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Biology of Reproduction 65, 1150-1155 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Cellular Mechanisms by Which Oxytocin Mediates Ovine Endometrial Prostaglandin F2{alpha} Synthesis: Role of Gi Proteins and Mitogen-Activated Protein Kinases1

Patrick D. Burns2,a, Jose O.B. Mendes Jr.a, Robert S. Yemma, Colin M. Clayb, Scott E. Nelsonb, Susan H. Hayesc, and William J. Silviac

a Department of Animal Sciences b Animal Reproduction Laboratory, Department of Physiology, Colorado State University, Fort Collins, Colorado 80523 c Department of Animal Sciences, University of Kentucky, Lexington, Kentucky 40546

ABSTRACT

Oxytocin stimulates a rapid increase in ovine endometrial prostaglandin (PG) F2{alpha} synthesis. The overall objective of these experiments was to investigate the cellular mechanisms by which oxytocin induces endometrial PGF2{alpha} synthesis. The objective of experiment 1 was to determine whether Gi proteins mediate oxytocin-induced PGF2{alpha} synthesis. Uteri were collected from four ovary-intact ewes on Day 14 postestrus. Caruncular endometrial explants were dissected and subjected to in vitro incubation. Pertussis toxin, an inhibitor of Gi proteins, had no effect on the ability of oxytocin to induce PGF2{alpha} synthesis (P > 0.10). The objective of experiment 2 was to determine whether any of the three mitogen-activated protein kinases (MAPKs), extracellular signal regulated protein kinase (ERK1/2), c-Jun N-terminal/stress-activated protein kinase (JNK/SAPK), or p38 MAPK, mediate oxytocin-induced PGF2{alpha} synthesis. Eleven ovary-intact ewes were given an injection of oxytocin (10 IU; i.v.; n = 5) or physiological saline (i.v.; n = 6) on Day 15 postestrus. Uteri were collected 15 min after injection and caruncular endometrium was dissected. Endometrial homogenates were prepared and subjected to Western blotting. Membranes were probed for both total and phosphorylated forms of all three classes of MAPK. All classes of MAPK were detected in ovine endometrium, but oxytocin treatment had no effect on the expression of these proteins (P > 0.10). ERK1/2 was the only phosphorylated MAPK detected and its concentrations were higher in oxytocin-treated ewes (P < 0.01). The objective of experiment 3 was to further investigate the role of ERK1/2 during oxytocin-induced PGF2{alpha} synthesis. Uteri were collected from four ovary-intact ewes on Day 14 postestrus. Caruncular endometrial explants were dissected and subjected to in vitro incubation. PD98059, a specific inhibitor of ERK1/2 activity, blocked the ability of oxytocin to stimulate PGF2{alpha} synthesis in a dose-dependent manner (P < 0.05). These results indicate that the ovine oxytocin receptor is not coupled to Gi proteins. These results indicate that oxytocin induces phosphorylation of ERK1/2 and that this MAPK appears to mediate oxytocin-induced PGF2{alpha} synthesis in ovine endometrium.

kinases, mechanisms of hormone action, oxytocin, signal transduction, uterus

INTRODUCTION

Prostaglandins play an essential role in reproduction in domestic ruminants. In nonpregnant ewes, prostaglandin (PG) F2{alpha} is released from the uterus in a series of high-amplitude pulses late in the estrous cycle to cause regression of the corpus luteum [1, 2]. Oxytocin is an acute stimulant for PGF2{alpha} secretion from ovine endometrium and may be responsible for generating the luteolytic pulses of PGF2{alpha} late in the estrous cycle [3]. Oxytocin binds to a cell surface membrane receptor [4, 5] that activates a complex intracellular signaling pathway that ultimately leads to the activation of phospholipase (PL) A2 [6]. The cytosolic form of PLA2 (cPLA2) mobilizes arachidonic acid from membrane phospholipids [7, 8]. Once mobilized, free arachidonic acid is rapidly converted to PGH2 by the enzyme prostaglandin H2 endoperoxide synthase (PGHS)-2 [9]. In endometrial tissue, PGH2 is then converted to PGF2{alpha} by the enzyme PGF synthase. The cellular mechanisms that lead to the activation of PLA2 in ovine endometrium are not completely understood.

The ovine endometrial oxytocin receptor (OTR) has been cloned and sequenced. It belongs to the seven transmembrane domain G protein coupled receptor family [10]. Seven transmembrane domain receptors can interact with and activate Gs, Gi, Gq, or G12 proteins. The {alpha} subunit of Gq proteins typically interacts with and activates PLC [11, 12]. In ovine endometrium, oxytocin increases PLC activity [13, 14], suggesting that the receptor is coupled to Gq proteins in this tissue. However, in human and rat myometrial cells, the OTR appears to be coupled to both Gq and Gi proteins [15–17]. In this cell type, Gi proteins appear to aid in regulating free intracellular concentrations of Ca2+ and PG synthesis during parturition. Ca2+ plays a critical role in mediating oxytocin-induced PGF2{alpha} synthesis in both ovine [14] and bovine [18–20] endometrium. It is possible that the ovine OTR may be coupled to Gi proteins, and that this G-protein may regulate free intracellular Ca2+ during PGF2{alpha} synthesis. The objective of experiment 1 was to determine whether Gi proteins play a role in mediating oxytocin-induced PGF2{alpha} release in ovine endometrial tissue.

Activity of cPLA2 can be regulated at both the cellular and genomic levels [7, 8]. Acute oxytocin stimulation does not appear to increase endometrial synthesis of the cPLA2 message or protein [21]. In other tissues, cPLA2 requires an increase in free intracellular Ca2+ and phosphorylation of serine 505 for maximum activity, which is a substrate for mitogen-activated protein kinase (MAPK) [22]. There are three major classes of MAPK: 1) extracellular signal regulated protein kinase (ERK1/2), 2) c-Jun N-terminal/stress-activated protein kinase (JNK/SAPK), and 3) p38 MAPK [23–25]. All three classes of MAPK have been reported to phosphorylate cPLA2 [26–28].

Oxytocin has been shown to phosphorylate ERK2 in human breast Hs578T cells [29], Chinese hamster ovary cells transfected with the rat OTR [30] and human endometrial cells [31]. It is possible that ERK1/2, JNK/SAPK or p38 MAPK may mediate oxytocin-induced PGF2{alpha} release in ovine endometrium. The objective of experiments 2 and 3 was to determine whether oxytocin stimulates phosphorylation of MAPK during PGF2{alpha} synthesis and release.

MATERIALS AND METHODS

All experimental and surgical procedures involving animals were approved by the Agriculture Animal Care and Use Committee, University of Kentucky (animal use protocol 96-0011A) and Colorado State University (animal use protocol 99-023A-02).

Materials

Pertussis toxin and PD98059 were purchased from Calbiochem (La Jolla, CA). Acrylamide, N,N'-methylene-bis-acrylamide, Bio-Rad protein assay dye reagent, and Kaleidoscope prestained molecular weight markers were purchased from Bio-Rad Laboratories (Hercules, CA). Total ERK1/2 (sc-94), JNK/SAPK (sc-474), and p38 (sc-535) rabbit polyclonal MAPK, phospho-ERK (sc-7383), phospho-JNK/SAPK (sc-6254), and phospho-p38 (sc-7973) mouse monoclonal MAPK, and anti-rabbit immunoglobulin G (IgG) conjugated to horseradish peroxidase were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Nitrocellulose membrane was purchase from Schleicher and Schuell (Keene, NH). CL-XPosure film and SuperSignal West Femto Maximum Sensitivity Substrate were purchased from Pierce (Rockford, IL). All other reagents were purchased from Sigma (St. Louis, MO).

Experiment 1

On Day 14 postestrus, four mature ovary-intact ewes were killed with sodium pentobarbital, uteri were collected, and caruncular endometrial explants were dissected. We have reported that both caruncular and intracaruncular endometrium will synthesize and release PGF2{alpha} in response to oxytocin [32]. However, caruncular endometrial tissue releases more PGF2{alpha} than intracaruncular endometrium. Therefore, caruncular endometrium was used in this and all subsequent experiments. Caruncular endometrial explants were placed in Kerbs-Hensleit buffered media (KHB; 118 mM NaCl, 4.7 mM KCl, 2.56 mM CaCl2, 1.13 mM MgCl2, 25 mM NaHCO3, 1.15 mM NaH2PO4, 5.55 mM glucose, 20 mM Hepes, and 0.013 mM phenol red; pH 7.4). Individual explants were weighed (30–50 mg), placed in 12 x 75 mm culture tubes, and subjected to in vitro incubation as described by Silvia and Homanics [14]. In brief, 32 endometrial explants from each uterus were subjected to a four-period incubation protocol lasting 90 (period 1), 60 (period 2), 5 (period 3), and 30 (period 4) min. Beginning at incubation period 2 and continuing through periods 3 and 4, explants were pretreated in the presence of 0, 0.01, 0.1, or 1 µg/ml pertussis toxin (an inhibitor of Gi proteins; n = 8 explants/dose). During incubation period 4, four explants from each dose of pertussis toxin were incubated in KHB (basal) or KHB containing oxytocin (10-7 M). Medium was collected at the end of incubation period 4, diluted 1:10 in PBS, and stored at -20°C until assayed for PGF2{alpha} by radioimmunoassay (RIA). Release of PGF2{alpha} was reported as ng PGF2{alpha}/g tissue per min.

Experiment 2

Eleven mature ovary-intact ewes were allowed to complete one estrous cycle prior to collection of data. On Day 15 postestrus of the second estrous cycle, each ewe was given an injection of oxytocin (10 IU; i.v.; n = 5) or physiological saline (i.v.; n = 6). Jugular blood samples were collected at -120, -60, -30, -15, 0, 5, 10, and 15 min postinjection and assayed for concentrations of 13,14-dihydro 15-keto prostaglandin F2{alpha} (the stable metabolite of PGF2{alpha}; PGFM). Uteri were collected 15 min after injection. Caruncular endometrial tissue was dissected, snap-frozen in liquid nitrogen, and stored at -80°C until subjected to Western analysis.

Experiment 3

Uteri were collected from four mature ovary-intact ewes on Day 14 postestrus. Caruncular endometrial explants were dissected and subjected to in vitro incubation as described in experiment 1. Thirty-two endometrial explants from each uterus were pretreated in the presence of 0, 0.2, 2, or 20 µM PD98059 (an inhibitor of ERK activity; n = 8 explants/dose) during incubation periods 2 through 4. During incubation period 4, four explants from each dose of PD98059 were incubated in KHB media with or without oxytocin (10-7 M). Medium was collected and assayed for PGF2{alpha} by RIA as described in experiment 1. At the termination of incubation period 4, the explants that were pretreated with 0 or 20 µM PD98059 and treated with or without oxytocin were pooled within each pretreatment-treatment group and stored at -80°C until subjected to Western analysis for the presence of phospho-ERK1/2.

Western Analysis

Approximately 0.5 g of tissue was homogenized in three volumes of ice-cold homogenization buffer (1x PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 10 µg/ml aprotinin, 1 mM PMSF, and 1 mM sodium orthovanadate). Crude homogenates were centrifuged at 800 x g for 10 min at 4°C to remove cellular debris. Supernatants were transferred to microtubes and centrifuged at 15 000 x g for 20 min at 4°C. The supernatant was stored at -80°C until determination of protein concentration and immunoblotting. Protein concentrations were determined by Bradford assay using BSA as the standard.

Proteins were separated by SDS-PAGE. Samples were diluted 1:1 in SDS gel loading buffer (17% glycerol, 1.25 mM ß-mercaptoethanol, 5.25% SDS, 0.22 M Tris-HCl pH 6.7, and 0.02% bromophenol blue), boiled for 3 min, and loaded onto 10% resolving gels containing a 4% stacking gel. After electrophoresis, proteins were electrophoretically transferred to nitrocellulose membranes. Following transfer, membranes were washed twice in 1x TBS-T (10 mM Tris-HCl pH 7.4, 140 mM NaCl, 0.1% Tween 20) for 5 min and blocked using a 5% instant nonfat dry milk TBS-T solution for 30 min. After blocking, membranes were then incubated for 1 h in anti-ERK1/2, anti-JNK/SAPK, anti-p38 MAP kinase, anti-phospho-ERK1/2, anti-phospho-JNK/SAPK, or anti-phospho-p38 MAP kinase antisera at 1:1000 in 5% milk TBS-T. Following incubations with the primary antibody, membranes were washed three times in 1x TBS-T for 5 min. After the final wash, membranes were incubated in a 5% milk-TBS solution containing anti-mouse or rabbit IgG conjugated to horseradish peroxidase at 1:3000 for 45 min. Following incubation, membranes were washed as described above. Lysates from bovine endometrial cells exposed to ultraviolet (UV) light were used as positive controls for detection of phospho-JNK and -p38 MAPK. Immunoreactive proteins were detected using chemiluminescence. Images were captured and relative concentration determined by using scion software (Scion Corporation, Frederick, MD).

Radioimmunoassays

Concentrations of serum PGFM were determined as described by Homanics and Silvia [33]. Samples were run in two assays and intraassay and interassay CVs were 10.4% and 7.8%, respectively. Sensitivity of the assay was 25 pg/ml. Concentrations of PGF2{alpha} in media samples were determined as described by Ellinwood et al. [34]. Within-assay and between-assay CVs across six assays for three media pools were 5.1% and 18.5%, respectively. Sensitivity of the assay was 0.05 pg/tube.

Statistical Analysis

In experiments 1 and 3, the effects of pretreatment and treatment on PGF2{alpha} release were analyzed using SAS PROC MIXED software [35]. The statistical model included ewe, pretreatment, treatment, pretreatment x treatment, and residual error. Ewe was considered a random effect within the statistical model. When main effects or interactions were significant (P < 0.05), individual means were compared using preplanned pairwise t-tests. In experiment 3, the effects of dose of PD98059 on basal and oxytocin-induced PGF2{alpha} release were further analyzed by least squares regression ANOVA. In experiment 2, PGFM data were analyzed by two-way ANOVA with repeated measures using SAS PROC MIXED software [35]. Residual error correlations between measurements on the same subject were assumed to decrease exponentially with the length of time between measurements. Fixed effects were treatment (oxytocin or saline), time, and the interaction. Individual time means were compared using the Bonferroni t-test. Ewe within treatment was a random effect. Effects of treatment on endometrial concentrations of total and phosphorylated MAPK were determined by t-test.

RESULTS

Experiment 1

Oxytocin stimulated PGF2{alpha} release above basal secretion in the absence of pertussis toxin (P < 0.05; Fig. 1). Pertussis toxin had no effect on the ability of oxytocin to stimulate PGF2{alpha} release (P > 0.10).



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  		FIG. 1. Effect of concentration of pertussis toxin on basal and oxytocin (10-7 M)-induced PGF2{alpha} release in ovine endometrial tissue. Lines above bars represent SEM. *Different from basal secretion (P < 0.05)

Experiment 2

Serum concentrations of PGFM did not differ between treatment groups prior to oxytocin or saline injection (P > 0.10). Oxytocin stimulated a rapid increase in serum concentrations of PGFM. Within 5 min postinjection, serum concentrations of PGFM were higher in oxytocin-treated ewes compared with saline-treated ewes (P < 0.01).

Total ERK1/2 (estimated as 44- and 42-kDa proteins, respectively), JNK/SAPK (estimated as a 54-kDa protein), and p38 (estimated as a 38-kDa protein) MAPK were detected in endometrial tissue collected from both saline and oxytocin-treated ewes (Fig. 2). Acute oxytocin stimulation had no effect on the expression of these proteins (P > 0.10). Phosphorylated JNK/SAPK and p38 MAPK were detected in lysates obtained from UV-treated bovine endometrial cells, but neither phosphorylated kinases were detected in endometrial tissue (data not shown). However, phosphorylated ERK1/2 was detected in endometrium collected from both saline-treated and oxytocin-treated ewes (Fig. 3). Endometrial concentrations of phosphorylated ERK1/2 were three- to fourfold higher in oxytocin-treated ewes (P < 0.01).



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  		FIG. 2. Effect of saline and oxytocin on endometrial concentrations of total extracellular signal-regulated protein kinase. A) ERK1/2, estimated as 44- and 42-kDa proteins, respectively. B) JNK/SAPK, estimated as a 54-kDa protein. C) p38 Mitogen-activated protein, estimated as a 38-kDa protein as determined by Western blot analysis. Saline-treated ewes are represented in lanes 1–6 and oxytocin-treated ewes are represented in lanes 7–11



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  		FIG. 3. Effect of saline and oxytocin on endometrial concentrations of phosphorylated ERK1/2. A) Western blot analysis of endometrial tissue collected from saline-treated (lanes 1–6) and oxytocin-treated (lanes 7–11) ewes. B) Quantitative results from the Western blot analysis. *Saline-treated vs. oxytocin-treated ewes (P < 0.01). Lines above bars represent SEM

Experiment 3

Oxytocin stimulated PGF2{alpha} release above basal secretion in the absence of PD98059 (P < 0.05; Fig. 4). PD98059 blocked oxytocin-induced PGF2{alpha} release in a dose-dependent manner (P < 0.01). Regression of PGF2{alpha} on dose of PD98959 indicated there was a linear decrease for both basal (Y = 1.1 – 0.04x) and oxytocin (Y = 1.7 – 0.06x)-treated explants. PD98059 inhibited the ability of oxytocin to stimulate PGF2{alpha} release at doses of 2 µM and higher (P < 0.05). PD98059 inhibited basal release at 20 µM (P < 0.05). As in experiment 2, oxytocin stimulated an increase in endometrial concentrations of phosphorylated ERK1/2 (Fig. 4). PD98059 (20 µM) blocked the ability of oxytocin to stimulate phosphorylation of ERK1/2 (P < 0.01).



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  		FIG. 4. A) Effect of concentration of PD98059 on basal and oxytocin-induced (10-7 M) PGF2{alpha} release in ovine endometrial tissue. Lines above bars represent SEM. *Different from basal (P < 0.05). B) Effects of PD98059 (20 µM) on basal and oxytocin-induced (10-7 M) endometrial concentrations of phospho-ERK1/2 obtained from three ewes. Lanes 1–3 are pooled explants pretreated with 0 µM PD98059 and treated with 0 M oxytocin. Lanes 4–6 are pooled explants pretreated with 20 µM PD98059 and treated 0 M oxytocin. Lanes 7–9 are pooled explants pretreated with 0 µM PD98059 and treated with 10-7 M oxytocin. Lanes 10–12 are pooled explants pretreated with 20 µM PD98059 and treated 10-7 M oxytocin

DISCUSSION

In human [15, 16] and rat [17] myometrial cells, the OTR appears to be coupled to both Gq and Gi proteins. Pertussis toxin has been shown to attenuate oxytocin-induced increases in intracellular Ca2+ and PG synthesis in this cell type. Pertussis toxin has been shown to ribosylate an {alpha} subunit in ovine endometrium, presumably the {alpha} subunit of Gi proteins [36]. However, pertussis toxin had no effect on the ability of oxytocin to stimulate PLC activity in that study. In the present study, pertussis toxin had no effect on the ability of oxytocin to stimulate PGF2{alpha} synthesis. These results would indicate that the ovine endometrial OTR is not coupled to Gi proteins.

All three classes of MAPK were detected in ovine endometrium. Each protein migrated on SDS-PAGE gels to sizes that have been reported in other tissues and cells [23–25]. Oxytocin is an acute stimulant of uterine PGF2{alpha} synthesis both in vivo [33, 37, 38] and in vitro [14, 39]. Of the three classes of MAPK detected in ovine endometrium, only ERK1/2 appears to be phosphorylated in response to oxytocin. In the present study, oxytocin-induced PGF2{alpha} synthesis was associated with a three- to fourfold increase in endometrial concentrations of phosphorylated ERK1/2. Incubating endometrial explants in the presence of an inhibitor of ERK1/2 activity blocked the ability of oxytocin to phosphorylate ERK1/2 and stimulate PGF2{alpha} synthesis. Our data are in agreement with other studies in which oxytocin stimulates ERK activity and that ERK mediates PG synthesis [29–31].

Although phospho-JNK/SAPK and phospho-p38 MAPK were not detected in ovine endometrium, it is possible that these two MAPKs may still play an important role in mediating oxytocin-induced PGF2{alpha} synthesis. In the present study, tissue was collected at 15 min after oxytocin injection. It is possible that these two MAPKs may have been down-regulated or not activated at this time. Additional time points are required to determine whether JNK/SAPK or p38 MAPK mediate oxytocin-induced PGF2{alpha} synthesis in ovine endometrium.

The cellular and molecular mechanisms by which ERK1/2 mediate oxytocin-induced PGF2{alpha} synthesis in ovine endometrium remains to be determined. In other tissues and cells that actively secrete PGs in response to acute stimuli, an increase in phosphorylated ERK1/2 was correlated with an increase in cPLA2 activity and mobilization of arachidonic acid [22, 30]. Cytosolic PLA2 message and protein are present in ovine endometrial tissue [21, 40, 41]. Therefore, it is possible that ERK1/2 may either directly or indirectly phosphorylate cPLA2, resulting in an increase in arachidonic acid available for PGF2{alpha} synthesis.

Another possible way that ERK1/2 may promote PGF2{alpha} synthesis in ovine endometrium is by increasing PGHS-2 gene expression. We have reported that oxytocin-induced PGF2{alpha} synthesis was associated with a rapid increase in endometrial concentrations of PGHS-2 mRNA [42]. In several cells and tissues, ERK1/2 activation resulted in an increase in PGHS-2 gene expression [43, 44]. ERK1/2 has been reported to translocate to the nucleus, phosphorylate transcription factors [23, 24], and increase gene transcription [43, 44]. We suggest that ERK1/2 may increase PGF2{alpha} synthesis in ovine endometrium by either or both of these mechanisms.

The intracellular signaling mechanisms by which oxytocin stimulates uterine ERK1/2 activation and subsequent PGF2{alpha} synthesis are not completely understood. Oxytocin binds to a cell surface receptor and activates PLC [13, 45]. Once activated, PLC cleaves phosphatidylinositol bisphosphate into two second messengers, diacylglycerol and inositol trisphosphate. Diacylglycerol may increase PGF2{alpha} synthesis by increasing protein kinase C (PKC) activity. Protein kinase C then can activate any number of intracellular enzymes that may lead to the activation of cPLA2. One way PKC may stimulate PGF2{alpha} synthesis is by activating either Ras or Raf-1 activity. Both Ras and Raf-1 can phosphorylate and activate MAPK kinase (MEK1/2) [25]. Activated MEK1/2 could then phosphorylate ERK1/2, and thereby increase its activity. Activated ERK1/2 then phosphorylates cPLA2, thereby increasing its activity, and thus mobilizing arachidonic acid.

Inositol trisphosphate increases free intracellular Ca2+ concentration by binding to its receptor and opening Ca2+ channels on the endoplasmic reticulum. Calcium plays a critical role during oxytocin-induced PGF2{alpha} release in bovine endometrium [20]. The increase in free intracellular Ca2+ in response to an agonist may promote PG synthesis in several ways. Many intracellular regulatory proteins (e.g., PLC, PKC, and PLA2) have Ca2+ binding sites and require binding of Ca2+ for maximum activity [46]. The increase in free intracellular Ca2+ may be required for the activation of calmodulin and subsequent activation of ERK1/2 in ovine endometrium. Calmodulin plays a major role in signal transduction in many cells and tissues by interacting with and activating Ca2+/calmodulin-dependent (CaM) protein kinases. In rabbit aortic smooth muscle cells, norepinephrine has been reported to increase CaM kinase II{alpha} activity and subsequent mobilization of arachidonic acid [47]. CaM protein kinase II{alpha} appears to mediate its stimulatory effects through the activation of MAPK cascades [47, 48]. The precise signaling cascade and, specifically, the relative role of PKC or possibly CaM kinase II{alpha} in ERK1/2 phosphorylation in ovine and bovine endometrial cells remains to be determined.

In conclusion, the ovine oxytocin receptor does not appear to be coupled to Gi proteins in uterine endometrium. All three classes of MAPK were detected in ovine endometrial tissue. Of the three classes of MAPK, only ERK1/2 kinase appears to be phosphorylated in response to oxytocin and to mediate oxytocin-induced PGF2{alpha} synthesis.

ACKNOWLEDGMENTS

The authors thank Doug Yelton and Brent Broaddus for their extensive efforts during experiment 2. The authors also thank Drs. Terry Engle and Jason Bruemmer for their critical review of the manuscript.

FOOTNOTES

First decision: 18 September 2000.

1 Supported in part by grants from the U.S. Department of Agriculture (96-35203-3495 to P.D.B.), Colorado Agriculture Experiment Station, and Kentucky Agriculture Experiment Station. Presented in part at the 1999 and 2000 American Society of Animal Science Western Section meetings. Back

2 Correspondence. FAX 970 491 5326; pburns{at}ceres.agsci.colostate.edu Back

Accepted: May 21, 2001.

Received: August 28, 2000.

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