tYmn1trr (1992), 13. 429137
Immunoreactive Prostaglandin G/H Synthase Content Increases in Ovine Cotyledons During Late Gestation
G. E. RICE”, M. J. PAYNE, M. H. WONG & G. D. THORBURN Department of Ph,ysiolo.gy, Alonash 14zutralia 3168 ” To whom correspondence
I;niz~ersiy,
Clayton.
I i’ctoria.
should be addressed
Paper accepted 25.2. I992
SUMMARY In this studio, using gel electrophoresis and Western blotting, we have demonstrated that the content oj’irPGHS in orine placenta increases during late gestation prior to the onset of labour. This increase in PGHS tissue content may contribute to the corresponding increasedproduction ofprostaglandins by the ovineplacenta during this period of pregnancy. Although the mechanism by which PGHS tissue content is elevated at this time remains to be established, one possibilitywhich is currently being incestigated in our laborato y is that increased PGHS content rejpzts an increased level qfPGHS gene expression.
INTRODUCTION In all species studied to date, labour onset and/or progression is associated with an increase in the synthesis of prostaglandins by gestational tissues. The mechanisms which regulate intra-uterine prostaglandin (PG) synthesis and their role in the initiation and maintenance of labour, however, remain to be clearly elucidated (for reviews see: Thorburn, 1977; Mitchell, Ellwood and Brennecke, 1983; Liggins and Wilson, 1988; Rice, 1990; Olson et al, 1991). Previously, it has been suggested that intra-uterine prostaglandin synthesis may be suppressed by endogenous inhibitors during pregnancy and that the labour-associated increase in prostaglandin formation represents an escape or release from inhibition (Thorburn, 1977; Wilson, 1988). Consistent with this suggestion, various endogenous inhibitors of prostaglandin formation have been identified in plasma (Brennecke et al, 1982; Etienne, Gruber and Polonovski, 1984); amniotic fluid (Saeed et al, 1982; Wilson et al, 1985); endometrium (Gurpide, Markiewicz and Schatz, 1986); myometrium (Rice, Wong and Thorburn, 1987a); and allantoic fluid (Leach Harper and Thorburn, 1984; Rice et al, 1987b). In vivo (Fowden et al, 1987) and in vitro (Noden et al, 1981; Risbridger et al, 1985; 0143-4004/92/050429
+ 09 $08.00/O
CQ 1992 Baillike
Tindall l.td
430
Placenta(1994, Vol. 13
Moussard et al, 1986; Moussard, Alber and Henry, 1987; Rice, Wong and Thorburn, 1988; Rice et al, 1989) studies, however, have provided data that suggest the activity of the PG-forming enzyme, prostaglandin G/H synthase (PGHS) in intra-uterine tissues is not tonically suppressed but that the content and/or activity of this enzyme in gestational tissues gradually increases during the latter half of pregnancy and more rapidly as term approaches. These studies have primarily utilized indirect estimates of PGHS activity (e.g., the concentration of immunoreactive prostaglandins in plasma or incubation medium, or the formation of radiolabelled metabolites) or polarographic determination of enzyme activity (Rice et al, 1990). On the basis of the data obtained in studies that have focused upon the activity of PGHS it has not been possible to dissociate the effects of an increase in the tissue content of this enzyme from changes in the presence or absence of inhibitors and/or stimulators of this enzyme. The aim of this study, therefore, was to determine whether or not the previously reported increase in PG formation and PGHS activity which occur during late gestation and prior to the onset of labour in ewes are associated with an increase in the content of PGHS in gestational tissues. We have utilized Western immunoblot analysis to quantify immunoreactive PGHS (irPGHS) in placental tissue (cotyledons) obtained from ewes at 131-143 days of gestation.
MATERIALS
AND
METHODS
Materials Phenylmethylsulfonyl fluoride (PMSF), bovine serum albumin, Trizma base, sodium dodecylsulfate (SDS), and glycine were purchased from Sigma Chemical Co, St Louis, MO, USA. Tween 20, mercaptoethanol, glycerol, sodium chloride, bromophenol blue, methanol, and ammonium persulfate were obtained from BDH Chemicals Ltd, Poole, England. Ethylene diaminetetra-acetic acid (EDTA) and sucrose were obtained from May and Baker, Australia Pty Ltd, Victoria, Australia. TEMED and SDS PAGE molecular weight standards were purchased from Bio-Rad Laboratories, Richmond, CA, USA. Acrylamide and bisacrylamide were purchased from Gradipore Ltd, Pyrmont, Australia. Nitrocellulose membrane was obtained from Schleicher and Schuell, West Germany. Protein Alz51 was purchased from New England Nuclear, Boston, MA, USA. Tissue collection and preparation of microsomes Cotyledons were collected at post mortem, within 3-4 min of death, from Border-Leicester/ Merino cross ewes (n = 12) at 131 to 143 days of gestation prior to the onset of labour. Cotyledons were removed from the pregnant horn of the uterus following the administration of a lethal dose of pentabarbitone and were immediately placed in liquid nitrogen. The tissue was then stored at - 70°C until processed and assayed for irPGHS content (within 5 days of collection). Microsomes were prepared according to the method of Flower, Cheung and Cushman (1973) with minor modifications as follows. All procedures were conducted at 4°C. Frozen cotyledons were partially thawed and the capsule and haemophagous zone were quickly dissected and removed. Tissue (3-6 g wet weight) was minced using fine scissors before homogenisation in three volumes of Tris buffered saline (TBS; containing Tris 20 mmol/l; NaCl500 mmol/l; PMSF 0.1 mmol/l; and EDTA 5 mmol/l, pH 7.5). Homogenization was achieved by 2 x 20 set bursts of a metal-blade homogenizer (Ultra-Tarrax, Janke and Kunke GMBH, Ika-Werk, Stanfen). The tissue homogenates were centrifuged at 10 000 g
Rice et NI: Immunowuctire Prostaglandin Increases in Orine Co@edons
431
for 10 min at 4°C (Sorvall, Model RC-5B, Du Pont, Wilminton, Delaware, USA). The supernate was aspirated and centrifuged at 100 000 g for 60 min at 4°C (Ultracentrifuge, .Llodel L8-55, Beckman Instruments, Palo Alto, California, USA). The resulting pellet was resuspended in 20 ml TBS and recentrifuged at 100 000 g for 60 min at 4°C. The washed microsomal pellet was solubilised in Tween 20 (0.1 per cent) and PMSF (0.1 mmol/l) and stored at -70°C until assayed for protein (Bradford, 1976) and irPGHS content. Preparation of a rabbit polyclonal antibody to PGHS Purified PGHS from ram seminal vesicles was purchased from Oxford Biochemicals Co., Oxford, MI, CSA. The purity of this preparation was assessed by SDS-polyacrylamide gel electrophoresis on 6-15 per cent gradient gels. When 1 pug protein was loaded onto gels, PGHS migrated as a single major band (M, 72 000) and was found to be greater than 99 per cent pure. Female New Zealand White rabbits (1.5 kg body weight, n = 3) were immunized by multiple-site subcutaneous injection of PGHS [60 pg in 300 ~1 saline:Freund’s complete ndjuvant (1: 1 x-/v) per rabbit], three times at intervals of 2 weeks. Booster immunizations were administered, thereafter, at intervals of 6 weeks. Six months after the initial immunization. rabbits were bled, serum was collected and stored at -20°C. The antiserum was characterized by Western blot analysis of ram seminal vesicle PGHS and ovine placental homogenate. SDS-polyacrylamide gel electrophoresis \licrosomal proteins were separated on a 6-15 per cent gradient SDS-polyacrylamide gel electrophoresis (PAGE) system using a discontinuous buffer system following modifications to the methods described by Shoeman and Schweiger (1982). Samples (150 ,ug protein) together with PGHS (Oxford Biochemicals) and Bio-Rad molecular weight markers were applied to gels in 20-30 ~1 of gradient gel loading buffer [Tris-HCl 100 mmol/l, pH 9.2; SDS 2 per cent (w/v), EDTA 2 mmol/l; glycerol 10 per cent (L-/V);bromophenol blue 0.05 per cent (w/v)]. Electrophoresis was conducted for approximately 20 h at 15 mA using a discontinuous buffer system employing Tris-borate (0.04 mol/l; pH 8.6), SDS (1 per cent w/v) in the upper resen oir and Tris-HCl (0.42 ~1; pH 9.2) in the lower reservoir. Western blot analysis lmmunoblotting of the resolved proteins was achieved using a semi-dry electrophoretic technique and graphite electrodes (Kyhse-Andersen, 1984). Following the transfer of tissue proteins to nitrocellulose paper, the blot was blocked b! incubating in TTBS [Tris-HCl(20 mmol/l; pH 7.5), NaCl500 mmol/l, Tween 20 0.5 per cent] for 0.5 h at room temperature. The blot was then incubated in TTBS containing antiPGHS IgG at a dilution of 1: 1000 for 1 h, washed in TTBS for 30 min and finally incubated for 2 h in TTBS containing 5 x 1O4 Bq ml -’ “‘I-protein A. The membrane was washed for 1 h and dried before apposition to Kodak X-AR film (with intensifying screens) for 24-48 h at - 70°C. Immune complexes were visualized following development of the exposed film. lllolecular weight protein standards were separately visualized by amido black staining (Kuro and Kihara, 1967). The molecular weights of electrophoretically resolved proteins were estimated from their migratory rates relative to that of the standards. Nitrocellulose membranes were overlaid with autoradiographs to localise irPGHS activity. .-\ section of nitrocellulose membrane (1.2 x 1 cm) containing the region of activity which
432
Placenta (1992), ZOI.13
co-migrated with ram seminal vesical standard was excised from the membrane and placed in a 12 X 75 mm plastic tube. Radioactivity was quantified by gamma scintillation spectrometry. To establish the relationship between bound ct/min and PGHS, ram seminal vesicle PGHS standard (1 to 500 ng/lane) was subjected to SDS-PAGE and radioimmunoblot analysis. Protein assay The protein content of microsomal preparations was determined using a protein dye-binding method (Bio-Rad Laboratories) using bovine serum albumin as a reference standard (Bradford, 1976). Statistical analysis The data presented in this paper represent the mean + standard error of the mean. Mean values were compared using two-tailed Student’s t-tests. Variation in irPGHS over the period of gestation examined was assessed by correlation analysis.
RESULTS Western immunoblot for ovine PGHS To determine the specificity of the polyclonal ram seminal vesicle PGHS anti-serum, Western immunoblot analysis of solubilized microsomes prepared from ovine cotyledons was performed (Figure 1). The antibody bound to a single major protein band (with an estimated M, = 70 800) which co-migrated with ram seminal vesicle standard. A secondary immunoreactive band (M, = 53 700) was also identified but represented
Immunoreactive Prostaglandin G/H Synthase Content Increases in Ovine Cotyledons During Late Gestation
G. E. RICE”, M. J. PAYNE, M. H. WONG & G. D. THORBURN Department of Ph,ysiolo.gy, Alonash 14zutralia 3168 ” To whom correspondence
I;niz~ersiy,
Clayton.
I i’ctoria.
should be addressed
Paper accepted 25.2. I992
SUMMARY In this studio, using gel electrophoresis and Western blotting, we have demonstrated that the content oj’irPGHS in orine placenta increases during late gestation prior to the onset of labour. This increase in PGHS tissue content may contribute to the corresponding increasedproduction ofprostaglandins by the ovineplacenta during this period of pregnancy. Although the mechanism by which PGHS tissue content is elevated at this time remains to be established, one possibilitywhich is currently being incestigated in our laborato y is that increased PGHS content rejpzts an increased level qfPGHS gene expression.
INTRODUCTION In all species studied to date, labour onset and/or progression is associated with an increase in the synthesis of prostaglandins by gestational tissues. The mechanisms which regulate intra-uterine prostaglandin (PG) synthesis and their role in the initiation and maintenance of labour, however, remain to be clearly elucidated (for reviews see: Thorburn, 1977; Mitchell, Ellwood and Brennecke, 1983; Liggins and Wilson, 1988; Rice, 1990; Olson et al, 1991). Previously, it has been suggested that intra-uterine prostaglandin synthesis may be suppressed by endogenous inhibitors during pregnancy and that the labour-associated increase in prostaglandin formation represents an escape or release from inhibition (Thorburn, 1977; Wilson, 1988). Consistent with this suggestion, various endogenous inhibitors of prostaglandin formation have been identified in plasma (Brennecke et al, 1982; Etienne, Gruber and Polonovski, 1984); amniotic fluid (Saeed et al, 1982; Wilson et al, 1985); endometrium (Gurpide, Markiewicz and Schatz, 1986); myometrium (Rice, Wong and Thorburn, 1987a); and allantoic fluid (Leach Harper and Thorburn, 1984; Rice et al, 1987b). In vivo (Fowden et al, 1987) and in vitro (Noden et al, 1981; Risbridger et al, 1985; 0143-4004/92/050429
+ 09 $08.00/O
CQ 1992 Baillike
Tindall l.td
430
Placenta(1994, Vol. 13
Moussard et al, 1986; Moussard, Alber and Henry, 1987; Rice, Wong and Thorburn, 1988; Rice et al, 1989) studies, however, have provided data that suggest the activity of the PG-forming enzyme, prostaglandin G/H synthase (PGHS) in intra-uterine tissues is not tonically suppressed but that the content and/or activity of this enzyme in gestational tissues gradually increases during the latter half of pregnancy and more rapidly as term approaches. These studies have primarily utilized indirect estimates of PGHS activity (e.g., the concentration of immunoreactive prostaglandins in plasma or incubation medium, or the formation of radiolabelled metabolites) or polarographic determination of enzyme activity (Rice et al, 1990). On the basis of the data obtained in studies that have focused upon the activity of PGHS it has not been possible to dissociate the effects of an increase in the tissue content of this enzyme from changes in the presence or absence of inhibitors and/or stimulators of this enzyme. The aim of this study, therefore, was to determine whether or not the previously reported increase in PG formation and PGHS activity which occur during late gestation and prior to the onset of labour in ewes are associated with an increase in the content of PGHS in gestational tissues. We have utilized Western immunoblot analysis to quantify immunoreactive PGHS (irPGHS) in placental tissue (cotyledons) obtained from ewes at 131-143 days of gestation.
MATERIALS
AND
METHODS
Materials Phenylmethylsulfonyl fluoride (PMSF), bovine serum albumin, Trizma base, sodium dodecylsulfate (SDS), and glycine were purchased from Sigma Chemical Co, St Louis, MO, USA. Tween 20, mercaptoethanol, glycerol, sodium chloride, bromophenol blue, methanol, and ammonium persulfate were obtained from BDH Chemicals Ltd, Poole, England. Ethylene diaminetetra-acetic acid (EDTA) and sucrose were obtained from May and Baker, Australia Pty Ltd, Victoria, Australia. TEMED and SDS PAGE molecular weight standards were purchased from Bio-Rad Laboratories, Richmond, CA, USA. Acrylamide and bisacrylamide were purchased from Gradipore Ltd, Pyrmont, Australia. Nitrocellulose membrane was obtained from Schleicher and Schuell, West Germany. Protein Alz51 was purchased from New England Nuclear, Boston, MA, USA. Tissue collection and preparation of microsomes Cotyledons were collected at post mortem, within 3-4 min of death, from Border-Leicester/ Merino cross ewes (n = 12) at 131 to 143 days of gestation prior to the onset of labour. Cotyledons were removed from the pregnant horn of the uterus following the administration of a lethal dose of pentabarbitone and were immediately placed in liquid nitrogen. The tissue was then stored at - 70°C until processed and assayed for irPGHS content (within 5 days of collection). Microsomes were prepared according to the method of Flower, Cheung and Cushman (1973) with minor modifications as follows. All procedures were conducted at 4°C. Frozen cotyledons were partially thawed and the capsule and haemophagous zone were quickly dissected and removed. Tissue (3-6 g wet weight) was minced using fine scissors before homogenisation in three volumes of Tris buffered saline (TBS; containing Tris 20 mmol/l; NaCl500 mmol/l; PMSF 0.1 mmol/l; and EDTA 5 mmol/l, pH 7.5). Homogenization was achieved by 2 x 20 set bursts of a metal-blade homogenizer (Ultra-Tarrax, Janke and Kunke GMBH, Ika-Werk, Stanfen). The tissue homogenates were centrifuged at 10 000 g
Rice et NI: Immunowuctire Prostaglandin Increases in Orine Co@edons
431
for 10 min at 4°C (Sorvall, Model RC-5B, Du Pont, Wilminton, Delaware, USA). The supernate was aspirated and centrifuged at 100 000 g for 60 min at 4°C (Ultracentrifuge, .Llodel L8-55, Beckman Instruments, Palo Alto, California, USA). The resulting pellet was resuspended in 20 ml TBS and recentrifuged at 100 000 g for 60 min at 4°C. The washed microsomal pellet was solubilised in Tween 20 (0.1 per cent) and PMSF (0.1 mmol/l) and stored at -70°C until assayed for protein (Bradford, 1976) and irPGHS content. Preparation of a rabbit polyclonal antibody to PGHS Purified PGHS from ram seminal vesicles was purchased from Oxford Biochemicals Co., Oxford, MI, CSA. The purity of this preparation was assessed by SDS-polyacrylamide gel electrophoresis on 6-15 per cent gradient gels. When 1 pug protein was loaded onto gels, PGHS migrated as a single major band (M, 72 000) and was found to be greater than 99 per cent pure. Female New Zealand White rabbits (1.5 kg body weight, n = 3) were immunized by multiple-site subcutaneous injection of PGHS [60 pg in 300 ~1 saline:Freund’s complete ndjuvant (1: 1 x-/v) per rabbit], three times at intervals of 2 weeks. Booster immunizations were administered, thereafter, at intervals of 6 weeks. Six months after the initial immunization. rabbits were bled, serum was collected and stored at -20°C. The antiserum was characterized by Western blot analysis of ram seminal vesicle PGHS and ovine placental homogenate. SDS-polyacrylamide gel electrophoresis \licrosomal proteins were separated on a 6-15 per cent gradient SDS-polyacrylamide gel electrophoresis (PAGE) system using a discontinuous buffer system following modifications to the methods described by Shoeman and Schweiger (1982). Samples (150 ,ug protein) together with PGHS (Oxford Biochemicals) and Bio-Rad molecular weight markers were applied to gels in 20-30 ~1 of gradient gel loading buffer [Tris-HCl 100 mmol/l, pH 9.2; SDS 2 per cent (w/v), EDTA 2 mmol/l; glycerol 10 per cent (L-/V);bromophenol blue 0.05 per cent (w/v)]. Electrophoresis was conducted for approximately 20 h at 15 mA using a discontinuous buffer system employing Tris-borate (0.04 mol/l; pH 8.6), SDS (1 per cent w/v) in the upper resen oir and Tris-HCl (0.42 ~1; pH 9.2) in the lower reservoir. Western blot analysis lmmunoblotting of the resolved proteins was achieved using a semi-dry electrophoretic technique and graphite electrodes (Kyhse-Andersen, 1984). Following the transfer of tissue proteins to nitrocellulose paper, the blot was blocked b! incubating in TTBS [Tris-HCl(20 mmol/l; pH 7.5), NaCl500 mmol/l, Tween 20 0.5 per cent] for 0.5 h at room temperature. The blot was then incubated in TTBS containing antiPGHS IgG at a dilution of 1: 1000 for 1 h, washed in TTBS for 30 min and finally incubated for 2 h in TTBS containing 5 x 1O4 Bq ml -’ “‘I-protein A. The membrane was washed for 1 h and dried before apposition to Kodak X-AR film (with intensifying screens) for 24-48 h at - 70°C. Immune complexes were visualized following development of the exposed film. lllolecular weight protein standards were separately visualized by amido black staining (Kuro and Kihara, 1967). The molecular weights of electrophoretically resolved proteins were estimated from their migratory rates relative to that of the standards. Nitrocellulose membranes were overlaid with autoradiographs to localise irPGHS activity. .-\ section of nitrocellulose membrane (1.2 x 1 cm) containing the region of activity which
432
Placenta (1992), ZOI.13
co-migrated with ram seminal vesical standard was excised from the membrane and placed in a 12 X 75 mm plastic tube. Radioactivity was quantified by gamma scintillation spectrometry. To establish the relationship between bound ct/min and PGHS, ram seminal vesicle PGHS standard (1 to 500 ng/lane) was subjected to SDS-PAGE and radioimmunoblot analysis. Protein assay The protein content of microsomal preparations was determined using a protein dye-binding method (Bio-Rad Laboratories) using bovine serum albumin as a reference standard (Bradford, 1976). Statistical analysis The data presented in this paper represent the mean + standard error of the mean. Mean values were compared using two-tailed Student’s t-tests. Variation in irPGHS over the period of gestation examined was assessed by correlation analysis.
RESULTS Western immunoblot for ovine PGHS To determine the specificity of the polyclonal ram seminal vesicle PGHS anti-serum, Western immunoblot analysis of solubilized microsomes prepared from ovine cotyledons was performed (Figure 1). The antibody bound to a single major protein band (with an estimated M, = 70 800) which co-migrated with ram seminal vesicle standard. A secondary immunoreactive band (M, = 53 700) was also identified but represented
