Regulation of Preimplantation Development of Bovine Embryos by Interleukin-1 1

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1 BIOLOGY OF REPRODUCTION 59, (1998) Regulation of Preimplantation Development of Bovine Embryos by Interleukin-1 1 F.F. Paula-Lopes, A.A.S. de Moraes, 3 J.L. Edwards, 4 J.E. Justice, and P.J. Hansen 2 Department of Dairy & Poultry Sciences, University of Florida, Gainesville, Florida ABSTRACT Experiments were performed to determine the actions of recombinant bovine interleukin-1 (IL-1 ) on the growth of preimplantation embryos. In the first series of studies, IL-1 was added at 8 10 h after insemination, and the percentage of oocytes developing to the blastocyst stage was evaluated. IL-1 increased development to the blastocyst stage when embryos were cultured at high density ( embryos/drop) but decreased or had no effect on development when cultured at low density ( 10 embryos/drop). Thus, the positive effect of IL-1 depends upon some other embryo-derived product. The effect of IL-1 on embryonic development was maintained in completely denuded embryos, indicating that cumulus cells do not mediate the actions of IL-1. Maximum development of embryos cultured at 25 30/drop occurred at ng/ml; 10 ng/ml was less effective. Addition of IL-1 to groups of embryos/drop at 8 10 h after insemination also increased embryo cell number at Day 5 postinsemination by increasing the proportion of embryos that reached the 9- to 16-cell stage. However, IL-1 had no effect on the proportion of blastocysts when added at Day 5 postinsemination. Thus, IL-1 probably acts to increase blastocyst numbers by exerting actions on embryo growth before Day 5. In contrast to its effect on embryos, addition of IL-1 during oocyte maturation did not affect cumulus expansion, cleavage rate of oocytes, or subsequent development to the blastocyst stage. In conclusion, IL-1 can modulate growth of bovine embryos at early stages of development in a manner dependent upon embryo density. INTRODUCTION Interactions between the conceptus and the reproductive tract are the basis for embryo development and implantation. This dialogue is mediated by a host of regulatory signals that include cytokines [1, 2]. Among these are various members of the interleukin-1 (IL-1) family. This group of polypeptides includes two agonists, IL- and IL-1, and one antagonist, IL-1 receptor antagonist (IL-1RA) [3, 4]. During early pregnancy, there is evidence for production of IL-1 by both embryo and reproductive tract tissues. Immunoreactive IL-1, IL-1RA, and IL-1 receptor type I have been identified in the cytoplasm and plasma membranes of human oocytes and embryos at the 2 3, 4-, 6-, 8-, 10- cell, morula, and blastocyst stages [5]. Also, IL-1 bioactivity has been identified in medium conditioned by human cumulus-oocyte complexes (COCs) [6], and IL-1 and IL- 1 in medium conditioned by human embryos [6 9]. IL- 1 has also been found in human endometrial epithelium [10], subepithelial areas of mouse endometrium [11], en- Accepted July 24, Received May 12, This is Journal Series No. R of the Florida Agric. Exp. Sta. Research was supported in part by the Florida Milk Checkoff Program. 2 Correspondence. FAX: ; hansen@dps.ufl.edu 3 Current address: Faculdade de Medicina Veterinária, Universidade Federal do Rio Grande do Sul, caixa postal 15094, CEP: , Porto Alegre, RS, Brazil. 4 Current address: Department of Animal Science, University of Tennessee, Knoxville, TN dometrial stromal cells of humans and mice [10 12], and uterine flushings of women [13] and cattle [14]. IL-1 expression in the pig appears to be different from that in other species studied because IL-1 was not detectable in the reproductive tract during early pregnancy although it was produced by the periimplantation conceptus [15]. The most studied animal regarding the role of IL-1 during pregnancy is the mouse. In this species, IL-1 mrna in uterine tissue peaks on Day 1 of pregnancy [16]. A second peak of expression and bioactivity immediately follows implantation at Day 5, after which concentrations continue to increase during the latter half of pregnancy and then decline markedly at parturition [17]. IL-1 mrna is expressed in preimplantation embryos from the 4-cell to the blastocyst stage. In one study, blockage of maternal endometrial IL-1 receptor type I with IL-1RA prevented implantation by interfering with embryonic attachment [18]. In another study, however, administration of IL-1RA had no effect on implantation although mice deficient in IL-1 receptor type I experienced a decrease in litter size at weaning [19]. There is also controversy as to whether IL-1 regulates preimplantation embryonic development. Administration of IL-1 to CBA/J DBA/2 mice promoted preimplantation development by increasing development to the morula stage [20]. On the other hand, it was reported [21] that IL-1 and IL-1 did not affect development of cultured mouse embryos to the blastocyst stage. The main objective of this study was to determine the actions of IL-1 on the regulation of preimplantation development using the cow as a model. An additional objective was to test whether IL-1 would promote oocyte maturation and cumulus expansion. This seems feasible since there is expression of IL-1 receptor type I in murine oocytes and cumulus [22] and IL-1 bioactivity is present in COCconditioned medium [6]. MATERIALS AND METHODS Materials Recombinant bovine IL-1 was donated by American Cyanamid (Princeton, NJ; purity 95% by SDS-PAGE; endotoxin contamination of 125 endotoxin units/mg protein; bioactivity of IU/mg in the murine LM-1 bioassay). BSA Fraction V, BSA (essentially fatty-acid free), and sheep hyaluronidase were purchased from Sigma Chemical Company (St. Louis, MO). Bovine steer serum and heat-treated fetal calf serum were purchased from Pel- Freez (Rogers, AR) and Atlanta Biologicals (Norcross, GA), respectively. Modified Tyrode s solutions were obtained from Specialty Media (Lavallette, NJ) to prepare HEPES- Tyrode s albumin lactate pyruvate (TALP), in vitro fertilization (IVF)-TALP, and Sperm-TALP [23]. Pituitaryderived FSH was purchased from Schering (Kenilworth, NJ), and Percoll from Pharmacia (Piscataway, NJ). Frozen semen from various bulls was obtained from American Breeders Service (Madison, WI) or Southeast Semen (Lake City, FL). The embryo culture medium, CR1aa (containing 3 mg/ml Fraction V BSA and 2.5 g/ml gentamicin), was

2 IL-1 STIMULATES EMBRYONIC DEVELOPMENT 1407 prepared as described [24] using reagents from Sigma. Hoechst dye was purchased from Sigma. Other reagents were purchased from Fisher Scientific (Pittsburgh, PA) or Sigma. In Vitro Production of Embryos Oocyte recovery and in vitro maturation. Ovaries were obtained from slaughtered cows and washed several times with sterile saline (0.9% [w:v] NaCl) containing 100 U/ml penicillin-g and 100 g/ml streptomycin at C to remove blood and debris. COCs were obtained by aspiration of 2- to 6-mm follicles (one experiment only) or by slicing the ovary (other experiments). Only COCs that had at least one layer of compact cumulus cells were used for subsequent steps. The COCs were washed three times in oocyte collection medium (Tissue Culture Medium [TCM] 199 supplemented with 2% [v:v] steer serum, 100 U/ml penicillin G, 100 g/ml streptomycin, and 0.04 USP U/ml heparin). Groups of 10 oocytes were placed in 50- l microdrops of oocyte maturation medium (TCM-199 with Earle s salts supplemented with 10% [v:v] steer serum, 22 g/ml pyruvate, 20 g/ml FSH, 2 g/ml estradiol, and 50 g/ml gentamicin) and covered with mineral oil. COCs were allowed to mature for 22 h at 38.5 C in an atmosphere of 5% CO 2 in humidified air. Sperm preparation. Frozen-thawed sperm (1 3 straws; each from a different bull when n 1) were purified by centrifugation on a Percoll gradient (3 ml of 45% Percoll [v:v] over 3 ml of 90% Percoll) at 2000 g for 20 min. The sperm pellet was collected at the bottom of the tube and placed in 10 ml of Sperm-TALP. Spermatozoa were centrifuged again at 1000 g for 5 min. The supernatant was removed, and the viable sperm pellet was resuspended in IVF-TALP to give an approximate concentration of 25 million spermatozoa/ml. IVF. Twenty-two hours after maturation, COCs were removed from maturation drops and washed once in HEPES- TALP. Groups of 30 oocytes were transferred to four well plates containing 600 l IVF-TALP per well. Oocytes were fertilized with 25 l of sperm suspension and 25 l ofphe (0.5 mm penicillamine, 0.25 mm hypotaurine, and 25 M epinephrine in 0.9% [w:v] NaCl) added to each well. After 8 10 h at 38.5 C and 5% CO 2 in humidified air, presumptive zygotes were removed from fertilization wells, placed in microcentrifuge tubes containing 40 l HEPES-TALP and vortexed for 4 5 min. Putative zygotes were washed 2 3 times in HEPES-TALP to remove remaining cumulus cells and associated spermatozoa. Even though embryo vortexing/washing removed most of the cumulus cells, in some cases a few cells were present during embryo culture. In vitro culture. Groups of 10 or presumptive zygotes were placed in 50- l drops of CR1aa containing 3 mg/ml of BSA and 2.5 g/ml gentamicin at 38.5 C and 5% CO 2 in humidified air. In one experiment, fetal calf serum (5 l/drop) was added to some drops at Day 5 after insemination. Determination of Cell Number Cell number was assessed using the protocol described by Pursel et al. [25]. Briefly, embryos were placed on a slide and covered with 10 l of 0.01% (w:v) Trypan blue dye for 1 min at room temperature. Trypan blue dye was removed, and embryos were then incubated in 10 g/ml Hoechst for 5 min at 37 C. Excess Hoechst dye was removed, coverslips were mounted with Permount (Fisher Scientific), and fluorescent staining of nuclei was visualized with an epifluorescence microscope (Leitz Laborlux K, Leica, Heerbruss, Switzerland). Experimental Design Effects of IL-1 added after fertilization on development of blastocysts. In the first experiment, using oocytes collected by aspiration, groups of presumptive zygotes were placed at 8 10 h after insemination in 50- l drops of embryo culture medium containing 0 or 10 ng/ml IL-1. Development proceeded until Day 9 postinsemination. The experiment was replicated 5 times using presumptive zygotes/treatment. The second experiment utilized a 3 2 factorial arrangement of treatments with main effects of IL-1 (0, 1, or 10 ng/ml) and fetal calf serum (0 or 5 l). When present, IL-1 was added 8 10 h after insemination, and fetal calf serum was added at Day 5 postinsemination. Presumptive embryos were cultured at 10/ drop (mean 9.3, SD 2.0). The experiment was replicated 5 times using presumptive zygotes/treatment. A third experiment using a 3 2 factorial design was conducted to test the main effects of IL-1 and number of embryos per drop. At 8 10 h postinsemination, groups of 10 (mean 9.9, SD 0.80) or (mean 26.9, SD 3.8) presumptive embryos were randomly assigned to drops of embryo culture medium containing 0, 1, or 10 ng/ml IL-1. The experiment was replicated 5 times using presumptive zygotes/treatment for embryos cultured in groups of 10/drop and for embryos cultured in groups of 25 30/drop. Effect of IL-1 on embryo cell number at Day 5 postinsemination. At 8 10 h postinsemination, groups of presumptive embryos were placed in 50- l drops of embryo culture medium containing 0 or 1 ng/ml IL-1. Cell number was assessed at Day 5 after insemination. The experiment was replicated 3 times with cell number performed on embryos/treatment, collected over two separate IVF runs. Effect of IL-1 added at Day 5 after insemination. Presumptive embryos were cultured (mean 24.7/drop, SD 2.8) in embryo culture medium. At Day 5, drops were supplemented with 5 l embryo culture medium (control), 5 l fetal calf serum, or 5 l IL-1 dissolved in embryo culture medium (final concentration 1 or 10 ng/ml). The experiment was replicated 6 times using presumptive zygotes/treatment. Effect of low concentrations of IL-1. This experiment was conducted to determine the lowest effective concentration of IL-1 for stimulating development. Eight to 10 h after insemination, presumptive embryos were placed (mean 25.8/drop, SD 3.2) in drops containing 0, 0.01, 0.1, 1, or 10 ng/ml IL-1. The experiment was replicated 5 times using presumptive zygotes/treatment. Effect of IL-1 on development of embryos completely denuded of cumulus cells. In many replicates, a few cumulus cells remained attached to presumptive zygotes after washing. To verify that effects of IL-1 were not mediated by the cumulus cells, an experiment was performed in which all cumulus cells were removed enzymatically. At 8 10 h postinsemination, presumptive embryos were placed in 40 l of Hepes-TALP containing 300 g/ml of hyaluronidase and vortexed for 5 min. Groups of (mean 26.4, SD 6.2) denuded presumptive embryos were placed in 50- l drops of embryo culture medium containing

3 1408 PAULA-LOPES ET AL. 0 or 1 ng/ml IL-1. The experiment was replicated 2 times using presumptive zygotes/treatment. Effect of IL-1 during oocyte maturation and cumulus expansion. Groups of 10 oocytes were randomly assigned to 50- l maturation drops. Drops consisted of oocyte maturation medium containing IL-1 (0, 1, or 10 ng/ml) and 10% (v:v) steer serum. After oocyte maturation, cumulus expansion was visually assessed under a stereomicroscope and graded from 0 3 (0 no expansion, 1 little expansion, 2 good expansion, 3 very good expansion). Oocytes were then fertilized, and embryos were placed in embryo culture medium (mean 22.6/drop, SD 4.2) as described earlier. The experiment was replicated 7 times using presumptive zygotes/treatment. Measurements. For each study (except when cell number was measured at Day 5), development to blastocyst stage was assessed from Day 7 to Day 9 postinsemination, and data were expressed as the percentage of inseminated oocytes that became blastocysts. Cleavage rate was recorded at Day 3 postinsemination and was expressed as the percentage of inseminated oocytes that developed to the 2-cell stage or greater. Statistical Analysis Data were analyzed by least-squares analysis of variance using the General Linear Models (GLM) procedure of SAS [26]. The experimental unit was considered the microdrop. Cleavage rate and embryonic development were recorded for each drop. Experiments were replicated on several different days using one or more drop of oocytes or embryos per treatment. Results were analyzed using three methods; the different analyses gave similar results for all experiments. For method 1, percentage cleaved or percentage developed to the blastocyst stage was considered a dependent variable. For method 2, percentage data were analyzed after arcsin transformation. For method 3, dependent variables were numbers of cleaved oocytes and total blastocysts in the drop; total number of presumptive embryos in the drop was used as covariate. This method avoids the nonestimable bias for percentage data caused by different numbers of observations per drop. Additionally, approaches 2 and 3 correct for problems of nonnormality associated with analysis of percentage data. Orthogonal contrasts and a means separation procedure of SAS called pdiff were performed when appropriate to determine differences between various levels of a treatment. The effect of IL-1 on embryo cell number at Day 5 postinsemination was analyzed by ANOVA using the GLM procedure of SAS, in which each embryo was considered the experimental unit. In addition, the effect of IL-1 on the distribution of embryos into various cell number classes was determined by CATMOD [26]. The effect of IL-1 on cumulus expansion was analyzed by ANOVA in which each oocyte was considered the experimental unit. RESULTS Effects of IL-1 Added after Fertilization on Development to Blastocyst Three experiments were conducted in which effects of IL-1 added 8 10 h after fertilization were evaluated (Fig. 1). In the first experiment, presumptive embryos were cultured in groups of 25 30/drop. IL-1 had no effect on cleavage rate but increased the proportion of presumptive embryos that developed to the blastocyst stage at Day 7 (p FIG. 1. Effect of IL-1 added after fertilization on development to the blastocyst stage. Results are least-squares means SEM. In experiment 1, IL-1 increased the proportion of presumptive zygotes that developed to the blastocyst stage at Day 7 (p 0.05) and Day 9 (p 0.06). In the second experiment, serum increased the proportion of presumptive embryos that developed to the blastocyst stage at Day 7 (p 0.01) and Day 9(p 0.05). IL-1 decreased development at Day 7 (0 vs. 1 and 10 ng/ ml, p 0.08) and Day 9 (main effect, p 0.01; 1 vs. 10, p 0.05). In the third experiment, there was an interaction between IL-1 and embryo density for development at Day 7 (p 0.05) and Day 9 (p 0.07). Further analysis of the data indicated that in presence of high embryo density, IL-1 increased the proportion of presumptive zygotes that developed to the blastocyst stage at Day 9 (main effect, p 0.05; 0 vs. 1 and 10, p 0.01; 1 vs. 10, p 0.08). For low embryo density there was no significant effect of IL ) and 9 (p 0.06) after insemination. In the second experiment, presumptive embryos were cultured in groups of 10/drop. Treatment with IL-1 (1 or 10 ng/ml) had no significant effect on cleavage rate. Development to the blastocyst stage was reduced by 1 and 10 ng/ml IL-1 at Day

4 IL-1 STIMULATES EMBRYONIC DEVELOPMENT 1409 FIG. 2. Effect of IL-1 added 8-10 h postinsemination on distribution of embryos at Day 5 postinsemination into different cell number classes. Open bars, control embryos; solid bars, embryos treated with IL-1. Treatment with 1 ng/ml IL-1 affected the distribution of embryos (p 0.05) by increasing the proportion of embryos that reached the 9- to 16-cell stage. FIG. 3. Effect of IL-1 added at Day 5 after insemination on the proportion of presumptive zygotes that developed to the blastocyst stage. Results are least-squares means SEM. Addition of 1 ng/ml IL-1 at Day 5 postinsemination had no effect on blastocyst development at Day 7 or 9. However, 10 ng/ml IL-1 tended to reduce development to the blastocyst stage at Day 9 (1 vs. 10, p 0.10). Serum increased the proportion of embryos that developed to the blastocyst stage at Days 7 (p 0.001) and9(p 0.001) after insemination. 7 (0 vs. 1 and 10 ng/ml, p 0.08) and by 10 ng/ml IL-1 at Day 9 (1 vs. 10 ng/ml, p 0.05). Addition of serum at Day 5 after insemination increased the proportion of oocytes that developed to blastocysts at Day 7 (p 0.01) and 9(p 0.05), but did not alter the effect of IL-1. A third experiment was conducted to determine whether the differential response to IL-1 in the first two experiments represented an effect of number of embryos per drop (Fig. 1). Cleavage rates were similar between groups (Fig. 1). There was an IL-1 embryo-per-drop interaction for development at Day 7 (p 0.05) and Day 9 (p 0.07). Further analysis of the data was done separately for 10 embryos/drop and embryos/drop. When embryos were cultured in large groups ( embryos/drop), IL- 1 increased the percentage of oocytes that developed to blastocysts at Day 9 (0 vs. 1 and 10 ng/ml, p 0.01), with 1 ng/ml being more effective than 10 ng/ml (1 vs. 10 ng/ ml, p 0.08). There was no significant effect of IL-1 on development at Day 7 or 9 when embryos were cultured in groups of 10/drop although development was numerically least for embryos cultured with 1 ng/ml IL-1. Development to the blastocyst stage did not differ between control embryos cultured at 10/drop or 25 30/drop. Effect of IL-1 on Embryo Cell Number at Day 5 Postinsemination Addition of 1 ng/ml IL-1 to embryos cultured at 25 30/drop at 8 10 h after insemination increased (p 0.05) embryo cell number at Day 5 postinsemination (12.6 vs cells for control and IL-1, respectively; SEM 0.9). Analysis of the distribution of embryos into various cell number classes revealed that IL-1 changed (p 0.05) the distribution (Fig. 2). In particular, IL-1 stimulated the proportion of embryos that reached the 9- to 16-cell stage. Effect of IL-1 Added at Day 5 after Insemination Addition of IL-1 to embryos cultured in groups of 25/ drop on Day 5 after insemination did not increase the percentage of embryos developing to blastocysts (Fig. 3). IL- 1 at the concentration of 1 ng/ml had no effect on development while 10 ng/ml IL-1 tended to reduce development at Day 9 (1 vs. 10 ng/ml, p 0.10). In contrast, addition of serum at Day 5 increased the proportion of embryos that developed to blastocysts at Days 7 (p 0.001) and9(p 0.001) after insemination. Effect of Low Concentrations of IL-1 When tested in embryos at 25 30/group and at concentrations of ng/ml, IL-1 had no effect on cleavage rate (results not shown). IL-1 stimulated development of oocytes to the blastocyst stage in a dose-dependent manner (Fig. 4). However, a concentration of 0.01 ng/ml had no significant effect on development although the percentage of oocytes developing to blastocysts at Day 9 was numerically greater than controls. Greatest development oc- FIG. 4. Effect of various concentrations of IL-1 on the proportion of oocytes that developed to the blastocyst stage (note the log scale on the x-axis). Results are least-squares means SEM. The main effect of IL-1 concentration approached significance at Day 7 (p 0.09) and was significant at Day 9 (p 0.01). Concentrations significantly different from 0 are indicated by (p 0.10), * (p 0.05), and ** (p 0.01).

5 1410 PAULA-LOPES ET AL. TABLE 1. Effect of IL-1 on development of embryos completely denuded of cumulus cells. a Parameter Number of replicates Number of oocytes Oocytes, % cleaved Oocytes, % blastocyst at Day 7 after insemination Oocytes, % blastocyst at Day 9 after insemination b a Data are least-squares means SEM. b Main effect of treatment, p IL-1 1 ng/ml curred at 0.1 ng/ml; there was a significant effect of this dose at Days 7 and 9. A concentration of 1 ng/ml was less effective, causing a significant increase at Day 9 but not at Day 7 after insemination. Addition of IL-1 at 10 ng/ml did not increase the proportion of oocytes developing to blastocysts at Days 7 and 9. Effect of IL-1 on Development of Embryos Completely Denuded of Cumulus Cells A few cumulus cells are often present on some embryos after fertilization. To rule out the possibility that the effect of IL-1 on embryonic development is mediated by cumulus cells, an experiment was conducted using only completely denuded embryos. Addition of 1 ng/ml IL-1 to denuded embryos cultured at 25 30/drop at 8 10 h after IVF increased the proportion of oocytes that developed to the blastocyst stage at Day 9 (p 0.05) after insemination (Table 1). Effect of IL-1 during Oocyte Maturation and Cumulus Expansion Addition of serum (p 0.001), but not IL-1, during oocyte maturation stimulated cumulus expansion (Fig. 5) by increasing the frequency of oocytes having the highest cumulus expansion score. Nevertheless, addition of neither serum nor IL-1 to oocyte maturation medium affected subsequent cleavage rate (77.0%, 74.0%, and 78.1%, respectively, for 0, 1, and 10 ng/ml IL-1 without serum, and 76.6%, 78.6%, and 76.7%, respectively, for 0, 1, and 10 ng/ml IL-1 with serum; SEM 1.90) or development of oocytes to the blastocyst stage (Fig. 6). DISCUSSION The present study demonstrated that IL-1 can regulate development of bovine embryos in vitro and that the nature of this regulation depends upon embryo density. When added to embryos at low density ( 10 embryos/drop), IL-1 either inhibited development or had no effect. However, when added to embryos cultured at a density of 25 30/ drop, concentrations of IL-1 as low as 0.1 ng/ml increased the proportion of embryos becoming blastocysts. This stimulatory effect of IL-1 on blastocyst development was exerted at early stages of development: addition of IL-1 at 8 10 h after insemination enhanced embryo cell number at Day 5 postinsemination by increasing the proportion of embryos that reached 9- to 16-cell stage, whereas addition of IL-1 at Day 5 postinsemination had no effect on development to the blastocyst stage. Since cultured bovine embryos can become blocked in FIG. 5. Effect of IL-1 during oocyte maturation on cumulus expansion. Results are least-squares means SEM. There was no effect of IL-1 on cumulus expansion. However, serum (p 0.001) altered the frequency distribution of cumulus expansion by increasing the frequency of oocytes having the highest cumulus expansion score. development at the 8- to 16-cell stage [27], it is likely that IL-1 promoted embryonic development by allowing more embryos to proceed through the 8-cell block. Furthermore, the 8- to 16-cell stage is also generally considered to be the time when embryonic genome activation takes place in cattle [28, 29]. Perhaps IL-1 regulates mechanisms that trigger gene expression. Other growth factors also have been reported to promote bovine embryonic development beyond the 8-cell block; these include a combination of transforming growth factor- and basic fibroblast growth factor [30] as well as platelet-derived growth factor [31]. The observation that addition of IL-1 to groups of embryos at Day 5 after insemination did not increase the percentage of oocytes that developed to the blastocyst stage means that, unlike some growth factors such as granulo- FIG. 6. Effect of IL-1 during oocyte maturation on the proportion of presumptive zygotes that developed to the blastocyst stage. Results are least-squares means SEM. There was no effect of IL-1 or serum on development of oocytes to the blastocyst stage at Day 7 or 9.

6 IL-1 STIMULATES EMBRYONIC DEVELOPMENT 1411 cyte-macrophage-colony stimulating factor [32], IL-1 does not appear to increase development to the blastocyst stage by acting at the morula stage or later. The fact that IL-1 increased the proportion of oocytes that reached the blastocyst stage when embryos were cultured in high-density groups but not in low-density groups may be indicative of several phenomena. One possibility is that IL-1 stimulates some embryo-derived growth factor that in turn stimulates embryonic development and that the concentrations of this second factor achieve stimulatory levels only in embryos cultured at high density. It is known that IL-1 can up-regulate leukemia inhibitory factor (LIF) expression in several cell populations, including bone marrow and synovial fibroblast-like cells [33]. In bovine embryos, LIF acts as a growth factor by increasing the mean cell number per blastocyst [34]. Perhaps IL-1 plays a stimulatory role in embryonic development through increased embryonic LIF secretion. It may also be that IL-1 needs another constitutively produced embryo factor to be stimulatory to development. Finally, it is possible that removal of some medium ingredient is required to observe effects of IL-1. The stimulatory effects of IL-1 on growth of embryos at high density was greatest at a concentration of 0.1 ng/ ml; higher concentrations were less effective. At the highest concentration (10 ng/ml), IL-1 stimulated development in the first experiment only. This experiment was performed using an older batch of IL-1, and a partial loss of biologic activity may have occurred over time, so that the effective concentration was lower than 10 ng/ml. There are several explanations as to why IL-1 is more active at lower concentrations, including the obvious one of receptor downregulation. In addition, IL-1 may activate a second, inhibitory receptor at higher concentrations. Alternatively, IL-1 may actually act to inhibit some metabolic pathway or gene transcription perhaps some inhibition is beneficial for development while more substantial inhibition reduces development. Cumulus cells can express IL-1 receptors [22], and thus effects of IL-1 on embryos could involve indirect actions mediated by cumulus. Nonetheless, at high embryo density, IL-1 stimulated development of completely denuded embryos, indicating that the beneficial effects of IL-1 are not mediated solely by cumulus cells. Rather, IL-1 appears to exert its effect directly on the embryo through a receptormediated system. Transcripts for IL-1 receptor type I are expressed in mouse embryos, and the highest frequency of positive embryos is observed during the blastocyst stage [35]. Cumulus cells are the site of action for growth molecules that regulate oocyte maturation [36, 37]. Even though IL- 1 has been implicated in ovarian function [38, 39], a role for this cytokine in oocyte maturation has not been established. In the present experiment, addition of IL-1 during in vitro maturation had no effect on cumulus expansion or development to the blastocyst stage following fertilization. Interestingly, serum, which is often added to maturation medium, had little beneficial effect on maturation. Although serum supplementation during oocyte maturation enhanced cumulus expansion, there was no improvement in the number of oocytes that developed into blastocysts. A more complete understanding of the role of the IL-1 system in cattle reproduction depends upon the knowledge of the family of polypeptides in the uterus, oviduct, and embryo. IL-1 has been detected in the luminal uterine fluid of cyclic and pregnant cows [14] and has been immunohistochemically localized to oviductal and endometrial cells [40]. Expression of other components of the IL-1 system in the bovine reproductive tract and embryo, such as IL-1 receptors and IL-1RA, remains to be determined. The variable nature of IL-1 actions on embryonic development (stimulatory at low concentration when embryo density is high and either less active or inhibitory at high concentration depending upon embryonic density) implies that IL-1 could exert actions on the embryo during inflammation, when local concentrations of IL-1 are high, that are different from those in the absence of local immune stimulation. ACKNOWLEDGMENTS The authors thank Dale E. Shuster, Ph.D., and American Cyanamid (Princeton, NJ) for donation of IL-1 and Tommy Bryan, owner of Central Packing Co. (Center Hill, FL), and his employees for their assistance collecting the ovaries. REFERENCES 1. Tabibzadeh S. Human endometrium: an active site of cytokine production and action. Endocr Rev 1991; 12: Robertson SA, Seamark RF, Guilbert LJ, Wegmann TG. 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