for by the increase in proportion of CC oocytes present at this time. The lower meiotic competence per CC oocyte noted during anestrus may be related to functional changes induced by absence of gonadotropins, or, possibly, to environmental influences on ovaries as they were recovered at the slaughterhouse during the winter.
Conclusions
In summary, the CC configuration was strongly associated with meiotic competence in horse oocytes. Meiotic competence increased with increasing follicle size both for oocytes with Cp and for those with Ex cumuli; however, in each follicle size category 20 mm, meiotic competence was greater for oocytes with Ex cumuli than for oocytes with Cp cumuli. In follicles > 20 mm, over 90% of nondegenerating, non-metaphase oocytes were in the CC configuration, indicating that this is the chromatin configuration of oocytes in preovulatory follicles prior to resumption of meiosis. The diffuse chromatin (FN) configuration was seen essentially only in oocytes with Cp cumuli from follicles 20 mm. We hypothesize that the FN configuration, which is meiotically incompetent, is maintained until the follicle is > 20-mm diameter in viable follicles that will progress toward ovulation, and that physiological acquisition of meiotic competence occurs after this follicle size. Condensation of chromatin in oocytes in smaller apparently viable follicles, while associated with acquisition of meiotic competence, may represent a pre-atretic change.
We conclude that there is no effect of season on the number of visible antral follicles present on horse ovaries, although the size distribution of these follicles changes over season. The proportion of viable follicles, and of follicles of > 10-mm diameter, increases during the ovulatory period. There was a significant effect of season on the proportions of CC oocytes and FN oocytes within the different follicle size/cumulus groups. While there was no significant effect of season on meiotic competence within any oocyte class, meiotic competence of CC oocytes may be depressed during anestrus but be balanced by an increased proportion of CC oocytes present at this time. The differences found in this study among follicle classes in initial chromatin configuration and meiotic competence point out the need for stating follicle and cumulus parameters in future studies on horse oocytes. More work is needed to evaluate physiological parameters of FN and CC oocytes, and the CC subtypes of tightly and loosely condensed chromatin, to determine their relationship to acquisition of meiotic competence in horse oocytes and to changes occurring during follicle atresia.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by USDA CRGO 90–37240–5757. 
2 Correspondence and current address: K. Hinrichs, Dept. of Physiology and Pharmacology, College of Veterinary Medicine, Texas A&M University, College Station, TX 77843-4466. FAX: 409 845 6544; khinrichs{at}cvm.tamu.edu
Accepted: December 14, 1999.
Received: April 1, 1999.
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