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Effects of indomethacin and prostaglandin F2α on ovulation and ovarian contractility in the rabbit
Effects of Indomethacin and Prostaglandin and Ovarian Contractility in the Rabbit*
F2~
on Ovulation
Augusto Diaz-lnfante, Jro, MoD., ** Karen H. Wright, B.S. and Edward Eo Wallach, MoD. Department of Obstetrics and Gynecology, Pennsylvania Hospital and University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19107 ABSTRACT The present investigation was carried out to determine whether inhibition of ovulation in the rabbit by administration of indomethacin can be correlated with any change in ovarian contractility at ovulation time and can be reversed by administration of prostaglandinso Indomethacin was adminstered intramuscularly using three different schedules in a dose of 5 mg/kg. A reduced number of ruptured follicles following HCG was noted in all groups treated with indomethacino Infusion of P G F 2 ~ into the aorta (1 ~g/kg/mino) could reverse this effect. Less pronounced ovarian contractility was observed after indomethacin treatment, but infusion of PGF2~ immediately enhanced contractility in ovaries from indomethacin treated rabbits. The inhibition of ovulation in the rabbit associated with indomethacin adminstration may be related to suppression of ovarian contractions° These data also suggest that prostaglandins may play a significant role in the mechanism of ovulation through an influence on ovarian contractility.
*Supported by the Population Council Grant M730103 **Ford Foundation Fellow in Reproductive Biology. Present address: Department of Obstetrics and Gynecology, School of Medicine, University of San Luis Potosi, San Luis Potosi, Mexico Send reprint requests to Edward Eo Wallach, MoDo, Pennsylvania Hospital, Philadelphia, Pennsylvania 19107 Acknowledgements The authors wish to thank Thomas Henry, Kendra Guernsey, Anne T. Barrett and Elaine Stevens for their assistance°
Accepted February 12, 1974.
PROSTAGLANDINS MARCH 25, 1974
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567
PROSTAGLANDINS
Indomethacin has been reported to block prostaglandin synthesis in several tissues (1). Recently, this drug was also found to inhibit ovulation in rats (2,3) and rabbits (4,5). In these studies, luteinization of ovarian follicles was not prevented, the ova were retained within the follicles, and the ovarian steroidogenic pattern appeared to be normal (4-6). Based upon the assumption that the antiovulatory action of indomethacin is related to its ability to inhibit prostaglandin biosynthesis, it has been suggested that prostag l a n d i n s a r e involved in the process of follicular rupture. Previous observations from our laboratory have demonstrated: (1) a prominent autonomic innervation of ovarian tissue (7); (2) smooth muscle fibers present in ovarian stroma (7,8); and (3) ovarian contractions, detected in several species, which seem to correlate with the stage of follicular development (9-12). Ovarian smooth muscle activity has been considered significant in the physical process of ovulation. Ovarian contractile patterns and their responses to adrenergic agents and to prostaglandin F2~ has consistently been found to enhance ovarian contractility, while prostaglandin E2 is inhibitory
(I0,II). The purposes of the present investigation were to determine whether inhibition of ovulation in the rabbit by the administration of indomethacin can be: (I) correlated with any change in ovarian contractility at the predicted time of ovulation; (2) related to the ability of indomethacin to inhibit prostaglandin biosynthesis; and (3) reversed by administration of exogenous prostaglandins. In attempting to determine whether indomethacin administation can bring about changes in ovarian contractility at the time of ovulation, it was also considered important to ascertain whether these alterations could be reversed by exogenous prostaglandins.
MATERIALS
AND
METHODS
Sexually mature New Zealand white female rabbits, weighing between 3 and 4 kg,, were housed individually for a minimum of three weeks and fed Purina rabbit chow and water ad libitum.
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PROSTAGLANDINS
Rabbits were maintained in a room with controlled temperature and light. Ovulation was induced with human chorionic gonadotropin (HCG)*, I00 IoUo via marginal ear vein. Ovulation in the rabbit predictably follows the intravenous injection of H C G by I0-14 hours (13). The time of HCG injection was designated "time zero" in all experiments. All other treatments were temporally related to this point. Ovulation studies: Twelve hours after receiving HCG, the animals were anesthetized with intravenous sodium pentobarbital (32 mg/kg), and anesthesia was maintained during a four-hour experiment. A midline laparotomy was performed, the viscera retracted, and the ovaries examined in situ for ovulation points with minimal manipulation. A 27 gauge needle attached to a polyethylene catheter #20 was inserted into the abdominal aorta just above the origin of the ovarian arteries. The distal end of the catheter was attached to a I0 ml syringe, inserted in a Harvard Multispeed Infusion Pump with the speed adjusted to deliver a total volume of 5 ml in four hours° The abdominal incision was approximated with towel clamps and four hours later (16 hours after HCG) the ovaries were observed again, the catheter removed, and the abdominal cavity closed. Thirty-six hours after HCG, the rabbits were anesthetized again, the abdomen reopened and both ovaries removed and examined with a dissecting m i c r o s c o p e . At each observation time (12 hours, 16 hours, and 36 hours after HCG), ovarian follicles were counted and classified using a modification of criteria previously described (14). Follicles were divided into the following stages of development: Mature - clear follicles, distinctly present, but not bulging above the ovarian surface. Graafian - large, ovarian surface.
clear
Vascular - similar surface vascularity.
to
follicles, Graafian
protruding
follicles
but
above with
the distinct
Ruptured - follicles which have already released contents, thus appearing hemorrhagic, less prominent, with a clear tear line on the surface.
their and
*APL,
Labora-
generously supplied by tories, Inc., New York,
M A R C H 25, 1974
V O L . 5 NO. 6
Garfield New York
Davies,
Ayerst
569
PROSTAGLANDINS
Hemorrhagic large, cystic follicles, with dark hemorrhagic antral fluid, and no surface signs of rupture° Often, the egg could be removed from these follicles when the follicle was punctured under the dissecting microscope. -
All drugs were reconstituted before each experiment. Prostaglandin F 2 ~ (PGF2~)* was diluted in isotonic saline to a concentration adequate to deliver a dose of 1 ~g/kg/min in a fixed volume (5 ml). Indomethacin ** (5 mg/kg/ was adminis a tered intramuscularly as a suspension in 1 ml of Emulphor*** i% in saline. To determine the most efficacious timing for ovulation inhibition, indomethacin was administered using three different schedules° Five groups of four rabbits each were studied in this fashion using the following drug schedules: Group 1 - HCG alone Group 2 - Indomethacin 12 hours before HCG Group 3 - Indomethacin at the time of HCG Group 4 - Indomethacin I0 hours after HCG Group 5 - Indomethacin 12 hours before HCG; twelve hours after HCG the intra-aortie infusion of PGF2~ was begun and maintained over a four-hour interval° (Groups 1-4 received an infusion of 5 ml of saline into the aorta during the same fourhour period of time.) Rabbits intramuscular hours before Ovarian
in group injection HCG.
1 served of the
Contractility
as controls vehicle for
and received indomethacin
an 12
Studies
In two additional groups of six rabbits, ovarian contractility studies were timed to span the anticipated interval of ovulation. The following schedule was used: Group A received HCG and an intramuscular injection of the vehicle 12 hours before HCG. These rabbits were used as controls° Group B served as the experimental group and received indomethacin 12 hours before administration of HCG. Twelve hours after HCG, a laparotomy was performed as described above, and ovarian contractility was recorded in vivo for four hours in a randomly selected ovary from each rabbit° In some cases the contralateral ovary was studied simultaneously in vitro. The technical details for recording
*Prostaglandins were supplied by John Pike, Ph.D., Upjohn Kalamazoo, Michigan **Indomethacin was supplied by Carl R° Stevenson, M.Do of Merck, Sharp & Dohme, West Point, Pennsylvania ***Emulphor, GAF Corporation, New York, New York
570
MARCH 25, 1974
Co.,
VOL. 5 NO. 6
PROSTAGLANDINS
contractions in vivo and in vitro in our laboratory have been previously described (10,~).-'-'-~ter adequate recordings of spontaneous contractility had been obtained in vivo, P G F ~ (Ipg/kg/min) was infused through the intraaor'~ic Catheter. Changes in the ovarian contractile pattern in response to this infusion were analyzed and the patterns in the experimental and control groups were compared. RESULTS Ovulation, The number of follicles at each stage of development was recorded by treatment group at each of the three observation times (Table I). A reduced number of ruptured follicles was noted in all groups treated with indomethacin. Comparison of group 3 (indomethacin at "time zero") and group 4 (indomethacin 12 hours after HCG) with the control group, also reveals fewer ruptured and a greater number of follicles in both of these indomethacin-treated groups at 12 and 16 hours after HCG° At 36 hours some of the vascular follicles progressed to rupture, while others became hemorrhagic. Ovaries of rabbits in which indomethacin was given 12 hours before HCG (group 2) contained the smallest number of ruptured follicles (8 ruptured follicles as compared with 30 in the control group)° This figure remained relatively constant up to 36 hours after HCGo Most of the vascular follicles in group 2 observed at 12 hours after HCG ultimately became hemorrhagic. Ovaries in these rabbits (group 2) had the largest number of hemorrhagic follicles of all five groups at 36 hours after HCG° With one exception, no significant differences were found in the numbers of early follicles (mature and Graafian) when groups 2, 3, and 4 were compared to the control group. Group 4 (indomethacin at +I0) provided the only exception, when at 36 hours after HCG, the number of early follicles was markedly increased. The most striking results were observed when indomethacin was administered 12 hours before HCG. This treatment schedule was thus used again for animals in group 5),which also received an infusion of P G F 2 ~ between 12 and 16 hours after HCG° As anticipated, at 12 hours after HCG, just prior to initiating the P G F 2 ~ infusion, the results were similar to those found in group 2. However, at 16 hours after HCG, when the PGF2~ infusion had been completed, a marked increase in the number of ruptured follicles (23 ruptured follicles)was observed° This figure differed significantly from the number of ruptured follicles (9 ruptured follicles) noted in group 2 which had not received P G F 2 ~ o The total number of \ ruptured follicles observed in group 5 (23 ruptured follicles) nonetheless was smaller than in the control group (39 ruptured
MARCH 25, 1974
VOL. 5 NO. 6
571
O~
o
< 0
=
d
1-4
m
g
2 0
Hemorrhagic
t
39
Ruptured
Vascular
53
Total
i. 4 r a b b i t s in e a c h g r o u p 2. HCG 100 i.u. i n t r a v e n o u s l y
2
8
Graafian
Mature
3
0
Vascular
Hemorrhagic
46 40
Total Ruptured
2
39
Total
Mature
2
3
4
Graafian
Mature
Graafian
3
0
Hemorrhagic
54
4
8
25 .
7.
29 l0
0
1
9
i0
9
26
0
2
13
3O
of
.
. .
.
Indomethacin
52
7
6
.
.
F2~
I.M.
.....
~,
I
84
22
l0
3
36 32
0
2
3
8
23
36
0
i0
0
20
-12 = 12 hs. b e f o r e H C G 0 = at the t i m e of H C G +12 = 12 hs. a f t e r H C G 1 mg/kg/min intraaortic infusion
5 mg/kg
67
18
15
14
0
.
0
. .... . 12.
.
0
9
5
8
18
39
0
I0
0
40 20
4. P r o s t a g l a n d i n
3.
.
17 12
38 27
2
5
2
3
26
37
2
7
2
6
20
Group 5 H C G at 0 Indo. at -12 PGF~ f r o m +12 to +16 ] 6 ..........
ovulation
Group 4 H C G at 0 Indo. at + 1 2
on HCG-induced
Group 3 H C G at 0 Indo. at 0
indomethacin
Group 2 HCG at 0 Indo. at -12
I - Effect
Vascular
FOLLICLES Ruptured
Group 1 H C G at 0 (control)
TABLE
>
©
PROSTAGLANDINS
follicles). At 36 hours after HCG, the number of ruptured follicles in the i n d o m e t h a c i n - - P G F 2 ~ group (group 5 ) s h o w e d a further increase (32 ruptured follicles), approximating more closely the number of ruptured follicles found in the control group (40 ruptured follicles). Fewer hemorrhagic follicles were found in this group at 36 hours (I0 hemorrhagic follicles) than in group 2 (25 hemorrhagic follicles) which did not receive P G F 2 ~ . This difference could be interpreted to indicate that more of the vascular follicles present at the earlier observation intervals had progressed to rupture instead of becoming hemorrhagic° At 36 hours, the number of early follicles in group 5 (39 early follicles) was strikingly greater than in group 2 which was not treated with P G F 2 ~ (12 early follicles), or in the control group (I0 early follicles)° This predominance of early follicles in group 5 (indomethacin-PGF2~) accounts for these rabbits having the largest total number of ovarian follicles among all the experimental groups. Group 4, which received indomethacin I0 hours after HCG, and only a saline infusion, but not P G F 2 = , was the only group in which a similar distribution of follicles was observed at 36 hours after HCG. An increase~in total number of follicles was observed between 16 and 36 hours after HCG in all indomethacin-treated groups, but was most pronounced in group 5 which received P G F 2 ~ in addition to indomethacin. The increase in follicles between 16 and 36 hours after HCG amounted to 15% in group l, 86% in group 2, 37~ in group 3, 67% in group 4, and 133% in group 5. Ovarian Contractility~ Recordings of spontaneous ovarian contractions demonstrated greater amplitude and more rhythmicity in the control rabbits than in indomethacin-treated animals. The contractile pattern did not change significantly in the control group during or immediately after the infusion of P G F 2 ~ (Fig° 1)o In the group previously treated with indomethacin, the infusion of P G F 2 ~ tended to provoke enhanced rhythmicity and increased amplitude° In general, no significant changes in contractile pattern were observed between 12 and 16 hours after HCGo In some indomethacin-treated rabbits in which the i n f u s i o n of P G F 2 ~ enhanced ovarian contractility, the new contractile pattern persisted even after P G F 2 ~ was discontinued (Fig. 2)° Similar findings were obtained in ovaries studied in vitro, namely, depressed spontaneous contractility in indo-meta-~ntreated rabbits, but an enhanced contractile response to P G F 2 ~ (Fig. 3 A&B). In contrast control ovaries, from animals not treated with indomethacin, showed either a minimal or no response to P G F 2 ~ in vitro (Fig° 3C).
MARCH 25, 1974
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573
PROSTAGLANDINS
RABBIT 9 IN VIVO
CONTROL
A
~t3hr 21min
post HCG
2"2
2"3
24
2"5"
2"6
2"7
2'8
B "i1 16min I"7 18 19 post HCG 12minutes post completion of PGF2¢ infusion 14hr
FIGURE
2"1
2"2
23
1
In vivo recording of ovarian not treated with indomethacino
574
20
contractions recorded
in Rabbit
9,
Ao
Spontaneous contractions minutes following HCGo
13 hours and 21
B.
Contractions recorded 12 minutes after completion of an intra-aortic infusion of PGF2~ (2 ~g/kg/min for 24 minutes)°
MARCH 25, 1974
VOL. 5 NO. 6
PROSTAGLANDINS
RABBIT 8 iN vIVO INDOMETHACIN 12 HOURS PRE HCG
A
l~'l~rnmin post HC,G
15
~;0
25
B PGF2~ INFUSION ~ .
.
.
.
1
.
13hr 5 0 r a i n
55
"
1,~hr
.
.
.
.
()5
"
post HCG
C
~4ht~l~i. " post HCG
FIGURE
B
~5
.
.
.
.
20
25
30
2
In vivo recordings of ovarian contractions in Rabbit 8 treated with indomethacin 12 hours prior to HCG administration. Ao
Spontaneous ovarian contractions recorded 13 hours and ll minutes after HCG.
Bo
Enhanced contractile pattern during a 30 minute intra-
aortic C.
infusion
of PGF2~
(I ~g/kg/min).
Recording of ovarian contractions immediately discontinuation Of PGF2~ infusion demonstrating existance of an enhanced contractile pattern.
MARCH 25, 1974
VOL. 5 NO. 6
following the
575
PROSTAGLANDINS
RABBIT 7 I~HACtN
IN VITRO 12 HOURS PRE HCG
12h~.~. . . . . posl HCG
4b
. . . .
4~
.
.
.
.
~o
'
B
12ht52min I~¢st HCG
RAB81T 2 CONTROL
55
13h~
05
;o
IN Vfl'RO
C ~IF2= ,;~,,sm;:'.....
post HCG
FIGURE
576
•
•
-
•
is . . . .
3'o . . . .
3's . . . .
40
3
Ao
In vitro recordings of ovarian contractions in Rabbit 7 treated with indomethacin 12 hours prior to HCG administration, demonstrating minimal contractility.
Bo
In the same preparation the addition of PGF2~ (i00 pg) at 12 hours and 55 minutes after HCG led to initiation of pronounced ovarian contractions.
Co
In vitro recording o f ovarian contractions in Rabbit 2 not treated with indomethacin. The addition of P G F 2 ~ (200 ~g) at 13 hours and 24 minutes after HCG had no appreciable effect on ovarian contractions.
MARCH
25,
1974
VOL.
5 NO.
6
PROSTAGLANDIMS
In order to express these results numerically, the area included by the c o n t r a c t i o n s in a I0 minute period of time was calculated by counting the square millimeters in the recording, using standard sensitivity and chart speed. With this method, a mean value could be obtained for spontaneous contractions as well as for contractions recorded during the P G F 2 ~ infusion° In each group of 6 rabbits the m e a ~ value for spontaneous contractions was compared. The mean value in the control group was 325°8 mm2, as compared with 182 mm2 in the indomethacin-treated group (85.8% of the control)° In the control group during the P G F 2 ~ i n f u s i o n the corresponding figure of 349°4 mm2, (an i n c r e a s e o f 7.2%) compares with 241 mm2 (an increase of 37o8~) in the indomethatin-treated group° DISCUSSION The ability of indomethacin to inhibit induced ovulation in the rat (2,3) and rabbit (4,5) has recently been described. The nature of this action of indomethacin, however, is still in question. In previous experiments carried out in the rabbit (5), a single intravenous dose of indomethacin given at the time of HCG failed to block HCG-induced ovulation° Supplemental indomethacin administered subcutaneously at 12 hour intervals for 48 hours after HCG led to inhibition of ovulation. However, because observations were not made at the expected time of ovulation but approximately 12 hours after HCG, a delay in ovulation attributable to indomethacin may have been missed. In the present experiment, a dose of indomethacin smaller than that given by O'Grady (5) and administered by the intramuscular route led to a decrease in number of ruptured follicles at the expected time of ovulation° Such was the case when indomethacin was given either at the same time as HCG or 10 hours later. The most striking effect was found when indomethacin was given 12 hours before HCG. These differences may be related to the intramuscular route of indomethacin administration yielding a prolonged absorption rate° M o r e p r o n o u n c e d depletion in endogenous prostaglandin levels might be anticipated in the group in which indomethacin was given 12 hours before HCG. Prostaglandin E 2 has been reported to overcome the inhibitory effect of indomethacin on ovulation in rats (6). In the present experiment, P G F 2 ~ reversed inhibition of ovulation brought about by indomethacin in the rabbit. These observations support the suggestion that the effect of indomethacin on ovulation is related to its inhibition of prostaglandin biosynthesis° Although indomethacin has been shown to block ovulation, it has not prevented follicular maturation, luteinization or progesterone secretion (4)° In fact, ova have been
MARCH 25, 1974
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577
PROSTAGLANDINS
described contained within luteinized follicles following indomethacin (5). Tsafriri, et al (6) postulated that indomethacin prevents only follicular rupture and that prostaglandins play an essential role in the m e c h a n s i m whereby LII leads to follicular rupture. Tsafriri also demonstrated that PGE2 is able to induce ovum maturation, although while not being essential to support this action of LHo Behrman (14) demonstrated that indomethacin has a prolonged effect, and that exogenous LH or gonadotropin releasing hormone fails to reverse its anti-ovulatory action while promoting progesterone secretion. The results in the present report concur with the hypothesis that the effect of indomethacin on ovulation is exerted locally at the ovarian level and that prostaglandins have an essential role in
the rupture of the mature follicle° The h e m o r r h a g i c f o l l i cles observed in our experiments probably correspond to those described by O'Grady (5) as exhibiting luteinization and containing entrapped ova; these structures represent follicles which were stimulated but failed to rupture. The marked increase in total number of follicles in indomethacintreated groups probably results from the disappearance of indomethacin from receptor sites, thus permitting the resumption of prostaglandin biosynthesis° Consequently, increasing local levels of prostaglandin support ovum maturation. This concept might also explain the markedly increased number of follicles in the group of animals pretreated with indomethacin and then infused with PGF2~. Earlier experiments in our laboratory have been directed to the demonstration of ovarian contractions in several species (9-12)o We have consistently found that PGF 2 enhances ovarian contractility and PGE 2 inhibits it (II). Prostaglandin E 2 has recently been shown to delay ovulation in the rabbit (14). Ovarian contractile patterns and ovarian responsiveness to prostaglandins also appear dependent upon the specific hormonal environmeht in the monkey (16). We have previously postulated that ovarian smooth muscle activity may play a significant role in the physical process of ovulation. In the second phase of the present study spontaneous ovarian contractility was found at the anticipated time of ovulation after HCG administration. However, the ovarian contractions recorded in the indomethacin-treated group lacked rhythmicity and demonstrated lower amplitude than in the control group~ Infusion of P G F 2 ~ had little effect on the ovarian contractile pattern in control animals, while in those rabbits pretreated with indomethacin, rhythmic contractions and an increase in amplitude were frequently provoked° One might postulate that the dose of P G F 2 ~ used in this experiment was only effective when endogenous
578
M A R C H 25, 1974
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PROSTAGLANDINS
prostaglandins had been depleted by indomethacin. This depletion of prostaglandins apparently impairs ovarian contraeilityo On the basis of these results we would propose that indomethacin acts locally on the rabbit ovary to prevent the rupture of ovarian follicles° This action would seem to depend on the ability of indomethacin to inhibit prostaglandin biosynthesis. It appears even more likely that prostaglandins play an important role in the m e c h a n i s m of ovulation, and possibly do so by influencing ovarian contractile patterns,
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PROSTAGLANDINS
REFERENCES
I.
Vane, JoR. Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs° Nature New Biol0~Y 231: 232, 1971.
2.
Armstrong, DoTo and Grinwieh, eous and LH-induced ovulation an inhibitor of prostaglandin glandins I: 21, 1972o
3.
Orczyk, GoPo and Behrman, H.Ro Ovulation blockade by aspirin or indomethacin - In vivo evidence for a role of prostaglandin in gonadotropin secretion. Prostag!andins I: 3, 1972.
4.
Grinwich, DoLo, Kennedy, ToG. and Armstrong, D.T. Dissociation of ovulatory and steroidogenic actions of luteinizing hormone in rabbits with indomethacin, an inhibitor of prostaglandin biosynthesis° Prostaglandins I: 89, 1972.
5.
O'Grady, J.P., Caldwell, B.V., Auletta, FoJ. and Speroff, The effects of an inhibitor of prostaglandin synthesis (Indomethacin) on ovulation, pregnancy, and pseudopregnancy in the rabbit° Prostaglandins I: 97, 1972o
6,
Tsafriri, A., Physiological of ovulation.
7.
Jacobowitz, D. and Wallach, E.E. Histochemical chemical studies of the autonomic innervation ovary. Endocrinology 81: 1132, 1967.
D.Lo Blockade of spontanin rats by indomethacin, biosynthesis. Prosta-
Lindner, H.Ro, Zor, U~ and Lamprecht, SoA. role of prostaglandins in the induction Prostaglandins 2: I, 1972o of
and the
8.
Okamura, Ho, Virutamasen, P., Wright, KoH. and Wallach, Ovarian smooth muscle in the human being, rabbit and cat. Histochemical and electron microscopic study. Amer. J. Obstet. Gynec. 112: 183, 1972o
9.
Rocereto, To, Jacobowitz, Do and Wallach, tions of spontaneous contractions of the vitro. Endocrinology 84: 1136, 1969o
I0o
580
L.
Virutamasen, P°, Wright, KoHo and Wallach, of catecholamines on ovarian contractility Obstet. Gynec~ 39: 225, 1972
E.E.
E.E. Observacat ovary i__~n E.E. Effects in the rabbit.
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PROSTAGLANDINS
II.
Virutamasen, P., Wright, K.H. and Wallach, E.E. Effects of prostaglandins E2 and F2~ on ovarian contractility in the rabbit. Fertil. Steril. 23: 675, 1972.
12o
Virutamasen, Po, Wright, K.H. and Wallach, E.E. Monkey ovarian contractility - its relationship to ovulation. Fertil. Sterilo 24: 763, 1973.
13o
Harper, M.J.Ko Ovulation in the rabbit: The time of follicular rupture and expulsion of eggs in relation to injection of luteinizing hormone. J. Endocrinol. 26: 307, 1963o
14.
Richman, KoA., Wright, K.Ho and Wallach, E.E. Local ovarian effects of prostaglandins E2 and F2~ on human chorionic gonadotropin - induced ovulation in the rabbit. Obstet. Gyne¢o 43: 203, 1974.
15.
Behrman, HOE°, Orczyk, G.P. and Greep, R.O. Effect of synthetic gonadotrophin - releasing hormone (Gn-RH) on ovulation blockade by aspirin and indomethacin. Prostaglandins I: 245, 1972.
16o
Diaz-Infante, A., Jr., Wright, K.H. and Wallach, Influence of estrogen and progesterone treatment ovarian contractility in the monkey (Unpublished
M A R C H 25, 1974
VOL. 5 NO. 6
E.E. on data).
581
F2~
on Ovulation
Augusto Diaz-lnfante, Jro, MoD., ** Karen H. Wright, B.S. and Edward Eo Wallach, MoD. Department of Obstetrics and Gynecology, Pennsylvania Hospital and University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19107 ABSTRACT The present investigation was carried out to determine whether inhibition of ovulation in the rabbit by administration of indomethacin can be correlated with any change in ovarian contractility at ovulation time and can be reversed by administration of prostaglandinso Indomethacin was adminstered intramuscularly using three different schedules in a dose of 5 mg/kg. A reduced number of ruptured follicles following HCG was noted in all groups treated with indomethacino Infusion of P G F 2 ~ into the aorta (1 ~g/kg/mino) could reverse this effect. Less pronounced ovarian contractility was observed after indomethacin treatment, but infusion of PGF2~ immediately enhanced contractility in ovaries from indomethacin treated rabbits. The inhibition of ovulation in the rabbit associated with indomethacin adminstration may be related to suppression of ovarian contractions° These data also suggest that prostaglandins may play a significant role in the mechanism of ovulation through an influence on ovarian contractility.
*Supported by the Population Council Grant M730103 **Ford Foundation Fellow in Reproductive Biology. Present address: Department of Obstetrics and Gynecology, School of Medicine, University of San Luis Potosi, San Luis Potosi, Mexico Send reprint requests to Edward Eo Wallach, MoDo, Pennsylvania Hospital, Philadelphia, Pennsylvania 19107 Acknowledgements The authors wish to thank Thomas Henry, Kendra Guernsey, Anne T. Barrett and Elaine Stevens for their assistance°
Accepted February 12, 1974.
PROSTAGLANDINS MARCH 25, 1974
VOL. 5 NO. 6
567
PROSTAGLANDINS
Indomethacin has been reported to block prostaglandin synthesis in several tissues (1). Recently, this drug was also found to inhibit ovulation in rats (2,3) and rabbits (4,5). In these studies, luteinization of ovarian follicles was not prevented, the ova were retained within the follicles, and the ovarian steroidogenic pattern appeared to be normal (4-6). Based upon the assumption that the antiovulatory action of indomethacin is related to its ability to inhibit prostaglandin biosynthesis, it has been suggested that prostag l a n d i n s a r e involved in the process of follicular rupture. Previous observations from our laboratory have demonstrated: (1) a prominent autonomic innervation of ovarian tissue (7); (2) smooth muscle fibers present in ovarian stroma (7,8); and (3) ovarian contractions, detected in several species, which seem to correlate with the stage of follicular development (9-12). Ovarian smooth muscle activity has been considered significant in the physical process of ovulation. Ovarian contractile patterns and their responses to adrenergic agents and to prostaglandin F2~ has consistently been found to enhance ovarian contractility, while prostaglandin E2 is inhibitory
(I0,II). The purposes of the present investigation were to determine whether inhibition of ovulation in the rabbit by the administration of indomethacin can be: (I) correlated with any change in ovarian contractility at the predicted time of ovulation; (2) related to the ability of indomethacin to inhibit prostaglandin biosynthesis; and (3) reversed by administration of exogenous prostaglandins. In attempting to determine whether indomethacin administation can bring about changes in ovarian contractility at the time of ovulation, it was also considered important to ascertain whether these alterations could be reversed by exogenous prostaglandins.
MATERIALS
AND
METHODS
Sexually mature New Zealand white female rabbits, weighing between 3 and 4 kg,, were housed individually for a minimum of three weeks and fed Purina rabbit chow and water ad libitum.
568
M A R C H 25, 1974
VOL. 5 NO. 6
PROSTAGLANDINS
Rabbits were maintained in a room with controlled temperature and light. Ovulation was induced with human chorionic gonadotropin (HCG)*, I00 IoUo via marginal ear vein. Ovulation in the rabbit predictably follows the intravenous injection of H C G by I0-14 hours (13). The time of HCG injection was designated "time zero" in all experiments. All other treatments were temporally related to this point. Ovulation studies: Twelve hours after receiving HCG, the animals were anesthetized with intravenous sodium pentobarbital (32 mg/kg), and anesthesia was maintained during a four-hour experiment. A midline laparotomy was performed, the viscera retracted, and the ovaries examined in situ for ovulation points with minimal manipulation. A 27 gauge needle attached to a polyethylene catheter #20 was inserted into the abdominal aorta just above the origin of the ovarian arteries. The distal end of the catheter was attached to a I0 ml syringe, inserted in a Harvard Multispeed Infusion Pump with the speed adjusted to deliver a total volume of 5 ml in four hours° The abdominal incision was approximated with towel clamps and four hours later (16 hours after HCG) the ovaries were observed again, the catheter removed, and the abdominal cavity closed. Thirty-six hours after HCG, the rabbits were anesthetized again, the abdomen reopened and both ovaries removed and examined with a dissecting m i c r o s c o p e . At each observation time (12 hours, 16 hours, and 36 hours after HCG), ovarian follicles were counted and classified using a modification of criteria previously described (14). Follicles were divided into the following stages of development: Mature - clear follicles, distinctly present, but not bulging above the ovarian surface. Graafian - large, ovarian surface.
clear
Vascular - similar surface vascularity.
to
follicles, Graafian
protruding
follicles
but
above with
the distinct
Ruptured - follicles which have already released contents, thus appearing hemorrhagic, less prominent, with a clear tear line on the surface.
their and
*APL,
Labora-
generously supplied by tories, Inc., New York,
M A R C H 25, 1974
V O L . 5 NO. 6
Garfield New York
Davies,
Ayerst
569
PROSTAGLANDINS
Hemorrhagic large, cystic follicles, with dark hemorrhagic antral fluid, and no surface signs of rupture° Often, the egg could be removed from these follicles when the follicle was punctured under the dissecting microscope. -
All drugs were reconstituted before each experiment. Prostaglandin F 2 ~ (PGF2~)* was diluted in isotonic saline to a concentration adequate to deliver a dose of 1 ~g/kg/min in a fixed volume (5 ml). Indomethacin ** (5 mg/kg/ was adminis a tered intramuscularly as a suspension in 1 ml of Emulphor*** i% in saline. To determine the most efficacious timing for ovulation inhibition, indomethacin was administered using three different schedules° Five groups of four rabbits each were studied in this fashion using the following drug schedules: Group 1 - HCG alone Group 2 - Indomethacin 12 hours before HCG Group 3 - Indomethacin at the time of HCG Group 4 - Indomethacin I0 hours after HCG Group 5 - Indomethacin 12 hours before HCG; twelve hours after HCG the intra-aortie infusion of PGF2~ was begun and maintained over a four-hour interval° (Groups 1-4 received an infusion of 5 ml of saline into the aorta during the same fourhour period of time.) Rabbits intramuscular hours before Ovarian
in group injection HCG.
1 served of the
Contractility
as controls vehicle for
and received indomethacin
an 12
Studies
In two additional groups of six rabbits, ovarian contractility studies were timed to span the anticipated interval of ovulation. The following schedule was used: Group A received HCG and an intramuscular injection of the vehicle 12 hours before HCG. These rabbits were used as controls° Group B served as the experimental group and received indomethacin 12 hours before administration of HCG. Twelve hours after HCG, a laparotomy was performed as described above, and ovarian contractility was recorded in vivo for four hours in a randomly selected ovary from each rabbit° In some cases the contralateral ovary was studied simultaneously in vitro. The technical details for recording
*Prostaglandins were supplied by John Pike, Ph.D., Upjohn Kalamazoo, Michigan **Indomethacin was supplied by Carl R° Stevenson, M.Do of Merck, Sharp & Dohme, West Point, Pennsylvania ***Emulphor, GAF Corporation, New York, New York
570
MARCH 25, 1974
Co.,
VOL. 5 NO. 6
PROSTAGLANDINS
contractions in vivo and in vitro in our laboratory have been previously described (10,~).-'-'-~ter adequate recordings of spontaneous contractility had been obtained in vivo, P G F ~ (Ipg/kg/min) was infused through the intraaor'~ic Catheter. Changes in the ovarian contractile pattern in response to this infusion were analyzed and the patterns in the experimental and control groups were compared. RESULTS Ovulation, The number of follicles at each stage of development was recorded by treatment group at each of the three observation times (Table I). A reduced number of ruptured follicles was noted in all groups treated with indomethacin. Comparison of group 3 (indomethacin at "time zero") and group 4 (indomethacin 12 hours after HCG) with the control group, also reveals fewer ruptured and a greater number of follicles in both of these indomethacin-treated groups at 12 and 16 hours after HCG° At 36 hours some of the vascular follicles progressed to rupture, while others became hemorrhagic. Ovaries of rabbits in which indomethacin was given 12 hours before HCG (group 2) contained the smallest number of ruptured follicles (8 ruptured follicles as compared with 30 in the control group)° This figure remained relatively constant up to 36 hours after HCGo Most of the vascular follicles in group 2 observed at 12 hours after HCG ultimately became hemorrhagic. Ovaries in these rabbits (group 2) had the largest number of hemorrhagic follicles of all five groups at 36 hours after HCG° With one exception, no significant differences were found in the numbers of early follicles (mature and Graafian) when groups 2, 3, and 4 were compared to the control group. Group 4 (indomethacin at +I0) provided the only exception, when at 36 hours after HCG, the number of early follicles was markedly increased. The most striking results were observed when indomethacin was administered 12 hours before HCG. This treatment schedule was thus used again for animals in group 5),which also received an infusion of P G F 2 ~ between 12 and 16 hours after HCG° As anticipated, at 12 hours after HCG, just prior to initiating the P G F 2 ~ infusion, the results were similar to those found in group 2. However, at 16 hours after HCG, when the PGF2~ infusion had been completed, a marked increase in the number of ruptured follicles (23 ruptured follicles)was observed° This figure differed significantly from the number of ruptured follicles (9 ruptured follicles) noted in group 2 which had not received P G F 2 ~ o The total number of \ ruptured follicles observed in group 5 (23 ruptured follicles) nonetheless was smaller than in the control group (39 ruptured
MARCH 25, 1974
VOL. 5 NO. 6
571
O~
o
< 0
=
d
1-4
m
g
2 0
Hemorrhagic
t
39
Ruptured
Vascular
53
Total
i. 4 r a b b i t s in e a c h g r o u p 2. HCG 100 i.u. i n t r a v e n o u s l y
2
8
Graafian
Mature
3
0
Vascular
Hemorrhagic
46 40
Total Ruptured
2
39
Total
Mature
2
3
4
Graafian
Mature
Graafian
3
0
Hemorrhagic
54
4
8
25 .
7.
29 l0
0
1
9
i0
9
26
0
2
13
3O
of
.
. .
.
Indomethacin
52
7
6
.
.
F2~
I.M.
.....
~,
I
84
22
l0
3
36 32
0
2
3
8
23
36
0
i0
0
20
-12 = 12 hs. b e f o r e H C G 0 = at the t i m e of H C G +12 = 12 hs. a f t e r H C G 1 mg/kg/min intraaortic infusion
5 mg/kg
67
18
15
14
0
.
0
. .... . 12.
.
0
9
5
8
18
39
0
I0
0
40 20
4. P r o s t a g l a n d i n
3.
.
17 12
38 27
2
5
2
3
26
37
2
7
2
6
20
Group 5 H C G at 0 Indo. at -12 PGF~ f r o m +12 to +16 ] 6 ..........
ovulation
Group 4 H C G at 0 Indo. at + 1 2
on HCG-induced
Group 3 H C G at 0 Indo. at 0
indomethacin
Group 2 HCG at 0 Indo. at -12
I - Effect
Vascular
FOLLICLES Ruptured
Group 1 H C G at 0 (control)
TABLE
>
©
PROSTAGLANDINS
follicles). At 36 hours after HCG, the number of ruptured follicles in the i n d o m e t h a c i n - - P G F 2 ~ group (group 5 ) s h o w e d a further increase (32 ruptured follicles), approximating more closely the number of ruptured follicles found in the control group (40 ruptured follicles). Fewer hemorrhagic follicles were found in this group at 36 hours (I0 hemorrhagic follicles) than in group 2 (25 hemorrhagic follicles) which did not receive P G F 2 ~ . This difference could be interpreted to indicate that more of the vascular follicles present at the earlier observation intervals had progressed to rupture instead of becoming hemorrhagic° At 36 hours, the number of early follicles in group 5 (39 early follicles) was strikingly greater than in group 2 which was not treated with P G F 2 ~ (12 early follicles), or in the control group (I0 early follicles)° This predominance of early follicles in group 5 (indomethacin-PGF2~) accounts for these rabbits having the largest total number of ovarian follicles among all the experimental groups. Group 4, which received indomethacin I0 hours after HCG, and only a saline infusion, but not P G F 2 = , was the only group in which a similar distribution of follicles was observed at 36 hours after HCG. An increase~in total number of follicles was observed between 16 and 36 hours after HCG in all indomethacin-treated groups, but was most pronounced in group 5 which received P G F 2 ~ in addition to indomethacin. The increase in follicles between 16 and 36 hours after HCG amounted to 15% in group l, 86% in group 2, 37~ in group 3, 67% in group 4, and 133% in group 5. Ovarian Contractility~ Recordings of spontaneous ovarian contractions demonstrated greater amplitude and more rhythmicity in the control rabbits than in indomethacin-treated animals. The contractile pattern did not change significantly in the control group during or immediately after the infusion of P G F 2 ~ (Fig° 1)o In the group previously treated with indomethacin, the infusion of P G F 2 ~ tended to provoke enhanced rhythmicity and increased amplitude° In general, no significant changes in contractile pattern were observed between 12 and 16 hours after HCGo In some indomethacin-treated rabbits in which the i n f u s i o n of P G F 2 ~ enhanced ovarian contractility, the new contractile pattern persisted even after P G F 2 ~ was discontinued (Fig. 2)° Similar findings were obtained in ovaries studied in vitro, namely, depressed spontaneous contractility in indo-meta-~ntreated rabbits, but an enhanced contractile response to P G F 2 ~ (Fig. 3 A&B). In contrast control ovaries, from animals not treated with indomethacin, showed either a minimal or no response to P G F 2 ~ in vitro (Fig° 3C).
MARCH 25, 1974
VOL. 5 NO. 6
573
PROSTAGLANDINS
RABBIT 9 IN VIVO
CONTROL
A
~t3hr 21min
post HCG
2"2
2"3
24
2"5"
2"6
2"7
2'8
B "i1 16min I"7 18 19 post HCG 12minutes post completion of PGF2¢ infusion 14hr
FIGURE
2"1
2"2
23
1
In vivo recording of ovarian not treated with indomethacino
574
20
contractions recorded
in Rabbit
9,
Ao
Spontaneous contractions minutes following HCGo
13 hours and 21
B.
Contractions recorded 12 minutes after completion of an intra-aortic infusion of PGF2~ (2 ~g/kg/min for 24 minutes)°
MARCH 25, 1974
VOL. 5 NO. 6
PROSTAGLANDINS
RABBIT 8 iN vIVO INDOMETHACIN 12 HOURS PRE HCG
A
l~'l~rnmin post HC,G
15
~;0
25
B PGF2~ INFUSION ~ .
.
.
.
1
.
13hr 5 0 r a i n
55
"
1,~hr
.
.
.
.
()5
"
post HCG
C
~4ht~l~i. " post HCG
FIGURE
B
~5
.
.
.
.
20
25
30
2
In vivo recordings of ovarian contractions in Rabbit 8 treated with indomethacin 12 hours prior to HCG administration. Ao
Spontaneous ovarian contractions recorded 13 hours and ll minutes after HCG.
Bo
Enhanced contractile pattern during a 30 minute intra-
aortic C.
infusion
of PGF2~
(I ~g/kg/min).
Recording of ovarian contractions immediately discontinuation Of PGF2~ infusion demonstrating existance of an enhanced contractile pattern.
MARCH 25, 1974
VOL. 5 NO. 6
following the
575
PROSTAGLANDINS
RABBIT 7 I~HACtN
IN VITRO 12 HOURS PRE HCG
12h~.~. . . . . posl HCG
4b
. . . .
4~
.
.
.
.
~o
'
B
12ht52min I~¢st HCG
RAB81T 2 CONTROL
55
13h~
05
;o
IN Vfl'RO
C ~IF2= ,;~,,sm;:'.....
post HCG
FIGURE
576
•
•
-
•
is . . . .
3'o . . . .
3's . . . .
40
3
Ao
In vitro recordings of ovarian contractions in Rabbit 7 treated with indomethacin 12 hours prior to HCG administration, demonstrating minimal contractility.
Bo
In the same preparation the addition of PGF2~ (i00 pg) at 12 hours and 55 minutes after HCG led to initiation of pronounced ovarian contractions.
Co
In vitro recording o f ovarian contractions in Rabbit 2 not treated with indomethacin. The addition of P G F 2 ~ (200 ~g) at 13 hours and 24 minutes after HCG had no appreciable effect on ovarian contractions.
MARCH
25,
1974
VOL.
5 NO.
6
PROSTAGLANDIMS
In order to express these results numerically, the area included by the c o n t r a c t i o n s in a I0 minute period of time was calculated by counting the square millimeters in the recording, using standard sensitivity and chart speed. With this method, a mean value could be obtained for spontaneous contractions as well as for contractions recorded during the P G F 2 ~ infusion° In each group of 6 rabbits the m e a ~ value for spontaneous contractions was compared. The mean value in the control group was 325°8 mm2, as compared with 182 mm2 in the indomethacin-treated group (85.8% of the control)° In the control group during the P G F 2 ~ i n f u s i o n the corresponding figure of 349°4 mm2, (an i n c r e a s e o f 7.2%) compares with 241 mm2 (an increase of 37o8~) in the indomethatin-treated group° DISCUSSION The ability of indomethacin to inhibit induced ovulation in the rat (2,3) and rabbit (4,5) has recently been described. The nature of this action of indomethacin, however, is still in question. In previous experiments carried out in the rabbit (5), a single intravenous dose of indomethacin given at the time of HCG failed to block HCG-induced ovulation° Supplemental indomethacin administered subcutaneously at 12 hour intervals for 48 hours after HCG led to inhibition of ovulation. However, because observations were not made at the expected time of ovulation but approximately 12 hours after HCG, a delay in ovulation attributable to indomethacin may have been missed. In the present experiment, a dose of indomethacin smaller than that given by O'Grady (5) and administered by the intramuscular route led to a decrease in number of ruptured follicles at the expected time of ovulation° Such was the case when indomethacin was given either at the same time as HCG or 10 hours later. The most striking effect was found when indomethacin was given 12 hours before HCG. These differences may be related to the intramuscular route of indomethacin administration yielding a prolonged absorption rate° M o r e p r o n o u n c e d depletion in endogenous prostaglandin levels might be anticipated in the group in which indomethacin was given 12 hours before HCG. Prostaglandin E 2 has been reported to overcome the inhibitory effect of indomethacin on ovulation in rats (6). In the present experiment, P G F 2 ~ reversed inhibition of ovulation brought about by indomethacin in the rabbit. These observations support the suggestion that the effect of indomethacin on ovulation is related to its inhibition of prostaglandin biosynthesis° Although indomethacin has been shown to block ovulation, it has not prevented follicular maturation, luteinization or progesterone secretion (4)° In fact, ova have been
MARCH 25, 1974
VOL. 5 NO. 6
577
PROSTAGLANDINS
described contained within luteinized follicles following indomethacin (5). Tsafriri, et al (6) postulated that indomethacin prevents only follicular rupture and that prostaglandins play an essential role in the m e c h a n s i m whereby LII leads to follicular rupture. Tsafriri also demonstrated that PGE2 is able to induce ovum maturation, although while not being essential to support this action of LHo Behrman (14) demonstrated that indomethacin has a prolonged effect, and that exogenous LH or gonadotropin releasing hormone fails to reverse its anti-ovulatory action while promoting progesterone secretion. The results in the present report concur with the hypothesis that the effect of indomethacin on ovulation is exerted locally at the ovarian level and that prostaglandins have an essential role in
the rupture of the mature follicle° The h e m o r r h a g i c f o l l i cles observed in our experiments probably correspond to those described by O'Grady (5) as exhibiting luteinization and containing entrapped ova; these structures represent follicles which were stimulated but failed to rupture. The marked increase in total number of follicles in indomethacintreated groups probably results from the disappearance of indomethacin from receptor sites, thus permitting the resumption of prostaglandin biosynthesis° Consequently, increasing local levels of prostaglandin support ovum maturation. This concept might also explain the markedly increased number of follicles in the group of animals pretreated with indomethacin and then infused with PGF2~. Earlier experiments in our laboratory have been directed to the demonstration of ovarian contractions in several species (9-12)o We have consistently found that PGF 2 enhances ovarian contractility and PGE 2 inhibits it (II). Prostaglandin E 2 has recently been shown to delay ovulation in the rabbit (14). Ovarian contractile patterns and ovarian responsiveness to prostaglandins also appear dependent upon the specific hormonal environmeht in the monkey (16). We have previously postulated that ovarian smooth muscle activity may play a significant role in the physical process of ovulation. In the second phase of the present study spontaneous ovarian contractility was found at the anticipated time of ovulation after HCG administration. However, the ovarian contractions recorded in the indomethacin-treated group lacked rhythmicity and demonstrated lower amplitude than in the control group~ Infusion of P G F 2 ~ had little effect on the ovarian contractile pattern in control animals, while in those rabbits pretreated with indomethacin, rhythmic contractions and an increase in amplitude were frequently provoked° One might postulate that the dose of P G F 2 ~ used in this experiment was only effective when endogenous
578
M A R C H 25, 1974
VOL. 5 NO. 6
PROSTAGLANDINS
prostaglandins had been depleted by indomethacin. This depletion of prostaglandins apparently impairs ovarian contraeilityo On the basis of these results we would propose that indomethacin acts locally on the rabbit ovary to prevent the rupture of ovarian follicles° This action would seem to depend on the ability of indomethacin to inhibit prostaglandin biosynthesis. It appears even more likely that prostaglandins play an important role in the m e c h a n i s m of ovulation, and possibly do so by influencing ovarian contractile patterns,
MARCH 25, 1974
VOL. 5 NO. 6
579
PROSTAGLANDINS
REFERENCES
I.
Vane, JoR. Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs° Nature New Biol0~Y 231: 232, 1971.
2.
Armstrong, DoTo and Grinwieh, eous and LH-induced ovulation an inhibitor of prostaglandin glandins I: 21, 1972o
3.
Orczyk, GoPo and Behrman, H.Ro Ovulation blockade by aspirin or indomethacin - In vivo evidence for a role of prostaglandin in gonadotropin secretion. Prostag!andins I: 3, 1972.
4.
Grinwich, DoLo, Kennedy, ToG. and Armstrong, D.T. Dissociation of ovulatory and steroidogenic actions of luteinizing hormone in rabbits with indomethacin, an inhibitor of prostaglandin biosynthesis° Prostaglandins I: 89, 1972.
5.
O'Grady, J.P., Caldwell, B.V., Auletta, FoJ. and Speroff, The effects of an inhibitor of prostaglandin synthesis (Indomethacin) on ovulation, pregnancy, and pseudopregnancy in the rabbit° Prostaglandins I: 97, 1972o
6,
Tsafriri, A., Physiological of ovulation.
7.
Jacobowitz, D. and Wallach, E.E. Histochemical chemical studies of the autonomic innervation ovary. Endocrinology 81: 1132, 1967.
D.Lo Blockade of spontanin rats by indomethacin, biosynthesis. Prosta-
Lindner, H.Ro, Zor, U~ and Lamprecht, SoA. role of prostaglandins in the induction Prostaglandins 2: I, 1972o of
and the
8.
Okamura, Ho, Virutamasen, P., Wright, KoH. and Wallach, Ovarian smooth muscle in the human being, rabbit and cat. Histochemical and electron microscopic study. Amer. J. Obstet. Gynec. 112: 183, 1972o
9.
Rocereto, To, Jacobowitz, Do and Wallach, tions of spontaneous contractions of the vitro. Endocrinology 84: 1136, 1969o
I0o
580
L.
Virutamasen, P°, Wright, KoHo and Wallach, of catecholamines on ovarian contractility Obstet. Gynec~ 39: 225, 1972
E.E.
E.E. Observacat ovary i__~n E.E. Effects in the rabbit.
MARCH 25, 1974
VOL. 5 NO. 6
PROSTAGLANDINS
II.
Virutamasen, P., Wright, K.H. and Wallach, E.E. Effects of prostaglandins E2 and F2~ on ovarian contractility in the rabbit. Fertil. Steril. 23: 675, 1972.
12o
Virutamasen, Po, Wright, K.H. and Wallach, E.E. Monkey ovarian contractility - its relationship to ovulation. Fertil. Sterilo 24: 763, 1973.
13o
Harper, M.J.Ko Ovulation in the rabbit: The time of follicular rupture and expulsion of eggs in relation to injection of luteinizing hormone. J. Endocrinol. 26: 307, 1963o
14.
Richman, KoA., Wright, K.Ho and Wallach, E.E. Local ovarian effects of prostaglandins E2 and F2~ on human chorionic gonadotropin - induced ovulation in the rabbit. Obstet. Gyne¢o 43: 203, 1974.
15.
Behrman, HOE°, Orczyk, G.P. and Greep, R.O. Effect of synthetic gonadotrophin - releasing hormone (Gn-RH) on ovulation blockade by aspirin and indomethacin. Prostaglandins I: 245, 1972.
16o
Diaz-Infante, A., Jr., Wright, K.H. and Wallach, Influence of estrogen and progesterone treatment ovarian contractility in the monkey (Unpublished
M A R C H 25, 1974
VOL. 5 NO. 6
E.E. on data).
581