Comp. Biochem. Physiol.Vol. 103B,No. I, pp. 217-220, 1992
0305-0491/92$5.00+ 0.00 © 1992PergamonPress Ltd
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PROGESTERONE STIMULATES PROSTAGLANDIN SYNTHESIS IN EGGSHELL GLAND MUCOSA OF ESTROGEN-PRIMED CHICKENS C. E. LUNDHOLM Department of Pharmacology, University of Linktping, Faculty of Health Sciences, S-581 85 Linktping, Sweden (Received 27 January 1992; accepted 6March 1992)
Abstract--1. Prostaglandins may be involved in calcium translocation in the avian shell gland, since indomethacin, administered at the beginning of shell formation, reduces eggshell thickness as well as 45Ca-uptake and prostaglandin synthesis by a homogenate of eggshell gland mueosa. 2. The stimulus for calcium transport in the shell gland during shell formation remains unknown. 3. The present study was undertaken to investigate the effects of progesterone on prostaglandin formation by the eggshell gland mucosa of the domestic fowl. 4. Progesterone significantly stimulated synthesis of PGFa, PGE2 and TXB2 by eggshell gland mucosa homogenate. 5. Progesterone treatment also induced the synthesis of the biotin-binding protein, avidin. 6. A microsomal fraction prepared from the eggshell gland mucosa had a high affinity for binding PGE2. 7. Progesterone treatment reduced the KD value of this binding without affecting the maximal number of binding sites. 8. Progesterone did not change the total calcium content of shell gland mueosa. 9. The role progesterone plays in prostaglandin formation and calcium transport in the eggshell gland mucosa is discussed.
INTRODUCTION
Prostaglandins play an important role in different reproductive processes, both in mammals and birds (Campbell, 1990). Involvement of prostaglandins in oviposition in birds has been extensively studied by, among others, Hertelendy (1972, 1974), Wechsung et aL (1978), Hammond et al. (1980) and Olson and Hertelendy (1981). Considerably less attention has been given to the effects of prostaglandins on other aspects of avian reproduction, such as eggshell formation. Hammond (1978) administered indomethacin (50 mg/kg), an inhibitor of prostaglandin synthesis, to egglaying domestic fowls 4-6 hr prior to oviposition. As a result of this treatment, eggs about to be laid were retarded in the shell gland. The shell thickness of these eggs was increased and plasma calcium was reduced by 30%. Lundholm (1985) administered indomethacin (100mg/bird daily) for three consecutive days to domestic fowls and observed that oviposition was delayed until the third day, at which time two eggs were laid, one shell-less and the other with a very thin calcified shell. Administration of two other prostaglandin synthesis inhibitors, naproxen and diclofenac (100mg daily per bird), for three consecutive days delayed oviposition and reduced shell thickness by 40%. Administration of a single oral dose of indomethacin (100 mg/bird) to egg-laying domestic fowls at 17:00, when the shell gland contained an egg with a shell beginning calcification, resulted in 21% shell thinning at the time of slaughter, that is 08:00 the next
day (Lundholm and Bartonek, in preparation). Prostagiandin production by shell gland mucosa was reduced by 62--66%, and the 45Ca-uptake by the homogenate of the shell gland mucosa and its subcellular fractions was reduced by 17-35%. These results indicate that, by influencing prostaglandin metabolism, indomethacin may influence eggshell calcification and oviposition as well. When studying avian reproductive physiology, it is difficult to identify the signalthat stimulates calcium translocation across the shell gland mucosa at the time of eggshell calcification. Addressing this problem, Eastin and Spaziani (1978a) Perfused the shell gland lumen of domestic fowls in situ and found that calcium translocation from blood to shell gland lumen only occurred during periods of active shell formation and was not initiated by distension of the shell gland caused by an artificial egg. These researchers concluded that the stimulus for calcium secretion was of a hormonal nature. Progesterone is known to stimulate the synthesis of egg-white proteins in the magnum region of the oviduct (O'Malley et al., 1969), and it probably also stimulates calcium translocation in the shell gland. It has been shown that by administering progesterone to domestic fowls, an egg about to be laid can be retarded in the shell gland, and the shell thickness of this egg is increased (Nys, 1987). The effects of progesterone on prostaglandin production by eggshell gland mucosa are not clear. Rzasa (1981) incubated homogenized domestic fowl uteri together with estradiol and estradioi progesterone
217
218
C.E. LUNDHOLM Prostaglandtn synthesis pmol/mg p r o t . / 3 0 r a i n .
It was observed that, alone, estradiol increased the p r o d u c t i o n o f prostaglandin-like material (as m e a s u r e d by a bioassay), whereas a c o m b i n a t i o n of estradiol a n d progesterone reduced this production. The present study was u n d e r t a k e n to further investigate the relationship between steroid h o r m o n e s a n d p r o s t a g l a n d i n m e t a b o l i s m in the shell gland mucosa.
Est*Prog Est
80 70 60 50 40
MATERIALS AND METHODS
30
Animals
20
Fourteen ten-week-old female chickens of the White Leghorn strain were obtained from a local poultry breeder. The birds were kept together in a single group indoors and allowed to acclimatize to the new environment for two days. Thereafter, all animals were given a daily i.m. injection of I mg estradiol in the form of estradiolbenzoate dissolved in 100/zl polyethylene glycol 400. After receiving estradiol injections for nine to 15 days, two of the birds were given 2 i.m. injections of I mg progesterone dissolved in 200/~1 polyethylene glycol 400: one injection was given 24 hr and one 12 hr prior to sacrifice. Two birds treated only with estradiol and the two treated with estradiol and progesterone were killed by a blow to the head and then exsanguinated. The magnum region of the oviduct of each animal was removed and frozen at - 2 0 ° C until analyzed for avidin. The remainder of each oviduct, and the rest of the shell glands, were immediately processed as described below.
10 0
PGF2a
PGE~
TxB2
Fig. 1. The effect of estrogen and estrogen + progesterone on the synthesis of prostaglandins F~, E 2 and thromboxane B2 by eggshell gland mucosa from sexually immature chickens. Mean + SEM, N = 14. Statistical significance is denoted by **P < 0.01, ***P < 0.001. poured through a Millipore filter (0.45 p m pore size) under vacuum. Each of the sample tubes was rinsed with 1 ml buffer, which was also filtered, and these filters were rinsed with 2 × 5 ml buffer Filters used for each sample were dissolved in 1 ml cellusolve, and a toluene-based scintillation cocktail was added and the samples were counted in a liquid scintillation counter.
Prostaglandin synthesis
After a fowl was slaughtered the eggshell gland was rapidly removed and chilled in buffer (Tris-HCl 100 mM, Na-EDTA 1 mM, pH 7.5) on ice. The mucosa was scraped from the muscular layer with a stainless steel knife. A 1.5 g portion of the mucosa was homogenized on ice in 10 ml of buffer in a glass/glass motor-driven homogenizer. The homogenizer and the pestle were rinsed, with 5 ml buffer, resulting in a ten-fold dilution of the tissue. Formation of prostaglandins, F2~, E2 and thromboxane B2 was determined by assaying the degradation of tritium-labelled arachidonic acid (5,6,8,9,11,12,14,15 3H-arachidonic acid, 180 Ci/mmol, NEN) and then separating the product by thin-layer chromatography (60 F2u 0.25 mm thick, 20 x 20 cm, Merck), as described in detail by Asboth et al. (1983) and Lundholm and Bartonek (1991).
Avidin assay
The content of avidin from the midportion of the frozen magnum regions was determined by the 14C-biotin method, as described by Elo et al. (1975). Protein
Protein was determined according to Lowry et al. (1951). Calcium content
Calcium content in shell gland mucosa was determined by atomic absorption spectroscopy, as described by Lundholm (1984).
RESULTS
Prostaglandin E 2 binding
Shell gland mucosa homogenates were prepared as described in the previous section and centrifuged at 2500 g for 20 min at 2°C. The supernatants were further centrifuged at l l0,000g for 60min, and the resulting pellets were homogenized (Teflon/glass) in 1 ml buffer to obtain membrane suspensions. Five 50-#1 aliquots of each of these eggshell gland suspension were added separately to 450 #1 portions of buffer containing different amounts of PGE 2 labelled with 3H-PGE2: PGE 2 concentrations of 0.31, 0.62, 1.25, 2.5 and 5 n M were used. To determine non-specific binding, a 100 x molar excess of non-labelled PGE 2 was added to a separate set of test tubes containing membrane suspensions. After incubation for 1 hr in a water-bath at 37°C, the samples were diluted with 1 ml ice-cold buffer and rapidly
Prostaglandin synthesis
T h e synthesis o f PGF2~, P G E 2 a n d T X B 2 in eggshell gland m u c o s a was significantly stimulated by progesterone, as can be seen in Fig. 1. In birds treated with b o t h estrogen a n d progesterone, values for PGF2~ synthesis increased f r o m 1 9 + 1.9 to 32 + 3.8 p m o l / m g protein/30 rain ( P < 0.01), as comp a r e d to the values for chickens treated with estrogen only. T h e c o r r e s p o n d i n g values for PGE2 synthesis r a n g e d f r o m 15 + 2.1 to 31 + 2.9 ( P < 0.001) a n d for T X B 2 from 40 + 6.6 to 84 + 10 ( P < 0.01) p m o l / m g protein/30 min.
Table 1. The effects o f estrogen and estrogen + progesterone treatment on the binding o f 3H-PGE2 to a microsomal m e m b r a n e fraction prepared f r o m eggshell gland m u c o s a o f chickens Treatment Estrogen Estrogen + progesterone
K D (pM)
Bmax ( p m o l / m g protein)
1.56 ± 0.14 1.00 _± 0.1 *
222 + 14 184 ± 23
K D = dissociation constant, Bmax = m a x i m a l n u m b e r M e a n + S E M , N = 12. Significance levels: * P < 0.05.
of
binding
sites.
Progesterone stimulates PG synthesis PGE2 binding
When calculating binding parameters according to Seatehard (1949; see Table 1), it was found that progesterone treatment significantly decreased the KD value. The maximal number of binding sites (Bm~) was not significantly changed by progesterone. Avidin synthesis
Treatment with estrogen alone resulted in very low levels (i.e. below the detection limit of the method; blank values) of avidin in oviduct magnum. After progesterone treatment, the content increased to 0.17 + 0.05/~g/g shell gland (i.e. 2.5 times the blank value). Calcium
The calcium content of the shell gland mucosa was not significantly changed by progesterone treatment (with estrogen 1257 + 101 #g/g dry weight and with estrogen + progesterone 1452 + 94 #g/g dry weight). The values were quite similar to those found for the shell gland mucosa of egg-laying birds (1279 _ 71 ttg/g dry weight). DISCUSSION
Administration of progesterone to estrogen-primed chickens significantly increased prostaglandin synthesis by homogenates of eggshell gland mucosa. This is not consistent with the results of Rzasa (1981) showing that estrogen + progesterone reduced the tissue level of smooth-muscle-contracting prostaglandins when homogenized shell glands were incubated in vitro with the steroid. Differences in experimental design might explain the divergent results. In the present study progesterone was administered in vivo and acted over a longer period of time (24 hr). Prostaglandin synthetase activity was determined but not the actual tissue level of the prostaglandins. The KD value for PGE2 was significantly reduced after progesterone treatment, which could indicate a higher tissue concentration of PGE2 and probably other prostaglandins as well. The increased level of avidin in the magnum region of the oviduct after progesterone administration is evidence of a progestogenic response (O'Malley et al., 1969). Studying chicken oviduct cell culture Niemel~i (1986) observed that PGF2~ and PGE2 stimulated avidin synthesis in much the same way progesterone did, but that the effects of progesterone and prostaglandins were not additive. It therefore seems probable that some of the effects of progesterone on the avian oviduct are mediated by prostaglandins. The involvement of prostaglandins in oviposition has, as mentioned above, been clearly demonstrated in several experiments. An increased prostaglandin synthesis could result in increased muscular contractions of the eggshell gland and premature expulsion of the egg. It is likely, however, that oviposition requires the additional influence of prostaglandins, arglnine--vasotocin, or of an unidentified ovipositioninducing factor released from the pre- and postovulatory follicles in the ovary, as discussed by Kelly et al. (1990). Another contributary effect could be that during oviposition prostaglandin synthesis in the
219
shell gland is directed towards the more muscle-contracting PGF2~, whereas during shell formation the synthesis is directed towards other prostaglandins. The role of prostaglandins in calcium transport across the eggshell gland mucosa has not been studied. HCO3- secretion from epithelial ceils of the duodenum is stimulated by PGE2 and inhibited by indomethacin (Flemstrrm et aL, 1982; Konturek et aL, 1983). Experiments performed by Eastin and Spaziani (1978b) and Pearson and Goldner (1973) suggest that part of the calcium transport across the shell gland is coupled to HCO3- secretion. Further evidence of this is provided by the results of later experiments (Lundholm, 1990) on eggshell gland mucosa from the domestic fowl; in these experiments eggshell thickness, carbanhydrase activity and 45Ca-uptake were all reduced by aeetazolamide. Prostaglandins stimulate the active transepithelial transport of Na ÷ in frog skin (Hall et al., 1976) and may also regulate transepithelial C1- transport (A1Bazzaz et al., 1981). Such a mechanism could be of importance during shell formation, since sodium from the egg is reabsorbed during shell formation (Mongin and Sauveur, 1970; Eastin and Spaziani, 1978b). Induction of prostaglandin synthesis by progesterone could also be of importance when elucidating the mechanism of the eggshell thinning effect of p,p'DDE. This compound is a potent inhibitor of prostaglandin synthesis in duck eggshell gland mucosa, both after addition in vitro and after prolonged ingestion of a dose that produces eggshell thinning (Lundholm and Bartonek, in preparation). In domestic fowls, however, p,p'-DDE caused no eggshell thinning, but did inhibit prostaglandin synthesis when added in vitro to eggshell gland mucosa homogenate to about the same level as in the corresponding preparation from the p,p'-DDE sensitive duck. Progesterone receptors prepared from eggshell gland mucosa of the domestic fowl had about 3 times higher affinity for binding progesterone than did receptors prepared from duck eggshell gland mucosa (Lundholm, 1988). It is probable that p,p'-DDE-induced eggshell thinning in the duck is the result of the combined effect of a direct inhibition of prostaglandin synthetase and an impairment of the binding of progesterone to its receptor, which would also inhibit the formation of prostaglandins.
REFERENCES
AI-Bazzaz F., Yaava V. P. and Westenfelder C. (1981) Modification of Na and CI transport in canine tracheal mucosa by prostaglandins. Am. J. Physiol. 240, F101-105. Asboth G., Toth M. and Hertelendy F. (1983) Conversion of arachidonic acid to prostanoids in the avian uterus. Biochim. biophys. Acta 750, 481--489. Eastin W. C. and Spaziani E. (1978a) On the control of calcium secretion in the avian shell gland (uterus). Biol. Reprod. 19, 493-504. Eastin W. C. Jr and Spaziani E. (1978b) On the mechanism of calcium secretion in the avian shell gland (uterus). Biol. Reprod. 19, 505-518. Elo H., Tuotrimaa P. and J~inne O. (1975) Cumulative superinduction of avidin in the chick oviduct by tissue
220
C.E. LUNDHOLM
damage and Actinomycin D. Molec. Cell Endocr. 2, 203-211. Flemstr6m G., Garner A., Nylander O., Hurst B. C. and Heylings J. R. (1982) Surface epithelial HCO3- transport by mammalian duodenum in vivo. Am. J. Physiol. 243, G348-358. Hall W. J., O'Donoghue J. P., O'Regan M. G. and Penny M. J. (1976) Endogenous prostaglandins, adenosine Y,5'monophosphate and sodium transport across isolated frog skin. J. Physiol., Lond. 258, 731-753. Hammond R. W. (1978) Effect of indomethacin on the laying cycle, plasma calcium and shell thickness in the laying hen. Poultry Sci. 57, 1141. Hammond R. W., Olson D. M., Frenkel R. B., Bieller H. V. and Hertelendy F. (1980) Prostaglandins and steroid hormones in plasma ovarian follicles during the ovulation cycle of the domestic hen. Gen. comp. Endocr. 195-202. Hertelendy F. (1972) Prostaglandin induced premature oviposition in the Coturnix quail. Prostaglandins 2, 269. Hertelendy F. (1974) Effects of prostaglandins, cAMP, seminal plasma, indomethacin and other factors on ov position in the Japanese quail. J. Reprod. Fert. 40, 87-93. Kelly J. D., Etches R. J. and Gu6men6 D. (1990) Follicular control of oviposition in the hen. Poultry Sci. 69, 288-29 I. Konturek S. J., Tasler J., Bilski J. and Kania J. (1983) Prostaglandins and alkaline secretion from oxyntic antral and duodenal mucosa of the dog. Am. J. Physiol. 245, G 539-546. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. Lundholm C. E. (1984) Calcium content of duck eggshell gland mucosa homogenate and the rate of Ca 2+ binding to its subcellular fractions during and after the formation of the eggshell. Camp. Biochem. Physiol. 77B, 655~563. Lundholm C. E. (1985) Studies of the effect of DDE on the calcium metabolism of the eggshell gland during formation of the eggshell in ducks and domestic fowls.
Link@ing University Medical Dissertations, No. 205, Link6ping, Sweden. Lundholm C. E. 0988) The effect of DDE, PCB and chlordane on the binding of progesterone to its cytoplasmic receptor in the eggshell gland mucosa of birds and the endometrium of the mammalian uterus. Comp. Biochem. Physiol. 89C, 361-368. Lundholm C. E. and Bartonek M. (1991) A study of the effects ofp,p'-DDE and other related chlorinated hydrocarbons on the inhibition of platelet aggregation. Arch. Toxicol. 65, 570-574. Mongin P. and Sauveur B. (1970) Composition du fluide uterin et de l'albumen durant le s6jour de l'oeuf dans l'ut6rus chez la poule domestique. C.-r. Acad. Sci., Paris 270D, 1715-1718. Niemelii A. O. (1986) Regulation of avidin accumulation by prostaglandins in chick oviduct cell culture. J. Steroid Biochem. 24, (3) 709 715. Nys Y. (1987) Progesterone and testosterone elicit increases in the duration of shell formation in domestic hens. Br. Poult. Sci. 28, 57~58. Olson D. M. and Hertelendy F. (1981) Plasma levels of 13,14-dihydro-15-keto prostaglandin F2~ in relation to oviposition in the domestic hen. Biol. Reprod. 24, 496-504. O'Malley B. W., McGuire P. O., Kohler P. O. and Korenman S. G. (1969) Studies on the mechanism of steroid hormone regulation of synthesis of specific proteins. Rec. Prog. Horm. Res. 25, 105-160. Pearson T. W. and Goldner A. M. (1973) Calcium transport across avian uterus I. Effects of electrolyte substitution. Am. J. Physiol. 225, 1505-1512. Rzasa I. (1981) Prostaglandin production by the hen oviduct in vivo and in vitro. In Recent Advances o f Avian Physiology. Advances in Physiologicol Science (Edited by Pethes G., P6czely P. and Rudas P.), Vol. 33. pp. 177-181. Wechsung L., Korteweg M., Verdonk G. and Houvenaghel A. (1978) Plasma levels of prostaglandin F~ related to oviposition in the domestic hen. Arch. Int. Pharmacodyn. 236, 331-333.
0305-0491/92$5.00+ 0.00 © 1992PergamonPress Ltd
Printed in Great Britain
PROGESTERONE STIMULATES PROSTAGLANDIN SYNTHESIS IN EGGSHELL GLAND MUCOSA OF ESTROGEN-PRIMED CHICKENS C. E. LUNDHOLM Department of Pharmacology, University of Linktping, Faculty of Health Sciences, S-581 85 Linktping, Sweden (Received 27 January 1992; accepted 6March 1992)
Abstract--1. Prostaglandins may be involved in calcium translocation in the avian shell gland, since indomethacin, administered at the beginning of shell formation, reduces eggshell thickness as well as 45Ca-uptake and prostaglandin synthesis by a homogenate of eggshell gland mueosa. 2. The stimulus for calcium transport in the shell gland during shell formation remains unknown. 3. The present study was undertaken to investigate the effects of progesterone on prostaglandin formation by the eggshell gland mucosa of the domestic fowl. 4. Progesterone significantly stimulated synthesis of PGFa, PGE2 and TXB2 by eggshell gland mucosa homogenate. 5. Progesterone treatment also induced the synthesis of the biotin-binding protein, avidin. 6. A microsomal fraction prepared from the eggshell gland mucosa had a high affinity for binding PGE2. 7. Progesterone treatment reduced the KD value of this binding without affecting the maximal number of binding sites. 8. Progesterone did not change the total calcium content of shell gland mueosa. 9. The role progesterone plays in prostaglandin formation and calcium transport in the eggshell gland mucosa is discussed.
INTRODUCTION
Prostaglandins play an important role in different reproductive processes, both in mammals and birds (Campbell, 1990). Involvement of prostaglandins in oviposition in birds has been extensively studied by, among others, Hertelendy (1972, 1974), Wechsung et aL (1978), Hammond et al. (1980) and Olson and Hertelendy (1981). Considerably less attention has been given to the effects of prostaglandins on other aspects of avian reproduction, such as eggshell formation. Hammond (1978) administered indomethacin (50 mg/kg), an inhibitor of prostaglandin synthesis, to egglaying domestic fowls 4-6 hr prior to oviposition. As a result of this treatment, eggs about to be laid were retarded in the shell gland. The shell thickness of these eggs was increased and plasma calcium was reduced by 30%. Lundholm (1985) administered indomethacin (100mg/bird daily) for three consecutive days to domestic fowls and observed that oviposition was delayed until the third day, at which time two eggs were laid, one shell-less and the other with a very thin calcified shell. Administration of two other prostaglandin synthesis inhibitors, naproxen and diclofenac (100mg daily per bird), for three consecutive days delayed oviposition and reduced shell thickness by 40%. Administration of a single oral dose of indomethacin (100 mg/bird) to egg-laying domestic fowls at 17:00, when the shell gland contained an egg with a shell beginning calcification, resulted in 21% shell thinning at the time of slaughter, that is 08:00 the next
day (Lundholm and Bartonek, in preparation). Prostagiandin production by shell gland mucosa was reduced by 62--66%, and the 45Ca-uptake by the homogenate of the shell gland mucosa and its subcellular fractions was reduced by 17-35%. These results indicate that, by influencing prostaglandin metabolism, indomethacin may influence eggshell calcification and oviposition as well. When studying avian reproductive physiology, it is difficult to identify the signalthat stimulates calcium translocation across the shell gland mucosa at the time of eggshell calcification. Addressing this problem, Eastin and Spaziani (1978a) Perfused the shell gland lumen of domestic fowls in situ and found that calcium translocation from blood to shell gland lumen only occurred during periods of active shell formation and was not initiated by distension of the shell gland caused by an artificial egg. These researchers concluded that the stimulus for calcium secretion was of a hormonal nature. Progesterone is known to stimulate the synthesis of egg-white proteins in the magnum region of the oviduct (O'Malley et al., 1969), and it probably also stimulates calcium translocation in the shell gland. It has been shown that by administering progesterone to domestic fowls, an egg about to be laid can be retarded in the shell gland, and the shell thickness of this egg is increased (Nys, 1987). The effects of progesterone on prostaglandin production by eggshell gland mucosa are not clear. Rzasa (1981) incubated homogenized domestic fowl uteri together with estradiol and estradioi progesterone
217
218
C.E. LUNDHOLM Prostaglandtn synthesis pmol/mg p r o t . / 3 0 r a i n .
It was observed that, alone, estradiol increased the p r o d u c t i o n o f prostaglandin-like material (as m e a s u r e d by a bioassay), whereas a c o m b i n a t i o n of estradiol a n d progesterone reduced this production. The present study was u n d e r t a k e n to further investigate the relationship between steroid h o r m o n e s a n d p r o s t a g l a n d i n m e t a b o l i s m in the shell gland mucosa.
Est*Prog Est
80 70 60 50 40
MATERIALS AND METHODS
30
Animals
20
Fourteen ten-week-old female chickens of the White Leghorn strain were obtained from a local poultry breeder. The birds were kept together in a single group indoors and allowed to acclimatize to the new environment for two days. Thereafter, all animals were given a daily i.m. injection of I mg estradiol in the form of estradiolbenzoate dissolved in 100/zl polyethylene glycol 400. After receiving estradiol injections for nine to 15 days, two of the birds were given 2 i.m. injections of I mg progesterone dissolved in 200/~1 polyethylene glycol 400: one injection was given 24 hr and one 12 hr prior to sacrifice. Two birds treated only with estradiol and the two treated with estradiol and progesterone were killed by a blow to the head and then exsanguinated. The magnum region of the oviduct of each animal was removed and frozen at - 2 0 ° C until analyzed for avidin. The remainder of each oviduct, and the rest of the shell glands, were immediately processed as described below.
10 0
PGF2a
PGE~
TxB2
Fig. 1. The effect of estrogen and estrogen + progesterone on the synthesis of prostaglandins F~, E 2 and thromboxane B2 by eggshell gland mucosa from sexually immature chickens. Mean + SEM, N = 14. Statistical significance is denoted by **P < 0.01, ***P < 0.001. poured through a Millipore filter (0.45 p m pore size) under vacuum. Each of the sample tubes was rinsed with 1 ml buffer, which was also filtered, and these filters were rinsed with 2 × 5 ml buffer Filters used for each sample were dissolved in 1 ml cellusolve, and a toluene-based scintillation cocktail was added and the samples were counted in a liquid scintillation counter.
Prostaglandin synthesis
After a fowl was slaughtered the eggshell gland was rapidly removed and chilled in buffer (Tris-HCl 100 mM, Na-EDTA 1 mM, pH 7.5) on ice. The mucosa was scraped from the muscular layer with a stainless steel knife. A 1.5 g portion of the mucosa was homogenized on ice in 10 ml of buffer in a glass/glass motor-driven homogenizer. The homogenizer and the pestle were rinsed, with 5 ml buffer, resulting in a ten-fold dilution of the tissue. Formation of prostaglandins, F2~, E2 and thromboxane B2 was determined by assaying the degradation of tritium-labelled arachidonic acid (5,6,8,9,11,12,14,15 3H-arachidonic acid, 180 Ci/mmol, NEN) and then separating the product by thin-layer chromatography (60 F2u 0.25 mm thick, 20 x 20 cm, Merck), as described in detail by Asboth et al. (1983) and Lundholm and Bartonek (1991).
Avidin assay
The content of avidin from the midportion of the frozen magnum regions was determined by the 14C-biotin method, as described by Elo et al. (1975). Protein
Protein was determined according to Lowry et al. (1951). Calcium content
Calcium content in shell gland mucosa was determined by atomic absorption spectroscopy, as described by Lundholm (1984).
RESULTS
Prostaglandin E 2 binding
Shell gland mucosa homogenates were prepared as described in the previous section and centrifuged at 2500 g for 20 min at 2°C. The supernatants were further centrifuged at l l0,000g for 60min, and the resulting pellets were homogenized (Teflon/glass) in 1 ml buffer to obtain membrane suspensions. Five 50-#1 aliquots of each of these eggshell gland suspension were added separately to 450 #1 portions of buffer containing different amounts of PGE 2 labelled with 3H-PGE2: PGE 2 concentrations of 0.31, 0.62, 1.25, 2.5 and 5 n M were used. To determine non-specific binding, a 100 x molar excess of non-labelled PGE 2 was added to a separate set of test tubes containing membrane suspensions. After incubation for 1 hr in a water-bath at 37°C, the samples were diluted with 1 ml ice-cold buffer and rapidly
Prostaglandin synthesis
T h e synthesis o f PGF2~, P G E 2 a n d T X B 2 in eggshell gland m u c o s a was significantly stimulated by progesterone, as can be seen in Fig. 1. In birds treated with b o t h estrogen a n d progesterone, values for PGF2~ synthesis increased f r o m 1 9 + 1.9 to 32 + 3.8 p m o l / m g protein/30 rain ( P < 0.01), as comp a r e d to the values for chickens treated with estrogen only. T h e c o r r e s p o n d i n g values for PGE2 synthesis r a n g e d f r o m 15 + 2.1 to 31 + 2.9 ( P < 0.001) a n d for T X B 2 from 40 + 6.6 to 84 + 10 ( P < 0.01) p m o l / m g protein/30 min.
Table 1. The effects o f estrogen and estrogen + progesterone treatment on the binding o f 3H-PGE2 to a microsomal m e m b r a n e fraction prepared f r o m eggshell gland m u c o s a o f chickens Treatment Estrogen Estrogen + progesterone
K D (pM)
Bmax ( p m o l / m g protein)
1.56 ± 0.14 1.00 _± 0.1 *
222 + 14 184 ± 23
K D = dissociation constant, Bmax = m a x i m a l n u m b e r M e a n + S E M , N = 12. Significance levels: * P < 0.05.
of
binding
sites.
Progesterone stimulates PG synthesis PGE2 binding
When calculating binding parameters according to Seatehard (1949; see Table 1), it was found that progesterone treatment significantly decreased the KD value. The maximal number of binding sites (Bm~) was not significantly changed by progesterone. Avidin synthesis
Treatment with estrogen alone resulted in very low levels (i.e. below the detection limit of the method; blank values) of avidin in oviduct magnum. After progesterone treatment, the content increased to 0.17 + 0.05/~g/g shell gland (i.e. 2.5 times the blank value). Calcium
The calcium content of the shell gland mucosa was not significantly changed by progesterone treatment (with estrogen 1257 + 101 #g/g dry weight and with estrogen + progesterone 1452 + 94 #g/g dry weight). The values were quite similar to those found for the shell gland mucosa of egg-laying birds (1279 _ 71 ttg/g dry weight). DISCUSSION
Administration of progesterone to estrogen-primed chickens significantly increased prostaglandin synthesis by homogenates of eggshell gland mucosa. This is not consistent with the results of Rzasa (1981) showing that estrogen + progesterone reduced the tissue level of smooth-muscle-contracting prostaglandins when homogenized shell glands were incubated in vitro with the steroid. Differences in experimental design might explain the divergent results. In the present study progesterone was administered in vivo and acted over a longer period of time (24 hr). Prostaglandin synthetase activity was determined but not the actual tissue level of the prostaglandins. The KD value for PGE2 was significantly reduced after progesterone treatment, which could indicate a higher tissue concentration of PGE2 and probably other prostaglandins as well. The increased level of avidin in the magnum region of the oviduct after progesterone administration is evidence of a progestogenic response (O'Malley et al., 1969). Studying chicken oviduct cell culture Niemel~i (1986) observed that PGF2~ and PGE2 stimulated avidin synthesis in much the same way progesterone did, but that the effects of progesterone and prostaglandins were not additive. It therefore seems probable that some of the effects of progesterone on the avian oviduct are mediated by prostaglandins. The involvement of prostaglandins in oviposition has, as mentioned above, been clearly demonstrated in several experiments. An increased prostaglandin synthesis could result in increased muscular contractions of the eggshell gland and premature expulsion of the egg. It is likely, however, that oviposition requires the additional influence of prostaglandins, arglnine--vasotocin, or of an unidentified ovipositioninducing factor released from the pre- and postovulatory follicles in the ovary, as discussed by Kelly et al. (1990). Another contributary effect could be that during oviposition prostaglandin synthesis in the
219
shell gland is directed towards the more muscle-contracting PGF2~, whereas during shell formation the synthesis is directed towards other prostaglandins. The role of prostaglandins in calcium transport across the eggshell gland mucosa has not been studied. HCO3- secretion from epithelial ceils of the duodenum is stimulated by PGE2 and inhibited by indomethacin (Flemstrrm et aL, 1982; Konturek et aL, 1983). Experiments performed by Eastin and Spaziani (1978b) and Pearson and Goldner (1973) suggest that part of the calcium transport across the shell gland is coupled to HCO3- secretion. Further evidence of this is provided by the results of later experiments (Lundholm, 1990) on eggshell gland mucosa from the domestic fowl; in these experiments eggshell thickness, carbanhydrase activity and 45Ca-uptake were all reduced by aeetazolamide. Prostaglandins stimulate the active transepithelial transport of Na ÷ in frog skin (Hall et al., 1976) and may also regulate transepithelial C1- transport (A1Bazzaz et al., 1981). Such a mechanism could be of importance during shell formation, since sodium from the egg is reabsorbed during shell formation (Mongin and Sauveur, 1970; Eastin and Spaziani, 1978b). Induction of prostaglandin synthesis by progesterone could also be of importance when elucidating the mechanism of the eggshell thinning effect of p,p'DDE. This compound is a potent inhibitor of prostaglandin synthesis in duck eggshell gland mucosa, both after addition in vitro and after prolonged ingestion of a dose that produces eggshell thinning (Lundholm and Bartonek, in preparation). In domestic fowls, however, p,p'-DDE caused no eggshell thinning, but did inhibit prostaglandin synthesis when added in vitro to eggshell gland mucosa homogenate to about the same level as in the corresponding preparation from the p,p'-DDE sensitive duck. Progesterone receptors prepared from eggshell gland mucosa of the domestic fowl had about 3 times higher affinity for binding progesterone than did receptors prepared from duck eggshell gland mucosa (Lundholm, 1988). It is probable that p,p'-DDE-induced eggshell thinning in the duck is the result of the combined effect of a direct inhibition of prostaglandin synthetase and an impairment of the binding of progesterone to its receptor, which would also inhibit the formation of prostaglandins.
REFERENCES
AI-Bazzaz F., Yaava V. P. and Westenfelder C. (1981) Modification of Na and CI transport in canine tracheal mucosa by prostaglandins. Am. J. Physiol. 240, F101-105. Asboth G., Toth M. and Hertelendy F. (1983) Conversion of arachidonic acid to prostanoids in the avian uterus. Biochim. biophys. Acta 750, 481--489. Eastin W. C. and Spaziani E. (1978a) On the control of calcium secretion in the avian shell gland (uterus). Biol. Reprod. 19, 493-504. Eastin W. C. Jr and Spaziani E. (1978b) On the mechanism of calcium secretion in the avian shell gland (uterus). Biol. Reprod. 19, 505-518. Elo H., Tuotrimaa P. and J~inne O. (1975) Cumulative superinduction of avidin in the chick oviduct by tissue
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C.E. LUNDHOLM
damage and Actinomycin D. Molec. Cell Endocr. 2, 203-211. Flemstr6m G., Garner A., Nylander O., Hurst B. C. and Heylings J. R. (1982) Surface epithelial HCO3- transport by mammalian duodenum in vivo. Am. J. Physiol. 243, G348-358. Hall W. J., O'Donoghue J. P., O'Regan M. G. and Penny M. J. (1976) Endogenous prostaglandins, adenosine Y,5'monophosphate and sodium transport across isolated frog skin. J. Physiol., Lond. 258, 731-753. Hammond R. W. (1978) Effect of indomethacin on the laying cycle, plasma calcium and shell thickness in the laying hen. Poultry Sci. 57, 1141. Hammond R. W., Olson D. M., Frenkel R. B., Bieller H. V. and Hertelendy F. (1980) Prostaglandins and steroid hormones in plasma ovarian follicles during the ovulation cycle of the domestic hen. Gen. comp. Endocr. 195-202. Hertelendy F. (1972) Prostaglandin induced premature oviposition in the Coturnix quail. Prostaglandins 2, 269. Hertelendy F. (1974) Effects of prostaglandins, cAMP, seminal plasma, indomethacin and other factors on ov position in the Japanese quail. J. Reprod. Fert. 40, 87-93. Kelly J. D., Etches R. J. and Gu6men6 D. (1990) Follicular control of oviposition in the hen. Poultry Sci. 69, 288-29 I. Konturek S. J., Tasler J., Bilski J. and Kania J. (1983) Prostaglandins and alkaline secretion from oxyntic antral and duodenal mucosa of the dog. Am. J. Physiol. 245, G 539-546. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. Lundholm C. E. (1984) Calcium content of duck eggshell gland mucosa homogenate and the rate of Ca 2+ binding to its subcellular fractions during and after the formation of the eggshell. Camp. Biochem. Physiol. 77B, 655~563. Lundholm C. E. (1985) Studies of the effect of DDE on the calcium metabolism of the eggshell gland during formation of the eggshell in ducks and domestic fowls.
Link@ing University Medical Dissertations, No. 205, Link6ping, Sweden. Lundholm C. E. 0988) The effect of DDE, PCB and chlordane on the binding of progesterone to its cytoplasmic receptor in the eggshell gland mucosa of birds and the endometrium of the mammalian uterus. Comp. Biochem. Physiol. 89C, 361-368. Lundholm C. E. and Bartonek M. (1991) A study of the effects ofp,p'-DDE and other related chlorinated hydrocarbons on the inhibition of platelet aggregation. Arch. Toxicol. 65, 570-574. Mongin P. and Sauveur B. (1970) Composition du fluide uterin et de l'albumen durant le s6jour de l'oeuf dans l'ut6rus chez la poule domestique. C.-r. Acad. Sci., Paris 270D, 1715-1718. Niemelii A. O. (1986) Regulation of avidin accumulation by prostaglandins in chick oviduct cell culture. J. Steroid Biochem. 24, (3) 709 715. Nys Y. (1987) Progesterone and testosterone elicit increases in the duration of shell formation in domestic hens. Br. Poult. Sci. 28, 57~58. Olson D. M. and Hertelendy F. (1981) Plasma levels of 13,14-dihydro-15-keto prostaglandin F2~ in relation to oviposition in the domestic hen. Biol. Reprod. 24, 496-504. O'Malley B. W., McGuire P. O., Kohler P. O. and Korenman S. G. (1969) Studies on the mechanism of steroid hormone regulation of synthesis of specific proteins. Rec. Prog. Horm. Res. 25, 105-160. Pearson T. W. and Goldner A. M. (1973) Calcium transport across avian uterus I. Effects of electrolyte substitution. Am. J. Physiol. 225, 1505-1512. Rzasa I. (1981) Prostaglandin production by the hen oviduct in vivo and in vitro. In Recent Advances o f Avian Physiology. Advances in Physiologicol Science (Edited by Pethes G., P6czely P. and Rudas P.), Vol. 33. pp. 177-181. Wechsung L., Korteweg M., Verdonk G. and Houvenaghel A. (1978) Plasma levels of prostaglandin F~ related to oviposition in the domestic hen. Arch. Int. Pharmacodyn. 236, 331-333.
