- Email: [email protected]
Relationship between prostaglandins and cyclic-AMP in the process of implantation in rats
Prostaglandins
RELATIONSHIP IMPLANTATION
and
Medicine
3:
l-6,
BETWEEN PROSTAGLANDINS IN RATS.
1979
AND CYCLIC-AMP
S.K. Garg, Ajanta N. DeSouza, R.R. Chaudhury. Postgraduate Institute of Medical Education India. (reprint requests to SKG)
and
IN THE PROCESS OF
Department Research,
of Pharmacology, Chandigarh,
ABSTRACT The effect of alloxan, a knowninhibitor of adenylate cyclase, on the levels of prostaglandins E and F was investigated in pregnant rats. Animals were treated intraluminally with alloxan in normal saline or alloxan plus cyclicAMP in normal saline or normal saline alone on day 4 of pregnancy. The uteri were collected on days 5 and 6 of pregnancy and the prostaglandins E and F were extracted and measured by a radioimmunoassay technique. In the alloxan treated rats, a significant reduction in PGE and PGF levels was found on days 5 and 6 of pregnancy as compared to untreated control group, whereas in the alloxan plus cyclic-AMP treated group the levels of PGE and PGF were These findings suggest comparable to those of the untreated control group. a possible relationship between prostaglandins and cyclic-AMP in the process of implantation in rats.
I NTRODUCTI ON It has been well established that estrogen is necessary for decidualization at the onset of implantation in rats (1). Considerable evidence indicates that increased estrogen levels lead to increased levels of uterine prostaglandins (2,3) and cyclic-AMP (4). Indomethacin, a potent inhibitor of prostaglandin (PG) biosynthesis (5) has been reported to inhibit implantation in mice (6) and cause resorption of implants in rabbits (7,8). A delays implantation single injection of indomethacin on day 5 of pregnancy in rats (9). Prostaglandins also play an important role in the induction of decidualization in rats (lo), which has been confirmed by blocking the decidual reaction in rats by indomethacin (11). On the other hand, it has been shown that dibutyryl cyclic-AMP (12) and cyclic-AMP (13) both are capable of inducing implantation in ovariectomized, progesterone treated mice. It has also been suggested that changes in cyclic nucleotide concentrations following estrogen treatment are involved in the decidual response of implantation in rats (4). Recently, Ebert & Fox (14) have demonstrated essential rolesof cyclic-AMP and cyclic-GMP in the process of implantation in rats using alloxan, a known inhibitor of cyclic-AMP synthesis (15). From the above mentioned studies it seems that both 1
cyclic nucleotides and prostaglandins may be involved simultaneously in the process of implantation. This paper describes the effect of alloxan on uterine prostaglandin E and F levels in pregnant rats and the possible relationship between cyclic-AMP and prostaglandins in the process of implantation. MATERI ALS AND METHODS rats of known fertility weighing between 175-225 g were Adult fema e albino male rats of proven fertility in the proestrous phase al lowed to mate with Next morning the vaginal smear was examined microof the oes t rous cycle. of thick clumps of spermatozoa which indicates scopical ly for the presence successful mating and this day was designated as-day 1 of pregnancy ( 16). The pregnant rats were divided into the following three groups, each consisting of ten rats, to determine the effect of alloxan on prostag landins E and F levels in the rat uterine tissue at the time of implantation. Animals received intraluminal saline on day 4 of pregnancy performing laparotomy under
injections of 40 ~1 norma 1 between 9.00-10.00 a.m. by 1 ight ether anaesthesia.
Group
I (control):
Group
II
:
Animals received intraluminal injections of 40 ul of 3.5 mM alloxan in normal saline on day 4 of pregnancy between 9.00-10.00 a.m. by performing laparatomy under 1 ight ether anaesthesia.
Group
III
:
Animals received intraluminal injections of 40 ~1 of 3.5 mM alloxan plus 10 ul of 8 mM cyclic-AMP in normal saline on day 4 of pregnancy between 9.00-10.00 a.m. by performing laparotomy under light ether anaesthesia.
Five rats from each groups were sacrificed on days 5 and 6 of pregnancy by cavity was opened a blow on the head and decapitation. The abdominal freed of fat, blood and weighed. and the uterine horns removed immediately, The tissue was homogenized in ice cold Krebs’ solution (NaCl, 117.95; KCl, 4.65; NaH2P04.2H20, 0.89; MgCl2.6H20, 0.57; NaHC03, 25.00; Glucose, 11.11; Fused CaCl2, 2.52 mM) using a teflon homogenizer to give a final tissue concentration of 50 mg/ml. 200 ul al iquots in duplicate were then removed from each tissue homogenate and to this was added nine volumes of 96% ice cold ethanol to prevent any further biosynthesis of prostaglandins (17). All tubes were centrifuged at 900 g for 20 minutes at 4oC, ethanol layer taken off and dried at 370C under a current of nitrogen. The dried residue was dissolved in 300 ~1 of Tris-HCl buffer (pH 7.4) and extracted twice with 1.2 ml of ethyl acetate at pH 4.0. Theethyl acetate layer was separated and dried in a current of nitrogen at 37OC. The residue was redissolved in 2.0 ml of diluent buffer (pH 7.2) and assayed simultaneously for prostaglandins E and F by radioimmunoassay technique as described earlier (18). The average recovery of PGE and PGF from the uterine tissue ranged between The sensitivity of assay for PGEl is 100 pg/ml and for PGE 20 pg/ 75-80%. ml . The intra-assay coefficient of variation is 7.29% for PGE and 7.83% for PGF. The results of the experiments were analysed statistically using student’s
“t”
test.
2
RESULTS The rat alloxan untreated
uterine PGE and PGF content on days 5 and 6 of treated group, alloxan plus cyclic-AMP treated control group have been shown in Table 1.
Table PROSTAGLANDINS
Group
I
(Control)
-_-_--__--
pregnancy group and
1
E AND F IN THE PREGNANT RAT UTERINE Day -----_-__ 5 of Pregnancy __-_
in the the
______-_-________
TISSUE
Day _____--__ 6 of Pregnancy ____
_----_-
PGE levels
PGF levels
PGE 1 eve1 s
PGF levels
(q/IO0 Ttisue
(@I/~00 T&&e
mg f SE)
(W/100 T&hue
(q/100 T&hue
111.08
+ 2.97
63.78 + 1.79
65.68 + 3.48
44.88 + 1.63;:
17.64 + 1.48*
67.28 1: 1.83
53.04 + 1.75
mg + SE)
82.50 + 2.52
II(Alloxan)
54.62 f 3.30fi
III(Alloxan
76.74 + 2.59
44.88 + 3.36" 107.94 + 2.72
mg * SE)
mg f
t CyclicAMP
The PGE and PGF levels in alloxan treated rats on both the days of pregnancy i.e. days 5 and 6 have been significantly reduced as compared to that in the control group, while the PGE and PGF levels return back to those of control group in the alloxan plus cyclic-AMP treated group. In the untreated control group, the levels of both PGE and PGF were higher on day 5 than on day 6 of pregnancy. The decrease in the prostaglandin levels in the alloxan treated group though significant (p < 0.01) on both the days of pregnancy as compared to that in the untreated control group, was observed to be more marked on day 5 of pregnancy. The difference in the PGE and PGF levels in the untreated control group was noted to be significant on day 5 of pregnancy, whereas on day 6 no significant d i fference was observed. In the alloxan treated rats, however, the difference in the PGE and PGF levels was more marked on day 6 as compared to day 5 of pregnancy. In the alloxan plus cyclic-AMP treated back to control group values and the statistically significant as compared days of pregnancy.
group the increase in to alloxan
PGE and PGE and treated
PGF levels return PGF levels was group on both the
SE)
DISCUSSION The role of ovarian decidualization at be acting as first that the effect of through cyclic-AMP.
hormones, estrogen and progestins in the process of the onset of implantation is undisputed and claimed to messenger (1,lp). Rosenfeld and O’Malley (20) suggested estrogen in the process of implantation is mediated
The possible role of prostaglandins in the process of implantation is not clearly understood. Chatterjee (21) reported that indomethacin at a dose of 15 mg/kg intraperitoneally inhibited implantation when administered in rats. either on day 2 or 3 of pregnancy This effect may be either due to modification in the ovum transport by prostaglandins or the toxic effect of high dose of indomethacin used (22). lntravaginal administration of indomethacin at a dose of 25 mg/kg on day 5 of pregnancy also inhibits implantation in rats (23). However, the role of prostaglandins in the decidualization in the rat has been confirmed (10,ll). Considerable evidence is available to suggest that prostaglandins stimulate cyclic,-AMP levels in ovarian sl ices of rat (24) and rabbit corpus luteum uterine tissue, PGEl and PGE2 showed significant ele(25). In the hamster vation in cyclic-AMP concentration, while in pseudopregnant hamsters, PGEPcr, PGEl and PGE2 failed to increase the cyclic-AMP concentration significantly Reports are also available, indicating that in some tissues prosta(26). glandins inhibit the cyclic-AMP system and in others are stimulatory (27). In view of the conflicting evidence, however, it is difficult to define precisely the role of prostaglandins as stimulators of cyclic-AMP. Alloxan a known potent inhibitor of adenyl cyclase was shown to inhibit reversal of which was brought about by administering implantation in rats, The suggested role of cycl ic-GMP as third messenger in the cycl ic-GMP (14). process of implantation, however, is doubtful as the reversal of inhibition of implantation by simultaneous administration of cyclic-AMP is not reported by these workers. Our results demonstrate that alloxan when injected intraluminally on the morning of day 4 of pregnancy in rats, just prior to the estrogen surge (28) at a dose which has been reported to inhibit implantation in rats (14), 5 and 6 of pregnancy in significantly reduces PGE and PGF levels on days the uterine tissue as compared to untreated control group. This effect levels on days 5 and 6 of pregnancy was of alloxan on the prostaglandin reversed by administering alloxan plus cyclic-AMP on day 4 of pregnancy, suggesting thereby that this effect may be mediated through cyclic-AMP as alloxan is known to depress cyclic-AMP levels through an inhibitory This fall in prostaglandin levels by effect on adenylate cyclase (29). alloxan on the day of blastocyst attachment (30), indicates possible involvement of prostaglandins in the process of implantation in rats and The levels of inter-relationship between prostaglandins and cyclic-AMP. PGE and PGF on days 5 and 6 of pregnancy in the control group were observed to be higher than the levels of prostaglandins in normal pregnant rats on the same days (Garg, unpublished data). The increased prostaglandin levels on days 5 and 6 in the untreated control group could be attributed to the release of prostaglandins at the injection site as a result of inflammatory response to trauma (31).
From the above prostaglandins
findings it is and cyclic-AMP
suggested that in the process
there is a relationship of implantation in rats.
between
ACKNOWLEDGMENTS Authors glandin gift of
are grateful antisera and prostaglandins.
to to
Dr. Dr.
S.C. J.E.
Sharma, Pike of
Dublin Upjohn
for the supply of Co., Michigan for
prostagenerous
REFERENCES In The Biology of Blastocyst. AV. of Chicago Press, Chicago, 1971.
383 (RJ Blandau
p
1.
Nalbandov Univ.
2.
Measurement Sharma SC, Garg SK. ine tissue and uterine fluid various doses of estradiol-17B.
3.
Castracane uterine Reprod
4.
Kuehl FA Jr, Ham EA, Zanetti ME, Sanford CH, Nicol Estrogen related increase in uterine guanosine phate levels. Proc Nat Acad Sci USA 71: 1866,
5.
Vane JR. Inhibition of aspirin like
6.
Lau
7.
Saksena SK, Harper MJK. Prostaglandins taglandins in the uterine horns of IUD. Fertil Steril 25: 121, 1974.
8.
El-Banna AA, transport
Sacher 8, Schilling E. and pregnancy in rabbits.
9.
Kennedy TG. blastocyst
Evidence for implantation
10.
of prostaglandins of ovariectomized J Reprod Fertil
VD, Jordan VC. The effect prostaglandin biosynthesis 13: 587, 1975.
of in
E and F in the uterrats treated with 51: 119, 1977.
estrogen and progesterone the ovariectomized rat.
of prostaglandin synthesis drugs. Nature New Biol 231:
Shanes tor
N, of
Baulieu ED, decidualization
on Biol
SE, Goldberg ND. 3’5’-cyclic monophos1974.
as a mechanism 232, 1971.
IF, Saksena SK, Chang MC. Pregnancy blockade by inhibitor of prostaglandin synthesis. Its reversal and progesterone in mice. Prostaglandins 4: 795,
a role of in rats.
ed)
of
action
indomethacin, an by prostaglandins 1973.
mediated action pregnant rabbits
of with
IUD. Prosunilateral
Effect of indomethacin J Reprod Fertil 46:
on egg 375, 1976.
prostaglandins Biol Reprod
in 16:
LeGoascongne C. Prostaglandins in rat uterus. Mol Cell
the 286,
inhibition 1977.
as inductive Endocrinol 6:
of
fac153
1976. 11.
Tobert JA. lization into the
A study of the possible role of prostaglandins using a non-surgical method for the instillation rat uterine lumen. J Reprod Fertil 47: 391,
12.
Webb FTC. c-AMP.
Implantation in J Reprod Fertil
ovariectomized 42: 511, 1975.
13.
Holmes PV, Bergstrom S. cyclic-AMP. J Reprod
14.
Ebert KM, Fox RA. Induction tides. Contraception 16:
mice
treated
Induction of blastocyst Fertil 43: 329, 1975. of implantation 149, 1977.
5
the
rat
deciduaof fluids
1976.
with
implant
in
in
in
dibutyryl
mice
by cyclic
by
nucleo.
15. Cohen yl
KL, Bitensky MW. cyclase. J Pharm
16. Garg
SK, carota Fertil
17. Anggard
Mathur Linn
Inhibitory Exp Ther
effects
alloxan
VS. Effect of chromatographic (seeds) on fertility in female
fractions of albino rats.
SK, Sharma SC. The effect of orciprenaline production of prostaglandins E and F by the Prostaglandins 13: 1179, 1977.
19. Lutwak-Mann
C. Carbonic anhydrase distribution and hormonal
ence, Rosenfeld
MG, O’Malley
BW.
activity
and adenosine 168: 253, 1970.
21.
Steroid
408,
in female dependance.
Daucus J Reprod
sulphate guinea pig
on the uterus.
in
hormones:
effect in
effect of in rats.
on adenyl
target
cyclase
tissue.
Sc i ence
PGF2u and indomethacin Proc Indian Nat Sci
in Acad
1973.
22.
Klaassen CD. indomethacin
23.
Gavin MA. Fernandez-Tejerina JCD, Heras MFMDL, Espana L. Effect of inhibition of prostaglandins biosynthesis on the rat implantation. Reproduction 1: 177, 1974.
Studies on toxicity.
24. Mapsom NR, Toomey Endocrine 25.
Jonsson HT Jr, prostaglandin
RE .
Society.
the mechanism of spirolactone protection 38: 127, 1976. Toxic01 Appi Pharmacol
Stimulation of San Francisco,
Shelton VL, in rabbit
Bagget corpus
c-AMP in rat June 1971.
B. Stimulation luteum. Biol
ovaries.
KT, Wyngarden LJ. In vitro alteration of uterine prostaglandins. Adv Biosci (Suppl) 9: 180, 1972.
Prostaglandins: Nakano J. appl icat ions. Trans 58: 184, 1972.
28. Yoshinaga ovary
Their Assoc Life
physiological Insurance
53rd
against
an
Meeting
of adenyl cyclase by Reprod 7: 107, 1972.
26. Ki rton 27.
v itro
reproductive tract. Occ urr13: 26,1955. J Endocrinol
3’5’-monophosphate
Chatterjee A. Some studies on the the physiology of pseudopregnancy 39:
aden-
prostaglandins from arachi3518, 1965. Chem 210:
18. Garg
20.
on mammalian
31: 143, 1972. E, Samuelsson B. Biosynthesis of acid in guinea pig lung. J Biol
donic
of
169: 80, 1969.
cyclic-AMP
roles and Med Directors
RA, Stocker JF. Estrogen secretion K, Hawkins in vivo during the estrus cycle and pregnancy.
by
therapeutic of America
by the rat Endocrinology
85: 103, 1969. 29. Rall
TW, Sutherland EW. Cellular regulatory Harbor Symp Quant Biol 25: 347, 1961.
30. Tachi
S, Tachi C, attachment and 29: 123, 1970.
31.
Piper PJ, tissues.
Linder HR. trophoblastic
Ultrastructural invasion
mechanisms.
in
features the rat.
Vane JR. The release of prostaglandins Ann NY Acad Sci 180: 363, 1971.
from
Cold
Spring
of blastocyst J Reprod Ferti
lungs
1
and other
RELATIONSHIP IMPLANTATION
and
Medicine
3:
l-6,
BETWEEN PROSTAGLANDINS IN RATS.
1979
AND CYCLIC-AMP
S.K. Garg, Ajanta N. DeSouza, R.R. Chaudhury. Postgraduate Institute of Medical Education India. (reprint requests to SKG)
and
IN THE PROCESS OF
Department Research,
of Pharmacology, Chandigarh,
ABSTRACT The effect of alloxan, a knowninhibitor of adenylate cyclase, on the levels of prostaglandins E and F was investigated in pregnant rats. Animals were treated intraluminally with alloxan in normal saline or alloxan plus cyclicAMP in normal saline or normal saline alone on day 4 of pregnancy. The uteri were collected on days 5 and 6 of pregnancy and the prostaglandins E and F were extracted and measured by a radioimmunoassay technique. In the alloxan treated rats, a significant reduction in PGE and PGF levels was found on days 5 and 6 of pregnancy as compared to untreated control group, whereas in the alloxan plus cyclic-AMP treated group the levels of PGE and PGF were These findings suggest comparable to those of the untreated control group. a possible relationship between prostaglandins and cyclic-AMP in the process of implantation in rats.
I NTRODUCTI ON It has been well established that estrogen is necessary for decidualization at the onset of implantation in rats (1). Considerable evidence indicates that increased estrogen levels lead to increased levels of uterine prostaglandins (2,3) and cyclic-AMP (4). Indomethacin, a potent inhibitor of prostaglandin (PG) biosynthesis (5) has been reported to inhibit implantation in mice (6) and cause resorption of implants in rabbits (7,8). A delays implantation single injection of indomethacin on day 5 of pregnancy in rats (9). Prostaglandins also play an important role in the induction of decidualization in rats (lo), which has been confirmed by blocking the decidual reaction in rats by indomethacin (11). On the other hand, it has been shown that dibutyryl cyclic-AMP (12) and cyclic-AMP (13) both are capable of inducing implantation in ovariectomized, progesterone treated mice. It has also been suggested that changes in cyclic nucleotide concentrations following estrogen treatment are involved in the decidual response of implantation in rats (4). Recently, Ebert & Fox (14) have demonstrated essential rolesof cyclic-AMP and cyclic-GMP in the process of implantation in rats using alloxan, a known inhibitor of cyclic-AMP synthesis (15). From the above mentioned studies it seems that both 1
cyclic nucleotides and prostaglandins may be involved simultaneously in the process of implantation. This paper describes the effect of alloxan on uterine prostaglandin E and F levels in pregnant rats and the possible relationship between cyclic-AMP and prostaglandins in the process of implantation. MATERI ALS AND METHODS rats of known fertility weighing between 175-225 g were Adult fema e albino male rats of proven fertility in the proestrous phase al lowed to mate with Next morning the vaginal smear was examined microof the oes t rous cycle. of thick clumps of spermatozoa which indicates scopical ly for the presence successful mating and this day was designated as-day 1 of pregnancy ( 16). The pregnant rats were divided into the following three groups, each consisting of ten rats, to determine the effect of alloxan on prostag landins E and F levels in the rat uterine tissue at the time of implantation. Animals received intraluminal saline on day 4 of pregnancy performing laparotomy under
injections of 40 ~1 norma 1 between 9.00-10.00 a.m. by 1 ight ether anaesthesia.
Group
I (control):
Group
II
:
Animals received intraluminal injections of 40 ul of 3.5 mM alloxan in normal saline on day 4 of pregnancy between 9.00-10.00 a.m. by performing laparatomy under 1 ight ether anaesthesia.
Group
III
:
Animals received intraluminal injections of 40 ~1 of 3.5 mM alloxan plus 10 ul of 8 mM cyclic-AMP in normal saline on day 4 of pregnancy between 9.00-10.00 a.m. by performing laparotomy under light ether anaesthesia.
Five rats from each groups were sacrificed on days 5 and 6 of pregnancy by cavity was opened a blow on the head and decapitation. The abdominal freed of fat, blood and weighed. and the uterine horns removed immediately, The tissue was homogenized in ice cold Krebs’ solution (NaCl, 117.95; KCl, 4.65; NaH2P04.2H20, 0.89; MgCl2.6H20, 0.57; NaHC03, 25.00; Glucose, 11.11; Fused CaCl2, 2.52 mM) using a teflon homogenizer to give a final tissue concentration of 50 mg/ml. 200 ul al iquots in duplicate were then removed from each tissue homogenate and to this was added nine volumes of 96% ice cold ethanol to prevent any further biosynthesis of prostaglandins (17). All tubes were centrifuged at 900 g for 20 minutes at 4oC, ethanol layer taken off and dried at 370C under a current of nitrogen. The dried residue was dissolved in 300 ~1 of Tris-HCl buffer (pH 7.4) and extracted twice with 1.2 ml of ethyl acetate at pH 4.0. Theethyl acetate layer was separated and dried in a current of nitrogen at 37OC. The residue was redissolved in 2.0 ml of diluent buffer (pH 7.2) and assayed simultaneously for prostaglandins E and F by radioimmunoassay technique as described earlier (18). The average recovery of PGE and PGF from the uterine tissue ranged between The sensitivity of assay for PGEl is 100 pg/ml and for PGE 20 pg/ 75-80%. ml . The intra-assay coefficient of variation is 7.29% for PGE and 7.83% for PGF. The results of the experiments were analysed statistically using student’s
“t”
test.
2
RESULTS The rat alloxan untreated
uterine PGE and PGF content on days 5 and 6 of treated group, alloxan plus cyclic-AMP treated control group have been shown in Table 1.
Table PROSTAGLANDINS
Group
I
(Control)
-_-_--__--
pregnancy group and
1
E AND F IN THE PREGNANT RAT UTERINE Day -----_-__ 5 of Pregnancy __-_
in the the
______-_-________
TISSUE
Day _____--__ 6 of Pregnancy ____
_----_-
PGE levels
PGF levels
PGE 1 eve1 s
PGF levels
(q/IO0 Ttisue
(@I/~00 T&&e
mg f SE)
(W/100 T&hue
(q/100 T&hue
111.08
+ 2.97
63.78 + 1.79
65.68 + 3.48
44.88 + 1.63;:
17.64 + 1.48*
67.28 1: 1.83
53.04 + 1.75
mg + SE)
82.50 + 2.52
II(Alloxan)
54.62 f 3.30fi
III(Alloxan
76.74 + 2.59
44.88 + 3.36" 107.94 + 2.72
mg * SE)
mg f
t CyclicAMP
The PGE and PGF levels in alloxan treated rats on both the days of pregnancy i.e. days 5 and 6 have been significantly reduced as compared to that in the control group, while the PGE and PGF levels return back to those of control group in the alloxan plus cyclic-AMP treated group. In the untreated control group, the levels of both PGE and PGF were higher on day 5 than on day 6 of pregnancy. The decrease in the prostaglandin levels in the alloxan treated group though significant (p < 0.01) on both the days of pregnancy as compared to that in the untreated control group, was observed to be more marked on day 5 of pregnancy. The difference in the PGE and PGF levels in the untreated control group was noted to be significant on day 5 of pregnancy, whereas on day 6 no significant d i fference was observed. In the alloxan treated rats, however, the difference in the PGE and PGF levels was more marked on day 6 as compared to day 5 of pregnancy. In the alloxan plus cyclic-AMP treated back to control group values and the statistically significant as compared days of pregnancy.
group the increase in to alloxan
PGE and PGE and treated
PGF levels return PGF levels was group on both the
SE)
DISCUSSION The role of ovarian decidualization at be acting as first that the effect of through cyclic-AMP.
hormones, estrogen and progestins in the process of the onset of implantation is undisputed and claimed to messenger (1,lp). Rosenfeld and O’Malley (20) suggested estrogen in the process of implantation is mediated
The possible role of prostaglandins in the process of implantation is not clearly understood. Chatterjee (21) reported that indomethacin at a dose of 15 mg/kg intraperitoneally inhibited implantation when administered in rats. either on day 2 or 3 of pregnancy This effect may be either due to modification in the ovum transport by prostaglandins or the toxic effect of high dose of indomethacin used (22). lntravaginal administration of indomethacin at a dose of 25 mg/kg on day 5 of pregnancy also inhibits implantation in rats (23). However, the role of prostaglandins in the decidualization in the rat has been confirmed (10,ll). Considerable evidence is available to suggest that prostaglandins stimulate cyclic,-AMP levels in ovarian sl ices of rat (24) and rabbit corpus luteum uterine tissue, PGEl and PGE2 showed significant ele(25). In the hamster vation in cyclic-AMP concentration, while in pseudopregnant hamsters, PGEPcr, PGEl and PGE2 failed to increase the cyclic-AMP concentration significantly Reports are also available, indicating that in some tissues prosta(26). glandins inhibit the cyclic-AMP system and in others are stimulatory (27). In view of the conflicting evidence, however, it is difficult to define precisely the role of prostaglandins as stimulators of cyclic-AMP. Alloxan a known potent inhibitor of adenyl cyclase was shown to inhibit reversal of which was brought about by administering implantation in rats, The suggested role of cycl ic-GMP as third messenger in the cycl ic-GMP (14). process of implantation, however, is doubtful as the reversal of inhibition of implantation by simultaneous administration of cyclic-AMP is not reported by these workers. Our results demonstrate that alloxan when injected intraluminally on the morning of day 4 of pregnancy in rats, just prior to the estrogen surge (28) at a dose which has been reported to inhibit implantation in rats (14), 5 and 6 of pregnancy in significantly reduces PGE and PGF levels on days the uterine tissue as compared to untreated control group. This effect levels on days 5 and 6 of pregnancy was of alloxan on the prostaglandin reversed by administering alloxan plus cyclic-AMP on day 4 of pregnancy, suggesting thereby that this effect may be mediated through cyclic-AMP as alloxan is known to depress cyclic-AMP levels through an inhibitory This fall in prostaglandin levels by effect on adenylate cyclase (29). alloxan on the day of blastocyst attachment (30), indicates possible involvement of prostaglandins in the process of implantation in rats and The levels of inter-relationship between prostaglandins and cyclic-AMP. PGE and PGF on days 5 and 6 of pregnancy in the control group were observed to be higher than the levels of prostaglandins in normal pregnant rats on the same days (Garg, unpublished data). The increased prostaglandin levels on days 5 and 6 in the untreated control group could be attributed to the release of prostaglandins at the injection site as a result of inflammatory response to trauma (31).
From the above prostaglandins
findings it is and cyclic-AMP
suggested that in the process
there is a relationship of implantation in rats.
between
ACKNOWLEDGMENTS Authors glandin gift of
are grateful antisera and prostaglandins.
to to
Dr. Dr.
S.C. J.E.
Sharma, Pike of
Dublin Upjohn
for the supply of Co., Michigan for
prostagenerous
REFERENCES In The Biology of Blastocyst. AV. of Chicago Press, Chicago, 1971.
383 (RJ Blandau
p
1.
Nalbandov Univ.
2.
Measurement Sharma SC, Garg SK. ine tissue and uterine fluid various doses of estradiol-17B.
3.
Castracane uterine Reprod
4.
Kuehl FA Jr, Ham EA, Zanetti ME, Sanford CH, Nicol Estrogen related increase in uterine guanosine phate levels. Proc Nat Acad Sci USA 71: 1866,
5.
Vane JR. Inhibition of aspirin like
6.
Lau
7.
Saksena SK, Harper MJK. Prostaglandins taglandins in the uterine horns of IUD. Fertil Steril 25: 121, 1974.
8.
El-Banna AA, transport
Sacher 8, Schilling E. and pregnancy in rabbits.
9.
Kennedy TG. blastocyst
Evidence for implantation
10.
of prostaglandins of ovariectomized J Reprod Fertil
VD, Jordan VC. The effect prostaglandin biosynthesis 13: 587, 1975.
of in
E and F in the uterrats treated with 51: 119, 1977.
estrogen and progesterone the ovariectomized rat.
of prostaglandin synthesis drugs. Nature New Biol 231:
Shanes tor
N, of
Baulieu ED, decidualization
on Biol
SE, Goldberg ND. 3’5’-cyclic monophos1974.
as a mechanism 232, 1971.
IF, Saksena SK, Chang MC. Pregnancy blockade by inhibitor of prostaglandin synthesis. Its reversal and progesterone in mice. Prostaglandins 4: 795,
a role of in rats.
ed)
of
action
indomethacin, an by prostaglandins 1973.
mediated action pregnant rabbits
of with
IUD. Prosunilateral
Effect of indomethacin J Reprod Fertil 46:
on egg 375, 1976.
prostaglandins Biol Reprod
in 16:
LeGoascongne C. Prostaglandins in rat uterus. Mol Cell
the 286,
inhibition 1977.
as inductive Endocrinol 6:
of
fac153
1976. 11.
Tobert JA. lization into the
A study of the possible role of prostaglandins using a non-surgical method for the instillation rat uterine lumen. J Reprod Fertil 47: 391,
12.
Webb FTC. c-AMP.
Implantation in J Reprod Fertil
ovariectomized 42: 511, 1975.
13.
Holmes PV, Bergstrom S. cyclic-AMP. J Reprod
14.
Ebert KM, Fox RA. Induction tides. Contraception 16:
mice
treated
Induction of blastocyst Fertil 43: 329, 1975. of implantation 149, 1977.
5
the
rat
deciduaof fluids
1976.
with
implant
in
in
in
dibutyryl
mice
by cyclic
by
nucleo.
15. Cohen yl
KL, Bitensky MW. cyclase. J Pharm
16. Garg
SK, carota Fertil
17. Anggard
Mathur Linn
Inhibitory Exp Ther
effects
alloxan
VS. Effect of chromatographic (seeds) on fertility in female
fractions of albino rats.
SK, Sharma SC. The effect of orciprenaline production of prostaglandins E and F by the Prostaglandins 13: 1179, 1977.
19. Lutwak-Mann
C. Carbonic anhydrase distribution and hormonal
ence, Rosenfeld
MG, O’Malley
BW.
activity
and adenosine 168: 253, 1970.
21.
Steroid
408,
in female dependance.
Daucus J Reprod
sulphate guinea pig
on the uterus.
in
hormones:
effect in
effect of in rats.
on adenyl
target
cyclase
tissue.
Sc i ence
PGF2u and indomethacin Proc Indian Nat Sci
in Acad
1973.
22.
Klaassen CD. indomethacin
23.
Gavin MA. Fernandez-Tejerina JCD, Heras MFMDL, Espana L. Effect of inhibition of prostaglandins biosynthesis on the rat implantation. Reproduction 1: 177, 1974.
Studies on toxicity.
24. Mapsom NR, Toomey Endocrine 25.
Jonsson HT Jr, prostaglandin
RE .
Society.
the mechanism of spirolactone protection 38: 127, 1976. Toxic01 Appi Pharmacol
Stimulation of San Francisco,
Shelton VL, in rabbit
Bagget corpus
c-AMP in rat June 1971.
B. Stimulation luteum. Biol
ovaries.
KT, Wyngarden LJ. In vitro alteration of uterine prostaglandins. Adv Biosci (Suppl) 9: 180, 1972.
Prostaglandins: Nakano J. appl icat ions. Trans 58: 184, 1972.
28. Yoshinaga ovary
Their Assoc Life
physiological Insurance
53rd
against
an
Meeting
of adenyl cyclase by Reprod 7: 107, 1972.
26. Ki rton 27.
v itro
reproductive tract. Occ urr13: 26,1955. J Endocrinol
3’5’-monophosphate
Chatterjee A. Some studies on the the physiology of pseudopregnancy 39:
aden-
prostaglandins from arachi3518, 1965. Chem 210:
18. Garg
20.
on mammalian
31: 143, 1972. E, Samuelsson B. Biosynthesis of acid in guinea pig lung. J Biol
donic
of
169: 80, 1969.
cyclic-AMP
roles and Med Directors
RA, Stocker JF. Estrogen secretion K, Hawkins in vivo during the estrus cycle and pregnancy.
by
therapeutic of America
by the rat Endocrinology
85: 103, 1969. 29. Rall
TW, Sutherland EW. Cellular regulatory Harbor Symp Quant Biol 25: 347, 1961.
30. Tachi
S, Tachi C, attachment and 29: 123, 1970.
31.
Piper PJ, tissues.
Linder HR. trophoblastic
Ultrastructural invasion
mechanisms.
in
features the rat.
Vane JR. The release of prostaglandins Ann NY Acad Sci 180: 363, 1971.
from
Cold
Spring
of blastocyst J Reprod Ferti
lungs
1
and other