Effects of Follicular Fluid during In Vitro Maturation of Bovine Oocytes on In Vitro Fertilization and Early Embryonic Development'

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1 BIOLOGY OF REPRODUCTION 55, (1996) Effects of Follicular Fluid during In Vitro Maturation of Bovine Oocytes on In Vitro Fertilization and Early Embryonic Development' A. Romero-Arredondo and G.E. Seidel, Jr. 2 Animal Reproduction and Biotechnology Laboratory, Colorado State University, Fort Collins, Colorado ABSTRACT In vivo, oocytes mature while bathed in preovulatory follicular fluid, so we studied maturation of oocytes in vitro in media supplemented with such fluid. Bovine oocytes were obtained from 2- to 6-mm follicles from slaughterhouse ovaries and were matured for 24 h in tissue culture medium % estrous cow serum supplemented with bovine follicular fluid (BFF) obtained from a preovulatory follicle either before (0 h) or 8 or 20 h after a GnRH-induced LH surge. After in vitro fertilization, oocytes matured in 10% or 40% 20-h BFF had higher cleavage rates (p < 0.05) than those matured in 10% or 40% 0-h BFF (80% and 86% vs. 46% and 51%, respectively); oocytes matured in 8-h BFF were intermediate. A greater percentage of oocytes showed cumulus expansion with 20-h BFF than with 0-h BFF (p < 0.05). In experiment 2, more (p < 0.05) embryos from oocytes matured in 20% or 40% 20-h BFF developed to blastocysts after culture of embryos compared to those matured in 20% or 40% 0-h BFF (29% and 29% vs. 7% and 14%, respectively) or those matured in no BFF (12%). Embryos from oocytes matured in a mixture of 0-h and 20-h BFF developed to the blastocyst stage similarly (27%) to those matured in 20-h BFF, suggesting that positive factors in 20-h BFF overcame any inhibitory effect of 0-h BFF. To summarize, bovine oocytes matured with BFF collected 20 h after LH surge result in higher cleavage rates after in vitro fertilization and more blastocysts than those matured in 0-h BFF. When the factors responsible are identified, they might be used to enhance maturation of oocytes in vitro. INTRODUCTION Oocytes from healthy follicles are in meiotic arrest at the dictyate stage of meiosis-i until released by atresia or the LH surge. Meiotic inhibitors occur both in porcine and murine follicular fluids [1-3] and are produced by cells of the bovine follicle [4]. After the LH surge, the oocyte within the dominant follicle is committed to resume meiosis. However, the mechanisms responsible for this event have not yet been determined. Currently two hypotheses have been proposed: 1) resumption of meiosis is due to decreased effects of meiotic inhibitor(s), mediated by a reduction in the number of gap junctions between granulosa cells [5]; or 2) a meiotic stimulator generated within the follicular milieu overcomes meiotic arrest [6, 7]. The experiments described here were designed to determine whether acceleration of oocyte maturation produced by bovine follicular fluid (BFF) collected 20 h after the LH surge [8] would result in improved rates of fertilization and embryo development. Accepted June 20, Received June 29, 'This work was supported in part by the Colorado State University Experiment Station through USDA Regional Project W-171. A. Romero- Arredondo was supported by a grant from CONACYT, Mexico. 2Correspondence. FAX: (970) MATERIALS AND METHODS Collection of Follicular Fluid Crossbred beef cows received injections of 25 mg prostaglandin F 2. (Lutalyse; Pharmacia and UpJohn Inc., Kalamazoo, MI) on Day 15 or 16 of the estrous cycle. Estrus detection started 42 h later and continued hourly for 48 h. Blood samples were collected from the tail vein of all cows at the first sign of mounting activity that usually precedes standing estrus and were repeated at 6-h intervals. At the beginning of standing estrus, another blood sample was collected, and the ovary with the largest follicle was identified by ultrasonography. This ovary was collected per vaginum with an craseur either immediately (0-h BFF) or 8.5 or 20.5 h after an injection with 100 Rg of GnRH (i.m.) administered at the onset of standing estrus (termed 8- and 20-h BFF, respectively). A blood sample also was obtained either 1 h after GnRH injection or immediately after removal of the ovary in cows not receiving GnRH (0-h BFF). Follicular fluid from the dominant follicle was aspirated, supplemented with Hepes buffer (to 25 mm) and pentostatin (20 plm) to inhibit further purine metabolism, and centrifuged at 4000 x g for 10 min; the supernatant was stored at -70C. Serum concentrations of LH were determined by RIA [9], and follicular fluid from cows showing no LH response to GnRH or showing a precocious LH surge was discarded. Concentrations of estradiol-17[ and progesterone were determined in follicular fluid by RIA [10, 11] and used to verify the dominant status [8] of the collected follicle; in a few cases the morphologically dominant follicle was atretic [8], and the follicular fluid was discarded. Collection and Selection of Oocytes Ovaries were collected at a local slaughterhouse from culled dairy and beef cows and transported to the laboratory in sterile 0.15 M NaCl containing 100 IU/ml penicillin-g and 50 pxg/ml streptomycin sulfate. Ovaries were maintained between 25 and 30 C for the 2-4 h needed for collection and transportation. At the laboratory, ovaries were handled aseptically in a warmed room (28-32 C), trimmed of excess tissue, rinsed with 0.15 M NaCl, immersed in 70% ethanol for 20 sec (which may not be beneficial), and rinsed again with 0.15 M NaCl. Ovaries were then placed into a beaker of 0.15 M NaCl until collection of oocytes. Cumulus-oocyte complexes were aspirated from antral follicles (2-6 mm in diameter) with a washed and sterilized plastic syringe (6 or 12 ml) and an 18-gauge needle containing ml of sterile modified Dulbecco's PBS [12], without macromolecules or serum. Aspirate was expelled into Petri dishes (#1008; Falcon Plastics, Los Angeles, CA) on a warming plate at 37C. A stereomicroscope at was used to select oocytes with homogeneous, evenly granulated cytoplasm, a compact cumulus mass of 4-5 or more layers, and a visible nucleus (germinal vesicle).

2 FOLLICULAR FLUID EFFECTS ON BOVINE OOCYTE MATURATION 1013 Media for Oocyte Maturation, Sperm Capacitation, Fertilization, and Culture of Embryos Oocytes were matured in Hepes-buffered tissue culture medium (TCM)-199 with Earle's salts (M 2520; Sigma Chemical Company, St. Louis, MO) plus 100 IU penicillin G and 50 pjg streptomycin sulfate/ml and 10% (v:v) heattreated (56 C, 30 min) serum from a cow in estrus (mtcm- 199). Estradiol-17{ (E-8875, lot 34F-0540; Sigma) was added to the maturation treatment controls of both experiments at 1 g/ml, but not to BFF treatments. This was accomplished by first dissolving estradiol-173 (10 mg/ml) in 95% ethanol (stored at -20 C); this solution was then mixed 1:100 with mtcm-199 and frozen; 10 u1l of this stock solution was added per milliliter of maturation media. Follicular fluid collected 0, 8, and 20 h after the LH surge was mixed with mtcm-199 at 10%, 20%, and 40% on a volume/volume basis. Capacitation of sperm was done in Sp-Talp, a modified Tyrode's medium containing 5 IU/ml heparin [13]. Fertilization in vitro was performed in Fert- TALP [13] plus 5 mm caffeine. Embryos were cultured in mtcm-199 diluted by adding 10% (v:v) deionized water (TCM- 199 D) and conditioned by incubation with oviductal epithelial cells before embryos were cultured. To condition media, oviducts collected at slaughter from cows at random stages of the estrous cycle were washed by the method described previously for ovaries, except that oviducts were rinsed in ethanol for 10 sec. Conditioned media were produced as described by Eyestone et al. [14]. Epithelial cells were isolated by scraping the outer surface of the oviduct with a sterile microscope slide while grasping one end with sterile forceps. Cells were forced out the lumen of the fimbrial end of the oviduct into a dish containing modified Dulbecco's PBS. Sheets of cells were disrupted by aspiration through progressively smaller needles until they could pass through a 26-gauge needle. Cells were washed four times by centrifuging 5 sec at 900 x g, and supernatant was replaced twice with fresh PBS and then twice with TCM-199 D. Cells were then cultured in plastic culture flasks for 48 h, at a 1:50 ratio of cells to TCM-199 D (usually 0.2 ml of cell suspension in 10 ml of medium). After culture, medium was poured into 15-ml centrifuge tubes, and cells were allowed to settle to the bottom. Medium was aspirated from settled cells, centrifuged at 900 x g for 10 min, and then filtered through a 0.22 m filter. Conditioned media were frozen in 1- or 2-ml aliquots and stored at -20 C until use. Before use, medium was warmed to 39C, and equilibrated in 5% CO 2 in air. Several batches of conditioned media were made and screened for efficacy. The best batch was used for all replicates. All media were sterilized by filtration (0.22 m filter) after addition of BSA (TALP media) or estrous cow serum (mtcm-199) and adjustment to a final ph of 7.4 with NaOH, which increased osmolarity 3-4 mosm. Culture media were prepared the night before use in Petri dishes (#1008; Falcon); droplets were covered with paraffin oil and placed in 5% CO 2 in air. Oocyte maturation culture drops were 100 ul1 (mtcm-199), while fertilization (Fert- TALP) and embryo culture drops (TCM-199 D) were 250 and 200,ul, respectively. Oocyte Maturation It was important to keep the interval short between isolation of oocytes from follicular fluid and placing them into treatment drops. Therefore, as oocytes were collected, they were washed in groups of five in modified Dulbecco's PBS plus 1% BSA, and transferred within 10 min of aspiration to the respective treatment culture drops. This was repeated until all oocytes for the replicate had been collected (usually 15 per treatment), after which they were washed a second time in respective treatment media and transferred to final maturation drops for 24 h. Sperm Capacitation Frozen semen in 0.5-ml straws from ejaculates of two bulls was thawed in a 35 C water bath for 30 sec and mixed in a 12 x 75-mm plastic tube (#2058; Falcon). With a fine sterile glass pipette, the semen mixture (0.1 ml/tube) was placed under 1 ml of Sp-TALP + 5 U/ml heparin in each of 10 plastic 12 x 75-mm Falcon tubes. Tubes were held at a 45 angle in a wire test tube rack for the duration of the swim-up. This technique resulted in a distinct layer of sperm in extender beneath the Sp-TALP. Tubes were placed in a 5% CO 2 incubator (39 C) for 1 h, allowing the sperm to swim out of the seminal plasma and extender into the Sp-TALP. The clear Sp-TALP layer (-0.95 ml) containing these motile sperm then was aspirated with a fine glass pipette, leaving the extender and most sperm at the bottom of the tube. Aspirates from all 10 tubes were mixed in a 15-ml centrifuge tube, counted by hemocytometer, and centrifuged 5 min at 900 x g. The supernatant was removed, and the volume was adjusted to give a final concentration of 107 sperm/mi. The sperm suspension was incubated (39 C, 5% CO 2 in air) an additional 2 h before fertilization. In Vitro Fertilization Matured oocytes were washed three times in Fert-TALP and placed into xl drops (15 oocytes/drop) of Fert- TALP plus 5 mm caffeine under paraffin oil. Sperm suspension (50 1l) was added to these drops to give a final sperm concentration of 2 x 106 sperm/mi. Gametes were coincubated 18 h in 5% CO 2 in air at 39 C. Design of Experiment Experiment 1 was designed to test effects of maturing oocytes in BFF collected 0, 8, and 20 h after the LH surge. The responses studied were the percentage of oocytes with cumulus expansion after 24 h of culture, and the percentage of cleavage after in vitro fertilization. Maturation treatments consisted of mixtures of follicular fluid and mtcm-199. Two concentrations (10% and 40%), and three collection times (0, 8, and 20 h after the LH surge) of follicular fluid were tested. Our standard oocyte maturation procedure, consisting of mtcm estradiol-173 (1 ixg/ml), was included as a positive control. After maturation, cumulusenclosed oocytes were observed under a stereomicroscope (x20), and the number of oocytes with an expanded cumulus was recorded. Oocytes were then fertilized in vitro. After fertilization, zygotes were washed and transferred to conditioned media for 48 h. Ova were then observed for cleavage, which was used as a determinant of fertilization. Parthenogenesis occurs in less than 5% of oocytes with these procedures. The experiment was replicated four times; the same batches of maturation medium, frozen semen, TALP media, and conditioned media were used for all replicates. However, follicular fluids from different cows were used for each replicate so that results could be generalized to the population. The percentages of oocytes with cumulus expansion and embryo cleavage were evaluated by analysis

3 1014 ROMERO-ARREDONDO AND SEIDEL TABLE 1. Percentage of oocytes with cumulus expansion and cleavage rates (experiment 1). mtcm-1 99 supplement n* Mean + SD Range Cumulus expansion E 2 (control) J % 0 h BFF b % 0 h BFF b % 8 h BFF b % 8 h BFF ,b % 20 h BFF % 20 h BFF a Cleavage E 2 (control) a % 0 h BFF ± 13.3b % 0 h BFF b % 8 h BFF a,b % 8 h BFF a % 20 h BFF a % 20 h BFF a * Cleavage was not evaluated for one of four replicates due to bacterial contamination. a,b Means without common superscripts differ (p < 0.05). of variance using replicates as blocks; significant differences among means were determined by Tukey's honestly significant difference procedure [15]. Design of Experiment 2 Experiment 2 was designed to evaluate effects of follicular fluid on cleavage rate and early embryo development. Maturation treatments consisted of follicular fluid (different cows for each replicate) mixed with mtcm-199. Two concentrations (20% and 40%), and two collection times (0 h and 20 h) of follicular fluid were evaluated along with a combined treatment of 20% 0-h plus 20% 20-h BFF to determine whether any stimulatory effect of 20-h BFF could overcome any inhibitory effect of 0-h BFE Other procedures including the control were as described for experiment 1 except that 9 days after fertilization, numbers of embryos reaching the blastocyst stage were recorded. Cleavage and blastocyst formation rates were evaluated by analysis of variance with replicates as blocks; significant differences among means were determined using Bonferroni's correction due to unequal numbers of observations [16]. RESULTS Experiment 1 Both 10% and 40% 0-h BFF as well as 10% 8-h BFF produced less cumulus expansion (p < 0.05) than other treatments (Table 1). The highest percentage of cumulus expansion was observed in 40% 20-h BFF The rate of fertilization in treatments with expanded cumului was higher than in those with compact cumulus masses. Both concentrations of 0-h BFF treatments had lower (p < 0.05) cleavage rates than did other treatments (Table ). The cleavage rate for 40% 8-h BFF was not different (p > 0.1) from the control treatment or the 20-h BFF treatments, and the cleavage rate for 10% 8-h BFF was not different (p > 0.1) from the 0-h or 20-h BFF treatments. Experiment 2 The highest cleavage rate was achieved in the control treatment (85%), but this was not significantly higher than TABLE 2. Percentages of oocytes that cleaved and formed blastocysts (experiment 2). mtcm-1 99 supplement n* Mean ± SD Range Cleavage E 2 (control) a % 0 h BFF ' % 0 h BFF ' h + 20 h BFFt b % 20 h BFF ,b % 20 h BFF a b Blastocysts per cleaved zygote E 2 (control) a % 0 h BFF a % 0 h BFF ,b h + 20 h BFFt b % 20 h BFF b % 20 h BFF b Blastocysts per oocyte E 2 (control) ' % 0 h BFF a % 0 h BFF ' h + 20 h BFFt b % 20 h BFF ± 8. 9 b % 20 h BFF b * All treatments were replicated six times except mtcm E 2, which was missing in one replicate. t 20% of each type. ab Means without common superscripts differ (p < 0.05). in the 20% or 40% 20-h BFF treatments (68% and 72%, respectively, Table 2). Maturation treatments containing 0-h BFF had lower cleavage rates (p < 0.05) than did the control or either 20-h BFF treatment. Interestingly, the combination of 0-h plus 20-h BFF was similar to 20-h BFF in cleavage rates. Even though the highest cleavage rate was achieved with the control treatment (Table 2), the lowest percentage of blastocyst formation per embryo cleaved (14%) resulted after this treatment. Treatments with 0-h BFF had the second lowest percentages of blastocyst formation (22% and 36% for 20% and 40% 0-h BFF, respectively), although the 40% 0-h treatment result was not significantly different (p > 0.1) from that of the treatments with 20-h BFE The highest percentages of blastocyst formation were obtained from oocytes matured in 20-h BFF, either alone or combined with 0-h BFF The inhibitory effect of 0-h BFF decreased when 20-h BFF was present (p < 0.05, Table 2). The percentages of blastocysts formed per oocyte also are presented in Table 2. Oocytes matured in 20-h BFF had a higher percentage of blastocysts (p < 0.05) than treatments without 20-h BFF. DISCUSSION Experiment 1 With 0-h BFF at both concentrations and with 10% 8-h BFF, cumulus expansion was less than with 20-h BFF and the control. In humans and mice it has been shown that fertilization rates of oocytes with expanded cumului are significantly greater than with oocytes with compact cumulus masses [17-19]. Furthermore, human follicular fluid collected h after the LH surge is capable of inducing cumulus expansion of cultured mouse oocytes [20]. Our results are in accord with these observations. The 0-h BFF treatments had the lowest cleavage rates

4 FOLLICULAR FLUID EFFECTS ON BOVINE OOCYTE MATURATION 1015 of all treatments (Table 1), possibly because of the presence of a meiotic inhibitor [1-3]. However, the cleavage rate for 20-h BFF was greater (p < 0.05) than that of 0-h or 8-h BFF but not different from the control. Similar observations regarding use of follicular fluid to culture oocytes and enhance fertilization rates have been reported in swine [21, 22]. Culturing pig oocytes with porcine follicular fluid (from medium-sized follicles) and FSH (0.12 IU/ml) produced the highest level of oocyte maturation and male pronucleus development. Experiment 2 The combination of 0-h and 20-h BFF was similar to 20-h BFF in cleavage and blastocyst formation rates, suggesting that a stimulator in the 20-h BFF overcame an inhibitor in 0-h BFF The concept of a stimulus that can overcome meiotic arrest has been presented in several studies; among the substances capable of overcoming hypoxanthine-induced meiotic arrest in mice are epidermal growth factor and FSH [6], mitogenic lectins [23], and transforming growth factor alpha [24]. These potent meiotic stimulators of mouse oocytes may elicit their effect by binding to the same receptor, as has been demonstrated by competitive binding assays [25, 26]. Other substances capable of inducing resumption of meiosis in hypoxanthine-inhibited mouse oocytes are insulin and fibroblast growth factor, although the levels required are much higher than those for epidermal growth factor [27]. Epidermal growth factor also promotes meiotic maturation of bovine oocytes from immature follicles [28]. Treatments were applied to oocytes only during the first 24 h of oocyte maturation, so everything else was kept constant (culture media, semen batch, conditioned media, etc.); therefore, observed differences in cleavage and blastocyst production probably are related to the follicular fluid used during maturation. One property of 20-h BFF is its much lower concentration of estradiol-1713 than 0-h BFF [8]. However, it is unlikely that this explains the observed findings [29]. These experiments demonstrate that events that occurred during oocyte maturation were carried over to fertilization and early embryo development, as has been shown previously [29]. In women with clomiphene-stimulated cycles, higher cleavage rates occur in oocytes collected 36 h after hcg injection (given 24 h after the last clomiphene injection) than in those collected at 0 or 12 h after hcg injection [30]. In that study, follicular fluid collected 36 h after hcg injection had a much higher level of meiosis-stimulating substance (measured by the fetal mouse testes bioassay) than at earlier times. It was also determined that none of the cleaved embryos came from follicles lacking meiosisinducing substance. In both of our present experiments, except for cumulus expansion there was no clear dose response, indicating that a threshold had already been reached at 10-20% BFF Fortuitously, this enables a clear interpretation of results from mixing 0-h and 20-h BFF in experiment 2. We have previously demonstrated that 0-h BFF contains meiotic inhibitor(s) and that 20-h BFF contains meiotic stimulator(s) [8]. Use of BFF from the preovulatory follicle at precise times after an induced LH surge enables more physiologically relevant inferences than use of BFF from follicles with an unknown history. In the current study, we demonstrate that exposure of oocytes to follicular fluid collected at different times after the LH surge profoundly affects cumulus expansion, cleavage rates, and rates of blastocyst formation. While molecules such as purines, growth factors, and sterols have been implicated as possible mediators of these effects in various species [6, 27, 28, 31], the active factors in BFF remain to be determined. ACKNOWLEDGMENTS We wish to acknowledge the excellent technical assistance of Zella Brink, Shari Olson, and Wendell Thomas, and the discussions and editorial assistance of Heywood Sawyer. The LH and FSH were generously donated through the National Hormone and Pituitary Program of NIDDK, NICHHD, and USDA. REFERENCES 1. Tsafriri A, Channing CP An inhibitory influence of granulosa cells and follicular fluid on porcine oocyte meiosis in vitro. Endocrinology 1975; 96: Downs SM, Coleman DL, Ward-Bailey PF, Eppig JJ. 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