Equine Oocyte Competence for Nuclear and Cytoplasmic In Vitro Maturation: Effect of Follicle Size and Hormonal Environment'

BIOLOGY OF REPRODUCTION 57, 232-245 (1997) Equine Oocyte Competence for Nuclear and Cytoplasmic In Vitro Maturation: Effect of Follicle Size and Hormonal Environment' Ghylene Goudet, 2 Jacqueline Bezard, Guy Duchamp, Nadine Gerard, and Eric Palmer I.N.R.A.-Haras Nationaux, Unit6 Reproduction Equine, F-37380 Nouzilly, France ABSTRACT Equine oocyte competence after in vitro maturation (IVM) was investigated in terms of the diameter of the follicle of origin and the stage of the estrous cycle, with three criteria of maturation: nuclear stage after DNA Hoechst staining, meiotic spindle morphology after tubulin immunocytochemical staining, and cortical granule localization after lectin labeling. Seven successive in vivo ultrasound-guided follicular punctures were performed on 10 cyclic saddle mares, alternatively at the end of the follicular phase (after induction of ovulation with a gonadotropin injection) and in midluteal phase (with or without a gonadotropin injection). Expanded cumulus-oocyte complexes (COCs) were stained at collection, and compact COCs were stained after in vitro culture. They were observed under a confocal microscope. Successive punctures on one mare provided 0.9 preovulatory COCs and 8 immature COCs per 22 days. Among the preovulatory oocytes, 55% had completed nuclear and cytoplasmic maturation, 86% of which displayed a normal meiotic spindle. Of the 262 oocytes cultured in vitro, 37% completed nuclear maturation. The nuclear and cytoplasmic maturation rate significantly increased with follicle diameter. The IVM rate tended to be higher in follicular phase and tended to increase in luteal phase with the gonadotropin injection. The meiotic spindle morphology was not significantly different between the classes of follicular diameters. This study provided the opportunity to increase the number of characterized oocytes collected per cycle and per mare. This is the first report showing the progressive acquisition of meiotic competence in the equine oocyte during antral follicle growth and is the only description of the equine meiotic spindle. INTRODUCTION In vitro maturation (IVM) and fertilization (IVF) are required methods for the study of the mechanisms involved in oocyte maturation, fertilization, and early embryo development. However, in the mare, such research has been slow to progress. In the horse as compared with other domestic species, IVF is far from being routinely used [1, 2], and the IVM rate is low. Several culture times and media have been evaluated without any great improvement [3-6]. Moreover, in vivo maturation conditions in the mare are different from those in other domestic mammals. The ovulatory LH surge is a progressive increase and decrease lasting many days, with a maximum concentration occurring 1 day after ovulation [7, 8]. The fine structure of the equine oocyte after IVM has not been well described; most studies only evaluate nuclear Accepted March 17, 1997. Received November 18, 1996. 'This work was supported by grants from the Institut National de la Recherche Agronomique, France, and the Haras Nationaux, France. Ghylene Goudet was supported by a fellowship from the Institut National de la Recherche Agronomique, France, and the Region Centre, France. 2Correspondence: Ghylene Goudet, I.N.R.A.-Haras Nationaux, Unite Reproduction Equine, station P.R.M.D., F-37380 Nouzilly, France. FAX: 33-2-47-42-77-43; e-mail: goudet@tours.inra.fr 232 maturation using chromatin labeling [3-6, 9]. Baka et al. [10] and Pickering et al. [11] reported meiotic spindle disorganization with normal chromosome morphology in human oocytes. Aged human oocytes [11] and mouse oocytes atypical in meiotic progression [12] often display changes in spindle length or organization. To our knowledge, the equine oocyte meiotic spindle organization has never been described. To obtain fertilizability, a final cytoplasmic maturation must run parallel to nuclear maturation. In mammalian oocytes, the migration of cortical granules (CG) is an important step in cytoplasmic maturation [13, 14], and has been used as a significant criterion in the assessment of maturity and fertilizability [15, 16]. In the horse, Neumann et al. [17] evaluated organelle distribution before and after IVM; however, they worked with a total of 14 oocytes. No other work on equine oocyte cytoplasmic maturation after IVM has been published. The lack of knowledge and studies in the mare can be largely explained by the limited availability of equine oocytes. Collection of oocytes from preovulatory follicles by transvaginal ultrasound-guided puncture yields only 0.7 0.1 oocytes per cycle [18]. Superovulation treatments increase the number of preovulatory follicles; but they have not been very efficient in the mare [19-21], and they damage the follicular environment [18]. Within the female ovary, the numerous immature follicles contain a reserve of immature oocytes. One way of acquiring mature oocytes is through collection of immature oocytes with subsequent IVM. Gametes may be collected from slaughterhouse animals by dissecting the follicles and scraping the follicle wall. Slaughterhouse material has the obvious disadvantages of lack of repeatability, delay in the time between oocyte collection and placement in culture medium, and lack of information about the stage of the cycle and follicular growth. To gain an understanding of the reasons for the low maturation rate in the mare, and to select oocyte populations able to mature in vitro, the in vivo ultrasound-guided transvaginal puncture is an interesting tool for oocyte collection. It allows collection of a well-characterized population of immature and mature oocytes: the follicles are individually punctured, follicular growth can be regularly checked by echography, and one can choose the day of the estrous cycle on which to perform puncture [22, 23]. Moreover, since one can perform repeated punctures on the same mare, the method allows comparison of several populations of oocytes within the same animal [22]. Our study using this tool was designed with two aims: 1) development of an optimal rhythm of successive punctures, adjusted to the follicular growth pattern, to maximize the oocyte yield, and 2) investigation of oocyte competence after IVM in terms of follicle size and stage of the estrous cycle. Fluorescent microscopy was used to examine chromatin, microtubules, and CG as criteria of oocyte maturation.

EQUINE OOCYTE COMPETENCE FOR IN VITRO MATURATION 233 MATERIALS AND METHODS Experimental Animals Ten adult cyclic mares (Selle Franqais) in good body condition, from 8 to 17 yr old, were kept indoors and fed with concentrates for use from April through July. Ovarian activity was assessed by routine rectal ultrasound scanning [24] using an Aloka 210 (Socit6 Bernard, Nantes, France) with a 5-MHz linear probe. Blood samples were withdrawn every 2 days. Plasma progesterone concentrations were measured using the RIA method described by Palmer and Jousset [25]. Animal Treatment During the collection procedure, mares were sedated with detomidine (0.8 mg/100 kg BW i.v. Domosedan; Smithkline & French, Courbevoie, France) and butorphanol (1.4 mg/100 kg BW i.v. Torbugesic; Willows Francis Veterinary, Crawley, England), and the rectum was relaxed with prifinium bromide (45 mg/100 kg BW i.v. Prifinial; Vtoquinol, Lure, France). After puncture, the mares received an antibiotic injection (Mixtencilline: 1 600 000 IU penicillin/100 kg BW, and 1.3 g dihydrostreptomycin/100 kg BW i.m.; Rh6ne M6rieux, Lyon, France). To induce ovulation, an injection (i.v.) of 25 mg of crude equine gonadotropin (CEG) [26] was given when the largest follicle reached 35 mm. Luteolysis was induced by a prostaglandin F 2. analogue injection (cloprostenol; 250 pig/ mare i.m.; Estrumate, Pitman-Moore, Meaux, France). Follicular Puncture and Oocyte Recovery At the beginning of the experiment, all follicles larger than 5 mm were punctured 34 h after induction of ovulation (i.e. just before ovulation) to render the ovaries free of atretic follicles and allow healthy follicles to develop. For each mare, seven successive ultrasound-guided punctures were performed alternately, three at the end of the follicular phase (group "F") and four during the luteal phase (group "L"). At the end of the follicular phase, all follicles larger than 5 mm were punctured 34 h after induction of ovulation. During the luteal phase, all follicles larger than 5 mm were punctured 34 h after the largest follicle reached 18 mm, i.e., before it reached the dominant stage. Just after the puncture in luteal phase, the corpus luteum was suppressed by a prostaglandin F 2. analogue injection. In order to test the effect of the CEG injection, two punctures in luteal phase were performed after an injection (i.v.) of 25 mg of CEG on the day the largest follicle reached 18 mm (group "L+"), and two punctures in luteal phase were performed without any injection (group "L-"). Five mares were punctured according to the pattern of L+, F, L-, F, L+, F, L- and five according to the pattern of L-, F, L+, F, L-, F, L+. The follicles were punctured using a transvaginal ultrasound-guided follicular aspiration technique [22] with a 7.5-MHz sectorial probe (Kretz; Soframed, Truchtersheim, France). To improve the recovery rate of oocytes, two types of aspiration needles were used [22]. A single-lumen needle (length, 600 mm; outer diameter, 1.8 mm; Thiebaud Freres, Jouvernex Margencel, France) was used for puncturing follicles > 25 mm, and a double-lumen needle (length, 700 mm; outer diameter, 2.3 mm; internal diameter, 1.35 mm; CASMED, Cheam Surrey, England) was used for follicles < 25 mm. After follicular fluid aspiration, the follicle was flushed with PBS (Dulbecco A; Unipath, Dardilly, France) and heparin (50 IU/ml; LEO S.A., St-Quentin Yvelines, France) at 37 C. The single-lumen system allowed five successive flushes with complete filling and emptying. The doublelumen system allowed continuous flushing of the follicle, but aspiration was interrupted five times so that the follicle could fill again. All aspirated fluids were individually examined for oocyte recovery. The follicular fluid from follicles larger than 30 mm was centrifuged (15 min at 2000 x g), and the supernatants were stored at -18 0 C until assayed. Oocyte Culture and Fixation At recovery, oocytes were individually classified and processed according to cumulus aspect (compact or expanded). Compact cumulus-oocyte complexes (COCs) were cultured individually in a humidified atmosphere (95% air:5% CO 2 ) at 38.5 0 C for 30 h in 500 jil of maturation medium: Medium 199 with Earle's salts, 2.2 g/l NaHCO 3, and L-glutamine (Gibco, Eragny, France) supplemented with 20% inactivated fetal calf serum (FCS; Gibco), antibiotics (100 IU/ml penicillin, 100 IU/ml streptomycin, and 0.25 RIg/ml fungizone; Gibco), CEG (9.5 jig/ml equine FSH and 15 jig/ml equine LH) [26], and estradiol-17 (1 jig/ml; Sigma, La Verpillere, France). After culture, the COCs were stripped with small glass pipettes in 500 jil of PBS solution supplemented with 525 IU/ml hyaluronidase (type III, 875 IU/mg; Sigma) at 37C. Totally denuded oocytes were rinsed in PBS with 1% FCS at 37 C, fixed for 20 min in 2.5% paraformaldehyde in PBS at 37 C, and rinsed again. The oocyte diameter was then measured. Oocytes were kept at 4 C in PBS containing 0.05% NaN 3 (sodium azide; Prolabo, Paris, France) and 1 mm PMSF (Serva, Heidelberg, Germany). Expanded COCs at recovery were stripped of their cumulus cells, fixed, and kept as just described for the compact COCs. Immunocytochemical Staining and Lectin Labeling The oocytes were permeabilized in Triton X-100, 0.1% in PBS, for 5 min at room temperature and washed in PBS containing 0.05% NaN 3 and 1 mm PMSE Microtubule localization was performed according to the following immunofluorescence protocol. 1) Nonspecific reactions were blocked by preincubation of oocytes for 2 h at room temperature in an incubation solution: PBS containing 2% BSA (A-7030; Sigma), 0.05% NaN 3, 0.05% saponin (Sigma), and 10% goat serum. 2) Incubation was conducted for one night at 4C with a mouse monoclonal anti-oa tubulin TU-01 antibody diluted 1:300 in incubation solution; this antibody was produced by Dr. V. Viklicky (Institute of Molecular Genetics, Praha, Czech Republic) [27]. The oocytes were then rinsed in a washing solution: PBS with 0.2% BSA, 0.05% NaN 3, and 0.05% saponin. 3) Incubation was performed for 1 h at room temperature with tetramethylrhodamine isothiocyanate-conjugated goat antimouse IgG (Biosys, Compiegne, France) diluted 1:150 in the incubation solution, followed by rinsing. Oocytes were then incubated for 30 min at room temperature in 100 jig/ml of fluorescein isothiocyanate-conjugated peanut agglutinin in washing solution to detect distribution of CG. After rinsing, oocytes were stained with 1 ig/ml bis-benzimide (Hoechst 33342; Sigma) in PBS for 6 min for DNA detection. They were then mounted between

234 GOUDET ET AL. TABLE 1. Oocyte recovery rate from nonpreovulatory follicles (< 35 mm) according to group.* Groups Luteal Follicular phase phase Item Luteal phase + CEG + CEG Follicular aspiration attempts (n) 20 20 30 Punctured follicles (n) 222 195 255 Recovered COCs (n) 94 72 110 COCs per follicle 0.42 0.37 0.43 COCs per attempt 4.7 3.6 3.7 * The recovery rate per follicle was not significantly different between the 3 groups. slide and cover slide in a mixture of Mowiol V4-88 (133 mg/ml; Hoechst, Frankfurt, Germany) and n-propyl gallate (5 mg/ml; Sigma). The slides were kept at 4 0 C in darkness until observation. Controls for immunofluorescence were performed using no primary antibody, and those for lectin labeling were performed by means of a previous incubation with 100 mm D-galactose in PBS. Confocal Laser Scanning Microscopy The oocytes were observed under a confocal laser scanning microscope (CLSM 310; Carl Zeiss, Thornwood, NY). Immunolabeling and lectin labeling were visualized in the confocal mode, whereas Hoechst staining was detected by conventional epifluorescence. For fluorescein, an argon ion laser adjusted at 488-nm wave length was used; for rhodamine, a helium-neon ion laser adjusted at 543 nm was used. The size of the meiotic spindles was measured along the longitudinal and the lateral axis, respectively, with Visilog software (version V4.1.4; Neosis, Orsay, France). Each measure was performed twice, with an error between the measures of 0.6 pxm. Follicular Fluid Endocrinology Steroid concentrations in the follicular fluid were determined by RIA without extraction according to the methods of Saumande et al. [28] for progesterone, of Terqui et al. [29] for estradiol-17[, and of Hochereau de Reviers et al. [30] for testosterone. Intraassay variability and limits of sensitivity were, respectively, 8.1% and 0.10 ng/ml for progesterone, 6.3% and 0.015 ng/ml for estradiol-1713, and 8.1% and 0.10 ng/ml for testosterone. For each steroid, all samples were analyzed in the same assay to avoid interassay variability. Statistical Analysis The chi-square test was used to compare oocyte recovery and maturation rate in the three groups (F, L+, and L-). Analysis of oocyte maturation after IVM according to follicle diameter was performed by logistical regression analysis. Nonparametric test (G-test = 21-test) was used for comparison of oocyte recovery, nuclear maturation, meiotic spindle morphology, and CG localization between the various classes of follicle diameters. Variance analysis was used to compare oocyte spindle sizes and oocyte diameters. Comparison of steroid concentrations in different follicle groups was performed by a nonparametric test (Kolmo 2). TABLE 2. Oocyte recovery rate from nonpreovulatory follicles (< 35 mm) according to follicular diameter.* Follicular diameter (mm) Item 5to9 10to14 15to19 20to24 25to29 30to34 Punctured follicles (n) 188 222 124 84 44 10 Recovered COCs (n) 88 92 50 26 16 4 COCs per follicle 0.47 0.41 0.40 0.31 0.36 0.40 * The recovery rate per follicle was not significantly different between the follicular diameters. RESULTS Oocyte Recovery In the 10 mares, 708 follicles larger than 5 mm were punctured, alternatively at the end of the follicular phase (3 attempts per mare) and in midluteal phase (4 attempts per mare: 2 attempts with CEG injection and 2 attempts without). At the end of the follicular phase, preovulatory (larger than 35 mm in diameter) and nonpreovulatory follicles were punctured. In the midluteal phase, all punctured follicles were nonpreovulatory; two follicles were larger than 35 mm, however, but these were luteinized follicles with echogenic walls and irregular shape, and filled with fluid just after puncture. Preovulatory follicles (larger than 35 mm). From the 30 attempts in follicular phase, 36 preovulatory follicles were flushed and 28 oocytes were collected. Seventeen preovulatory follicles could not be flushed because they ovulated before puncture. Averages of 0.9 oocytes per attempt and 0.78 oocytes per preovulatory follicle were obtained. Nonpreovulatory follicles. From the 70 puncture attempts (30 in follicular phase, 20 in luteal phase with CEG injection, and 20 in luteal phase without CEG injection), 672 nonpreovulatory follicles were flushed and 276 COCs were recovered. Averages of 4 COCs per attempt and 0.41 COCs per follicle were obtained. The recovery rate per follicle was not significantly different between the three groups (Table 1). It tended to decrease with increasing diameter of aspirated follicles (Table 2). The COC recovery rate per mare ranged from 32.6% (29 of 89) to 60.0% (33 of 55). There was a significant effect of mare on the COC recovery rate (p < 0.05). Oocyte yield per cycle. The interval between attempts was 10.4 + 3.6 days after puncturing in follicular phase, 12.1 + 2.3 days after puncturing in luteal phase without CEG injection, and 11.3 + 1.7 days after puncturing in luteal phase with CEG injection. These intervals were not significantly different. The mean interval between 2 attempts in the same cycle stage was 22.1 days, the duration of a cycle. Puncturing once in follicular phase and once in luteal phase yielded a mean of 8.9 COCs per 22 days. Moreover, the mean number of punctured follicles and the mean number of collected oocytes in each group showed no increase or decrease during the successive punctures. Plasma progesterone levels. Puncture of the preovulatory follicle was always followed by a rapid increase in plasma progesterone, secreted by the resulting corpus luteum (data not shown). The maximum level was 17.2 9.5 ng/ml. The corpus luteum was suppressed by the prostaglandin injection on the luteal-phase puncture day. In one mare, the increase of progesterone did not occur until 11 days after the puncture of the preovulatory follicle, and the maximum concentration was 2.6 ng/ml.

EQUINE OOCYTE COMPETENCE FOR IN VITRO MATURATION 235 FIG. 1. Equine COCs. A) Oocyte with expanded cumulus at collection; x1 7. B) Oocyte with compact corona radiata at collection; x 170. C) Oocyte with compact cumulus and cells from follicular wall at collection; x170. D) Denuded and degenerated oocyte without a cytoplasmic membrane; x255. During the luteal phase, the follicles were punctured 34 h after the largest follicle reached 18 mm. However, as the follicles do not grow at a constant rate, some punctured follicles were larger than expected. Nine nonpreovulatory follicles punctured in luteal phase led to plasma progesterone concentrations greater than 2 ng/ml. This was the case for 2 of I follicles 22-24 mm (18%), 5 of 11 follicles 25-27 mm (45%), and 2 of 2 follicles larger than 29 mm (100%). On average, the largest follicle diameter was 24 + 3.8 mm in puncture attempts during the luteal phase without CEG injection and 24 2.8 mm in puncture attempts during the luteal phase with CEG injection. Progesterone concentrations were greater than 2 ng/ml after 3 of 15 punctures in luteal phase without CEG injection and after 6 of 15 punctures in luteal phase with CEG injection; these rates were not different. Cumulus Aspect at Recovery At collection, the 304 recovered COCs were classified into three groups: expanded cumulus (Fig. A), compact corona radiata (CR) (Fig. B), and compact cumulus and cells from the follicular wall (W) (Fig. 1C). Expanded cumulus. In follicular phase, 138 COCs were collected. From preovulatory follicles (larger than 34 mm), 24 of 28 recovered COCs (86%) had an expanded cumulus. In follicles with diameters from 30 to 34 mm, diameters from 25 to 29 mm, and diameters 24 mm and smaller, 4 of 4, 4 of 9, and 6 of 97 COCs, respectively, had an expanded cumulus. In luteal phase, 166 COCs were obtained and only 4 were expanded. Compact cumulus. Of the 70 puncture attempts made in luteal and follicular phases, 55 CR (21%) and 207 W (79%) were recovered. No significant difference in the portion of

236 GOUDET ET AL. FIG. 2. Chromatin configurations in equine oocytes. A) Germinal vesicle with diplotene chromatin; B) dense chromatin; C) metaphase I; D) metaphase 11 (m) and polar body (gp). x400. COCs in classes CR and W was observed according to group Evaluation of Oocytes from Expanded COCs (respectively, 19% and 81% in follicular phase, 20% and 80% in luteal phase with CEG, 24% and 76% in luteal phase Chromatin configuration. Of the 42 expanded COCs colwithout CEG). No clear relationship between follicle size lected, 3 were lost during staining and 39 were analyzed for and compact cumulus aspect (CR or W) could be estab- nuclear maturation. Of these, 14 were degenerated (Fig. 1D), lished. 2 were immature (Fig. 2A), 9 had resumed meiosis (Fig. 2,

EQUINE OOCYTE COMPETENCE FOR IN VITRO MATURATION 237 FIG. 4. CG localization on serial optical sections in equine oocytes. A) CG migration achieved: the majority of the CG are lining the oolemma. B) No CG migration: the CG are located in the medullary zone, and no CG are lining the oolemma; x250. B and C), and 14 were mature (Fig. 2D). The chromatin configuration in relation to follicular size is presented in Table 3. In follicles 30 mm and larger, 52% of expanded COCs (2/4 + 12/23) enclosed a mature oocyte (metaphase II or telophase I). No oocyte from follicles 29 mm and smaller were mature. In total, from the 30 attempts in follicular phase, 14 mature oocytes with expanded cumulus were recovered, giving an average of 0.5 mature oocytes per attempt. Meiotic spindle morphology. The 14 mature oocytes (telophase I or metaphase II) were examined for meiotic

238 GOUDET ET AL. FIG. 5. Influence of diameter of the follicle of origin upon the chromatin configurations of equine oocytes after IVM. Mature: telophase I or metaphase II; resumption of meiosis: oocytes with dense chromatin or metaphase I; immature: germinal vesicle with diplotene chromatin; degenerated: oocytes without a cytoplasmic membrane or fragmented. The maturation rate significantly increased with the follicle diameter (p < 0.1%). The maturation rate in diameter class 5-9 mm was significantly less than the maturation rate in the other classes (p < 1%). spindle morphology. Eight oocytes (57%) had a barrelshaped spindle with two poles and distinct microtubules between poles; the chromosomes were aligned along the metaphase plate. This was defined as a normal spindle (Fig. 3A). One oocyte was in telophase I (Fig. 3B), with the two sets of chromosomes arranged at the two spindle poles. Three oocytes had a barrel-shaped spindle with two poles and disorganized microtubules between poles; the chromosomes were arranged into the meiotic spindle. This was defined as a disorganized spindle (Fig. 3C). Two oocytes showed a dense network but no poles and no distinct microtubules; the chromosomes remained on this network. This was defined as scattered tubulin (Fig. 3D). The 5 metaphase I oocytes from expanded COCs had a normal spindle. CG localization. Of the 14 mature oocytes (metaphase II or telophase I), 11 were analyzed for CG localization; 3 were not analyzed due to technical problems. CG migration was achieved or in progress in 11 of 11 oocytes: in 10 of the oocytes, most of the CG were lining the oolemma (Fig. 4A); in 1 of them, the CG had an uniform distribution between the oolemma and the medullary zone. In none of the oocytes was all the CG located in the medullary zone with no CG lining the oolemma (see description of CG localization after IVM, below; Fig. 4B). Ability of Compact COCs to Mature In Vitro Cumulus aspect after IVM. Oocytes with compact cumulus at recovery were cultured for 30 h. After IVM, 95% of the COCs had an expanded cumulus (249 of 262), and 5% remained compact. Meiotic maturation. From the 70 puncture attempts in luteal and follicular phase, 262 compact COCs were col- TABLE 3. Chromatin configuration of expanded COCs according to follicular diameter.* Follicular diameter (mm) Chromatin configuration' 5 to 24 25 to 29 30 to 34 35 to 50 Mature (n) 0 0 2 12 Resumption of meiosis (n) 1 3 1 4 Immature (n) 2 0 0 0 Degenerated (n) 6 0 1 7 Total (n) 9 3 4 23 * No statistical analysis was performed because of the small number of oocytes. ' Mature: telophase I or metaphase II; resumption of meiosis: oocytes with dense chromatin or metaphase I; immature: germinal vesicle with diplotene chromatin; degenerated: oocytes without a cytoplasmic membrane or fragmented. FIG. 6. Influence of diameter of the follicle of origin upon the chromatin configurations of equine oocytes after IVM, according to the hormonal environment. Mature: telophase I or metaphase II; resumption of meiosis: oocytes with dense chromatin or metaphase I; immature: germinal vesicle with diplotene chromatin; degenerated: oocytes without a cytoplasmic membrane or fragmented. Regardless of group, the nuclear maturation rate increased with the follicle diameter (p < 5%).

EQUINE OOCYTE COMPETENCE FOR IN VITRO MATURATION 239 FIG. 7. Influence of diameter of the follicle of origin on metaphase II and telophase I spindle morphology after IVM. The spindle morphology was not significantly different between the classes of follicle diameters. lected and cultured in vitro; 258 of these were analyzed for nuclear maturation after IVM and 4 were lost. Their meiotic stages according to diameter of the follicle of origin are shown in Figure 5. The maturation rate significantly increased with an increase in follicular diameter (p < 0.001). In follicles 5-9 mm, 20% (17 of 83) of the oocytes reached metaphase II or telophase I, whereas in follicles 10-50 mm, 45% (78 of 175) of the oocytes were mature; these two rates were significantly different (p < 0.01). The maturation rate did not vary significantly with the cumulus aspect before IVM: in class CR, 17 of 55 (31%) oocytes reached metaphase II or telophase I vs. 78 of 203 (38%) in class W. Of the 12 oocytes whose cumulus remained compact after IVM, 3 (25%) were in metaphase II. This maturation rate was not significantly different from that of oocytes whose cumulus was expanded after IVM (37%, 92 of 246). The maturation rate did not vary significantly between the different mares. Figure 6 shows the meiotic stage after IVM according to diameter of the follicle of origin and group (in follicular phase, in luteal phase with CEG injection, and in luteal phase without CEG injection). The results for follicle diameter classes from 20 to 50 mm were combined because of the small number of oocytes collected. Regardless of the group, the nuclear maturation rate increased with the follicular diameter (p < 0.05), and the maturation rate of oocytes from 5-9-mm follicles was less than that for the other follicles (not significant for follicular phase and for luteal phase without CEG; p < 0.05 for luteal phase with CEG). The maturation rate tended to be different among the three groups. Only 28% (25 of 90) of the oocytes recovered in luteal phase without CEG injection were able to mature in vitro. The CEG injection in luteal phase increased the maturation rate (38%, 27 of 71), especially for oocytes from follicles 10 mm and larger. The maturation rate of oocytes recovered in follicular phase with CEG injection was the highest (44%, 43 of 97). Nevertheless the difference between these three maturation rates was not significant. Oocytes with dense chromatin and oocytes in metaphase I, telophase I, or metaphase II resumed meiosis. Within all three groups, the rate of oocytes able to resume meiosis significantly increased with the follicle diameter (p < 0.001), and this rate was significantly lower in follicle diameter class 5-9 mm than in classes 10-50 mm (p < 0.01). Meiotic spindle morphology. After IVM, 95 oocytes were in metaphase II and telophase I, of which 93 were examined for meiotic spindle morphology. As outlined above, we defined spindles as normal spindles, telophase I spindles, disorganized spindles, and scattered tubulin (Fig. 3, A-D, respectively). After IVM, some oocytes had a multipolar spindle (Fig. 3E). The meiotic spindle morphology was analyzed in terms of the diameter of the follicle of origin (Fig. 7). In follicles smaller than 10 mm, 14 of 16 oocytes had a normal metaphase II or telophase I spindle (88%). In follicles 10 mm or larger, only 55 of 77 oocytes had a normal spindle (71%). Oocytes from follicles 5-9 mm tended to be different from oocytes from follicles 10-50 mm, but the difference was not significant. The oocyte number was too low to allow any analysis according to group. After IVM, 65 oocytes were in metaphase I. Among them, 41 had a normal metaphase spindle (63%). The meiotic spindle morphology was not significantly different between the classes of follicle diameters (Fig. 8). CG localization. Of the 258 oocytes analyzed for nuclear maturation after IVM, 196 were examined for CG localization in the ooplasm in relation to follicular size (Fig. 9). In 68% (40 of 59) of the analyzed oocytes recovered from follicles smaller than 10 mm, CG migration was achieved or in progress after IVM. In follicles 10 mm or larger, 89% (122 of 137) of the analyzed oocytes were in this state. These two follicular populations were significantly different (p < 0.01). CG localization after IVM according to nuclear stage is presented in Figure 10. CG migration was achieved or in progress after IVM in 95% (77 of 81) of mature oocytes, FIG. 8. Influence of diameter of the follicle of origin upon metaphase I spindle morphology after IVM. The spindle morphology was not significantly different between the classes of follicle diameters.

240 GOUDET ET AL. FIG. 9. Influence of diameter of the follicle of origin on CG localization after IVM. The percentage of oocytes with CG migration achieved or in progress in diameter class 5-9 mm was significantly different from the percentage in the other classes (p < 1%). in 89% (58 of 65) of oocytes showing resumption of meiosis, and in 54% (26 of 48) of immature oocytes. The oocyte number was too low to allow any analysis according to group. Meiotic Spindle Size Of the COCs analyzed at recovery, i.e., after in vivo maturation, 8 of 14 (57%) metaphase II and telophase I oocytes and 4 of 5 (80%) metaphase I oocytes had a normal spindle. Of the COCs cultured in vitro, 54 of 93 (58%) metaphase II and telophase I oocytes and 41 of 65 (63%) metaphase I oocytes had a normal spindle after IVM. Among these 107 oocytes, 2 were lost and only 105 were analyzed for spindle width and length (8, 3, 53, and 41 oocytes, respectively). Collectively, COCs analyzed after in vivo maturation and COCs cultured in vitro demonstrated metaphase I spindles that were significantly wider than metaphase II spindles (21.6 + 4.3 tlm vs. 14.5 + 3.0 pxm, respectively, p < 0.001). Also, when all metaphase I and metaphase II oocytes were grouped, spindles from oocytes cultured in vitro were significantly wider and longer than spindles from oocytes analyzed after in vivo maturation (width: 17.9 ± 5.1 Lm vs. 13.9 + 2.9 pxm, respectively, p < 0.001; length: 18.0 + 4.9 pxm vs. 12.0 + 3.8 pxm, respectively, p < 0.001). FIG. 10. Influence of nuclear stage on the CG localization after IVM. M II: metaphase II; T I: telophase I; M : metaphase I; D Ch: dense chromatin; GV: germinal vesicle with diplotene chromatin. Oocyte Diameter The oocyte diameter was measured in COCs analyzed after in vivo maturation and in COCs analyzed after IVM (Table 4). The oocytes from COCs analyzed after in vivo maturation were significantly smaller than those from COCs analyzed after IVM (p < 0.01). Among the COCs cultured in vitro, no clear relationship between diameter of the follicle of origin and oocyte diameter could be established. Overall oocyte diameter decreased as nuclear maturation progressed: the oocytes that remained in germinal vesicle stage were larger than the oocytes that resumed meiosis (p < 0.05), and the metaphase I oocytes were larger than the metaphase II oocytes (p < 0.001). Follicular Fluid Endocrinology The concentrations of steroids were measured in follicular fluid from follicles larger than 30 mm. The results ranged as follows: progesterone, 57-10 895 ng/ml with a mean of 900 ng/ml; estradiol, 161-3251 ng/ml with a mean of 1575 ng/ml; testosterone, 1-104 ng/ml with a mean of 14 ng/ml. No relationship was found between steroid concentrations and oocyte recovery rate or between steroid concentrations and oocyte nuclear maturation. DISCUSSION The first aim of this study was to find an optimal rhythm of successive punctures, adjusted to the follicular growth pattern, in order to maximize the oocyte yield. Early attempts at in vivo collection of equine oocytes were made via standing flank transcutaneous puncture [31-33]. In these studies, ovaries were aspirated only during estrus, and only the preovulatory follicle was punctured. The procedure did not allow visualization of tissues, and therefore aspirations were conducted blindly. Recovery rates were approximately 65%. Recently, efforts have been made to develop transvaginal ultrasound-guided follicular puncture in the mare [34]. Using this technique, recovery of oocytes from preovulatory follicles after induction of ovulation has been 50-85% [18, 35]. In this study, we performed transvaginal ultrasound-guided punctures at the end of the follicular phase after induction of ovulation. The recovery rate for oocytes from preovulatory follicles was 78%, a rate similar to previous results [18, 35], but the yield was only 0.9 preovulatory oocytes per cycle. The puncture of small follicles was first proposed in the cow [36] and

EQUINE OOCYTE COMPETENCE FOR IN VITRO MATURATION 241 TABLE 4. Oocyte diameter (m) according to nuclear stage and maturation conditions (mean + SD).* Nuclear stage t Maturation M II T I M I D Ch GV Deg Total In vitro 114 + 7 116 6 119 + 7 119 + 12 123 -+ 7 119 + 11 (80) (15) (65) (6) (63) (9) 1 18 + 8 d In vivo 110 ± 5 125 0 115 + 15 108 ± 13 120 ± 2 109 ± 13 112 + 11 e (13) (1) (5) (4) (2) (2) Total 114 7a 116 6 ab 118 8b 115 -- 13 ab 123 ± 7c 117 -- 12ab * Statistical analysis was performed on total values; sample size in parentheses. M II: metaphase II; T I: telophase I; M I: metaphase I; D Ch: dense chromatin; GV: germinal vesicle with diplotene chromatin; Deg: degenerated. abc Values with different superscripts differ significantly (p < 0.05). de Values with different superscripts differ significantly (p < 0.01). was adapted to the mare by Cook et al. [35]. Several studies were then conducted to further develop this method and investigate the influence of various parameters (different vacuums, single- or double-lumen needles with various diameters) [22, 23, 35, 37]. Recovery rates displayed high variability, ranging from as low as 12% [23, 37] to as high as 47% [38]. The technique used in this study was established in our laboratory by Duchamp et al. [22], who obtained an average of 0.29 COCs per nonpreovulatory follicle. In the present study, 0.41 COCs per nonpreovulatory follicles were collected. This higher yield is probably due to the increased experience of the technicians. The recovery rate in nonpreovulatory follicles tended to decrease with increasing follicle diameter, a finding that agrees with several previous studies [22, 23, 35]. This may be the case because it is easier to scrape the whole follicular wall when the surface is smaller. The recovery rate from preovulatory follicles was higher than from nonpreovulatory follicles because of the looser connections between the oocyte and the follicular wall during cumulus expansion caused by administration of exogenous gonadotropins. According to our results, exogenous hormones and the hormonal environment had no effect on nonpreovulatory follicles, as the recovery rate was not significantly influenced by the CEG injection or by the cycle stage. Additionally, the recovery rates for immature oocytes from mares are generally lower than those obtained in cattle, which range from 55% [36] to 70% [39]. Hawley et al. [40] reported that the cumulus of equine oocytes has a broader base and closer attachment to the follicle wall than that of bovine oocytes, and that unique cumulus cell projections into the thecal pad anchor the equine oocyte to the follicle wall. To improve the oocyte recovery potential for cyclic mares, several patterns of successives punctures have been tested. In 1993, Cook et al. [35] performed aspirations during estrus and diestrus on light-horse mares. An average of 1.5 oocytes were recovered per cycle. Two years later, Duchamp et al. [22] collected 3 oocytes from saddle mares and 1.8 oocytes from pony mares per 21 days with weekly punctures of all follicles larger than 8 mm. But they obtained irregular responses with alternating follicular growth in terms of follicle number and follicle size. Punctures on any occasion when a follicle had reached 15 mm allowed an average collection of 10 oocytes per 21 days [22]. However, some follicles had an irregular shape and echogenic dots, 27% of oocytes had an expanded cumulus, half of which had resumed meiosis. In our study, the puncture protocol was determined by follicular size rather than by day interval, and aspirations were performed during estrus and diestrus on all follicles larger than 5 mm in diameter; smaller follicles were too difficult to puncture. This puncture protocol improved the recovery rate to an average of 8.9 COCs per 22 days. This rhythm is adjusted to the follicular growth pattern so that we obtain well-characterized populations of oocytes. The puncture of preovulatory follicles results in an active corpus luteum [41]. The delay in the increase of progesterone in one mare may have been due to damage in the granulosa cells. As previously described [22], luteinization occurred after puncture in some follicles between 22 and 27 mm. The occurrence of luteinization was not increased by exogenous gonadotropins. Little information on steroid concentrations in equine preovulatory follicles has been published. Our results are similar to previous data on preovulatory follicles from pony mares [18]. The great variability between follicles and the low number of follicles did not allow us to find any relationship between steroid concentrations and oocyte recovery rate or oocyte nuclear maturation. At collection, the oocytes were surrounded either by an expanded cumulus, by a compact corona radiata, or by a compact cumulus attached to cells from the follicular wall. As in many mammals, induction of ovulation in the mare leads to cumulus expansion in the dominant follicle [1, 18]. In preovulatory follicles as well as in follicles with diameters from 30 to 34 mm, most recovered COCs had an expanded cumulus, in agreement with findings of Duchamp et al. [26]. Therefore, it appears that CEG injection caused follicular maturation and cumulus expansion in follicles whose diameter is around 30 mm or larger. In our study, only 4 follicles from 35 to 38 mm contained COCs with a compact cumulus. They were all contemporaneous with a preovulatory follicle that had ovulated or that contained a mature oocyte. Thus, they may not have been dominant follicles and, in spite of their large size, they may not be responsive to the exogenous gonadotropins. From 27 expanded COCs (follicles 30 mm and larger), 14 mature oocytes were collected (Table 3). Moreover, 17 preovulatory follicles ovulated before puncture. One can speculate that these follicles enclosed mature oocytes with expanded cumulus complexes. With these taken into account, 70% of expanded COCs enclosed a mature oocyte (14 + 17 / 27 + 17). This result is low compared to findings in pony mares [18, 42] and could be explained by the fact that more spontaneous multiple large follicles occur in large mares than in pony mares [43]. The unique large follicle in pony mares is undoubtedly the follicle that will ovulate and that contains a mature oocyte, whereas in large mares, not all the large follicles are intended for ovulation. They may grow, and cumulus expansion may occur, but the oocytes may be unable to complete maturation and ultimately will degenerate. In this study, 13 nonmature or degenerated oocytes surrounded by an expanded cumulus were collected from follicles 30 mm and larger. Of these,

242 GOUDET ET AL. 9 were contemporaneous with a large follicle that either ovulated or contained a mature oocyte, or with a follicle that was larger than 35 mm but whose oocyte could not be recovered. One of them came from a luteinized follicle. Only 3 oocytes were collected from a unique preovulatory follicle and were degenerated with no apparent reason. In humans, there have been reports of oocytes with an abnormal spindle but normal, compact chromosomes [44, 45]. Therefore, the analysis of nuclear stage by DNA staining does not allow detection of alterations in the spindle that may contribute to induction of aberrant embryonic development [46, 47]. Research on spindle structure may then be of considerable interest. The cytoskeletal organization of the oocyte has been described in many species and seems to be common in most mammals. However, to our knowledge, no description has been reported in the equine species. As a first step, we analyzed meiotic spindle morphology in the 14 metaphase II and telophase I oocytes collected with an expanded cumulus from follicles 30 mm and larger. Eight oocytes had a barrel-shaped spindle with two poles and distinct microtubules between poles, defined as a normal spindle. This equine spindle is similar to the normal spindle described in Xenopus [48], mouse [49, 50], human [45, 51], bovine [52], and porcine [53] oocytes. One oocyte was in telophase I with a spindle structure typical of that of Xenopus [48], mouse [49, 50], and porcine [53] oocytes. Three oocytes had a barrel-shaped spindle with two poles and disorganized microtubules between poles. This structure may be a normal step in microtubule dynamics during equine meiotic progression, as it looks like the prometaphase spindle described in Xenopus [48], mouse [50], and porcine [53] oocytes. However, one must be careful with this interpretation, as Pickering et al. [11, 51] obtained barrel-shaped oocytes with internal disorganization of spindle microtubules in aged or cooled oocytes. This disorganization was nevertheless often associated with chromosome dispersion, which was not observed in the present study. Among the 14 oocytes we analyzed, 2 had scattered tubulin. This is undoubtedly a sign of spindle degeneration, because no interphase network of microtubules is ever formed during the normal transition from metaphase I to metaphase II [48, 49] and such a network was observed in aged human oocytes [54] and in cooled human [45, 51] and bovine [52] oocytes. The presence of an abnormal spindle in in vivomatured oocytes has been previously reported in the human [11, 45]. Among the 14 metaphase II and telophase I oocytes collected with an expanded cumulus from follicles 30 mm and larger, 11 could be analyzed for CG localization as a criterion of cytoplasmic maturation. All apparently progressed through a normal maturation, as Bezard et al. [18] showed that in equine preovulatory oocytes collected just before ovulation, CG line the oolemma or lie in a homogeneous distribution in the ooplasm. In follicles smaller than 30 mm, 14 of 106 COCs had an expanded cumulus. Comparison of this rate with findings in other reports is not reliable, as the definition of an expanded cumulus is highly variable-from a corona radiata with partially dissociated cumulus [23, 55] to a gelatinous mass providing structural support to detached cumulus cells ([56], this report). Hinrichs [56] recovered most of the expanded COCs in atretic follicles. In the present study, 6 of 9 of the expanded COCs that were collected from follicles smaller than 25 mm contained a degenerated oocyte; therefore the follicle of origin may be atretic. Of the expanded COCs collected in follicles of 25- to 29-mm diameter in follicular phase, 3 of 3 were in metaphase I. The expansion of cumulus cells and resumption of meiosis may be due to the appearance of LH receptors sensitive to the exogenous LH. Of the 70 puncture attempts performed in luteal and follicular phases, 55 CR (21%) and 207 W (79%) were recovered. No significant difference in the proportion of COCs in classes CR and W was observed relative to the three groups or the follicular diameter. Comparison with cumulus morphology as reported by others is difficult-first because the classification systems used to characterize the cumulus are highly variable, and second because the recovery methods are different and some may damage the COCs. However, in most reports, the morphology of compact cumulus did not depend on follicle size up to 30 mm [55, 56]. COCs with compact cumulus at recovery were cultured for 30 h in the present study. After IVM, most COCs had an expanded cumulus. However, as previously reported [3, 6, 57], some COCs remained compact. The degree of cumulus expansion did not necessarily correlate with nuclear maturation, as the maturation rate of oocytes whose cumulus remained compact after IVM was not significantly different from that of oocytes whose cumulus became expanded during IVM. However, these two conditions are necessary, as Testart et al. [58] found that a higher proportion of human oocytes were able to cleave following IVF when cumulus expansion was complete. In this study, the IVM rate was about 37%. In several previous investigations, the maturation rate of equine oocytes reached 70% [6, 57, 59]. Our low maturation rate can be explained by the conditions of IVM and by the origin of the oocytes. The culture medium we used was developed in this laboratory [5] and seems to be quite well adapted to equine oocytes, as the positive effect of Tissue Culture Medium 199 containing serum, estradiol, and equine gonadotropins has been demonstrated (see [60] for review). However, equine gonadotropins are added as crude pituitary extract containing 91% unknown products [26] that could have a negative effect on oocyte maturation. Moreover, oocytes were cultured individually, whereas group culture is more often used [6, 57, 59]. In cattle, culture of oocytes individually, as compared with group culture, results in significantly reduced rates of morula formation [61]. Bruck et al. [62] cultured equine oocytes individually in medium supplemented with crude equine pituitary extract, and the IVM rate was about 38%. On the other hand, most experiments are carried out with oocytes obtained at slaughter. The ovaries are thus transported to the laboratory with a delay in the time between oocyte collection and placement in culture medium, leading to a heterogeneous population of oocytes from healthy and atretic follicles. A higher percentage of competent oocytes in follicles in early atresia has been suggested in cattle [61, 63]. Finally, oocytes are often selected before culture according to their cumulus morphology and the appearance of their cytoplasm [4, 59], and the maturation rate is calculated on selected oocytes only. In this study, all oocytes were used and the maturation rate was calculated over all the collected oocytes. We observed that the IVM rate significantly increased with antral follicle size. This is consistent with findings for ovine [64, 65], porcine [66, 67], bovine [68], human [69], and rhesus monkey [70] oocytes. On the other hand, in several rodent species, acquisition of meiotic competence is associated with antrum formation and maximum oocyte size [71-73], and developmental competence is acquired during antral follicular growth [74]. This difference be-

EQUINE OOCYTE COMPETENCE FOR IN VITRO MATURATION 243 tween species can be explained by oocyte size: rodent oocytes reach their full size before antrum formation, whereas porcine and bovine oocytes are still growing when follicles reach the early antral stage [68, 75]. In pigs and cattle, as in rodent species, the ability to resume meiosis is acquired during oocyte growth. In the current study, we were unable to establish any relationship between equine oocyte diameter and follicle size. Moreover, the oocytes that remained in germinal vesicle stage after IVM were larger than the oocytes that resumed meiosis. It seems that in the horse, as in the rhesus monkey [70], meiotic competence is not associated with maximum oocyte diameter. Oocytes that appear fully grown may still need to undergo cytoplasmic changes before they are competent for optimal maturation. The differences in oocyte diameter according to the nuclear stage after IVM may be linked to changes in the osmolarity of the cytoplasm, but whether these differences are detectable before IVM or whether they are due to differing behavior of the oocytes during IVM remains unknown. The expulsion of the polar body may account for a decrease in oocyte diameter. On the other hand, the smaller oocyte diameter after in vivo maturation versus IVM cannot be explained by a difference in osmolarity between the maturation medium and the follicular fluid, since both were evaluated (320 mos for the maturation medium and 290 mos for the follicular fluid). The nuclear maturation rate of oocytes collected in follicles from 5 to 9 mm was significantly lower than the maturation rate of oocytes from larger follicles. Similar results with equine oocytes obtained from slaughterhouse ovaries have been recently published [62]. Additionally, it was observed that CEG injection in the luteal phase tended to increase the IVM rate, but the increase was minimal in follicles smaller than 9 mm. On the other hand, data for prepubertal fillies 4-12 mo of age showed that, on average, the follicles did not exceed 10 mm [76]. Likewise, during deep anestrus, ovarian follicles usually do not develop above 10 mm [77]. Therefore, follicular populations less than 10 mm in diameter are relatively independent of the hormonal environment, and they represent the basal gonado-independent follicular growth. In these follicles, the cytoplasmic maturation rate of oocytes, using CG localization as a criterion, is significantly lower than that of oocytes from larger follicles. These results are not surprising, as CG localization seems to be linked with the stage of nuclear maturation. Finally, the rate of normal metaphase II or telophase I spindles in oocytes from follicles smaller than 9 mm tend to be higher from follicles larger than 10 mm, whereas they are less competent for IVM. It seems that, when they are able to mature, maturation takes place normally. On the other hand, the percentage of normal metaphase I spindles in oocytes cultured in vitro is low (63%) compared with the rate for normal metaphase II and telophase I spindles (74%). This can be explained by the aging of oocytes arrested in metaphase I. In this study, the mare's reproductive status did not significantly affect the percentage of metaphase II oocytes after in vitro culture. However, the hormonal environment in the follicular phase tended to increase the percentage of competent oocytes. Conflicting results are obtained with oocytes from slaughterhouse ovaries [62, 78]. The reason may be that the mares are slaughtered at different phases of the cycle, with cyclic stage determined relative to plasma progesterone concentrations and presence of corpora lutea on the ovaries. However, many other factors, not taken into account, bring about variations in the hormonal environment, such as the presence of a dominant follicle, of the LH rise, and of a persistent corpus luteum. The populations of oocytes are therefore heterogeneous. Repeated in vivo follicular punctures allow collection of well-characterized oocyte populations and lead to strict and reliable comparisons within the same animal. When we pooled oocytes with a metaphase II or metaphase I spindle at recovery and oocytes with a metaphase II or metaphase I spindle after IVM, metaphase I spindles were significantly wider than metaphase II spindles. As previously reported in the mouse oocyte [49], the size of the spindles is correlated with the number of chromatids present within them. Moreover, spindles obtained after IVM are significantly wider and longer than spindles obtained at recovery. It seems that a general change in the critical concentration of tubulin in the cytoplasm may induce an alteration in the size of the spindle [79]. In vitro culture may allow the same nuclear maturation as in vivo maturation but may induce a change in the concentration of cytoplasmic proteins, such as tubulin, and also other factors involved in regulation of the cell cycle. In conclusion, a systematic protocol for successive punctures that lead to well-characterized populations of oocytes was developed in this study. The protocol provides for an increase in the number of oocytes collected to an average of 8.9 COCs per 22 days. Moreover, our study demonstrates that the acquisition of meiotic competence in equine oocytes occurred progressively during antral follicle growth. 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