- Email: [email protected]
Ovulation blockade through synergism of cycloheximide with assorted anesthetics
CONTRACEPTION
OVULATION BLOCKADE THROUGH CYCLOHEXIMIDE WITH ASSORTED Zeev Dickmann
SYNERGISM VCS
OF
and Paul F. Terranova*
Departments of Gynecology & Obstetrics and *Physiology, and the Ralph L. Smith Research Center University of Kansas Medical Center Kansas City, Kansas 66103, U.S.A.
ABSTRACI This report addresses the following question: Can ovulation be blocked through a combined action of two drugs, which individually have no effect on ovulation. Ovulation blockade in hamsters was achieved through synergism of two drugs that individually had no effect on ovulation. Treatments were given at 18:OO h on pro-estrus. The keystone finding was that cycloheximide (CX), in a dose too small to affect ovulation, can be made to block ovulation by pretreating hamsters 15 min earlier with pentobarbital. Ovulation blockade was also achieved with phenobarbital + CX, ketamine + CX, and ether + CX. When given alone, none of the four anesthetics interferred with ovulation. The mechanism by which the anesthetic/CX synergism works is not known. The pituitary can be excluded as a target, because it is not necessary for ovulation after 15:30 on pro-estrus. It is speculated that CX reduces the synthesis of ovarian proteins needed for follicle rupture, and the anesthetics augment this process through action on either the ovary, the brain, the liver, or a combination thereof. INTRODUCDON Drugs able to block ovulation have been known for several decades (1,2). Although investigators have continued to search for additional ovulation-blocking drugs, we are not aware of any publication that has raised the question, “Is it is possible to block ovulation through the combined action of two different types of drugs, which individually have no effect on ovulation.” This question is addressed in the present report. Treating pro-estrous hamsters with cycloheximide (CX), a protein synthesis inhibitor, caused ovulation blockade (3,4). An experiment in our laboratory revealed that a dose of CX too smalJ to affect ovulation, can be made to block ovulation by pretreating hamsters Since ovulation with sodium pentobarbital (PB) -- a general, short-acting anesthetic. blockade through synergism of two drugs has not been reported for any animal species, this synergistic phenomenon was further investigated.
Correspondence
to: Dr. Z. Dickmann
This work was supported Submitted Accepted
by NIH grant HD 20389.
for publication for publication
September 25, 1989 October 24, 1989
FEBRUARY 1990 VOL. 41 NO. 2
189
CONTRACEPTION
MATERIAL
AND METHODS
Adult female golden hamsters (Mesocricetus auratus), weighing loo-130 g, were maintained in a room at 71-75°F. with lights on from 0500 to 19:00 h. Under this li.aht regimen, most ova are ovulated between24:30 and 02:OOh on estrus (5). Only animals exhibiting at least 2-3 consecutive 4-day cycles were used. Estrus was determined by the characteristic postovulatory vaginal discharge. On pro-estrus at 18:00h, animals were injected with either saline (controls), CX, PB, or PB followed 15 min later by CX. The hamsters were killed the next morning (Day 1) between 09:OO and 10:OOh. Their oviducts were excised and flushed with saline (9 g NaCI/L) to recover the ovulated ova which were embedded in granulosa cells. To enable accurate counting of ova, the granulosa cells surrounding them were dispersed with hyaluronidase. CX was purchased from Sigma Chemical Co., St. Louis, Missouri. It was dissolved in saline and injected s.c., in a concentration of 10 mg/ml. The amounts injected are indicated in the Tables. [The reader should note that the potency of different batches of CX may vary. Thus, in experiments not included in this report, we used CX which had a decidedly higher potency than the CX (Lot 68F-0638) used in the present work.] PB (Veterinary Laboratories, Inc., Lenexa, Kansas) in a concentration of 65 mg/ml was injected i.p. at a dose of 8.8 mg/lOO g body weight (B.W.), which is an anesthetic dose. Lower doses of PB were used in two experiments (Table II). Three additional anesthetics were tested alone, and in combination with CX. They were: 1) Phenobarbital (PH) (Elkins-Sinn, Inc., Cherry Hill, New Jersey) - a general, longacting anesthetic, which, like PB, belongs to the barbiturate group, it was injected i.p.; 2) Ketamine (KT) (Bristol Laboratories, Syracuse, New York) - a dissociative anesthetic, injected i.p.; and 3) Ether (ET) (Fisher Scientific, Fair Lawn, New Jersey) - an inhalant type anesthetic. Like PB, PH and KT were injected at 18:OOh on pro-estrus. In the PH/CX, KT/CX, and ET/CX treatments, CX was injected at 18:15h. ET anesthesia was begun at 18:00 h, and was maintained for one hour. The data were anlyzed by the student’s t test. Differences were considered statistically significant at the level of p 5 0.05. RESULTS Table I shows that, compared to controls, neither 2 mg nor 3 mg CX had a significant effect on ovulation, whereas 4 mg significantly (p
190
FEBRUARY 1990 VOL. 41 NO. 2
2 3 4 5
5
5
6
9
1
2
3
4
N.s.: Not significantly different from control * Significantly lower than control, p < 0.001
_-_
6
Cx me/100
Control (saline injected)
Exo. No.
No. Animals
Table 1. The number of ova ovulated by hamsters (CX) at lS:OOh, on pro-oestrus (Day 4)
g B.W.
treated
Ova Ovulated
1.77 kO.73 *
000112237 13 , , 7>93
l
NS
2.50 f 1.31
9.80 20.73
10.20 -c 1.24 N.S.
0,0,0,3,4,8
7,10,10,11,11
6,9,11,12,13
11.8 20.6
MeankS.E.M.
or with various doses of cycloheximide
10,11,11,12,13,14
Per animal
with saline (controls),
6
5
6
6
6
7
8
9
N.S.:
l
3
3
3
2
___
Treatment Cx me/100 e B.W.
Not significantly different from control Significantly lower than control, p < 0.001
5
No. animals
5
Exp. No. PB me/100
2.2
4.4
8.8
8.8
8.8
g B.W.
Ova ovulated
of pentobarbital
124777 , , I , ,
002248 , 1, , ,
O,O,O,O, 1
000117 7?, , 9
8,14,14,14,14
Per animal
Table II. The number of ova ovulated by hamsters treated with various dose-combinations (CX) at 18:OOh on pro-oestrus (Day 4)
1.2
N.S.
S.E.M
4.66 2 1.11 *
2.66 +- 1.23 *
0.20 -e 0.20 ’
1.50 5 1.12 *
12.80 f
Meant
(PB) + cycloheximide
6
5
6
5
5
8
11
12
13
14
15
16
different
_--
__-
** Significantly
higher than control, p < 0.02
__-
__-
8.0
15.0
8.0
___
___
KT mg/lOOe B.W.
from control
___
___
___
8.8
8.8
PH me/lOOe B.W
* Significantly lower than control, p < 0.001
N.s.: Not significantly
5
No. animals
10
Exp. No.
Treatment
yes
yes
___
___
_--
___
__-
ET for 1 hr
3.0
_--
3.0
_--
___
3.0
_--
0,0,0,0,1,2,3,3
12,13,13,14,15
00000 , 77.
8,10,10,13,14,15
12,13,14,14,15
0.16 f 0.16
00000 , 3 9 , 11
1.1 t 0.39 *
13.4 lr 0.50 N.S.
0.0 *
11.6 4 1.11 N.S.
13.6 + 0.50 N.S.
l
14.4 t 0.50 **
13,14,14,15,16
Per animal
Mean2S.E.M.
(KT), ether (ET) alone,
Ova ovulated
(PH), ketamine
cx me/lOOe B.W.
Table III. The number of ova ovulated by hamsters treated with either phenobarbital or in combination with 3 mg cycloheximide (CX), at 18:OOh on pro-oestrus (Day 4)
CONTRACEPTION
was followed by a 3 mg CX injection, the ovulation rates dropped very significantly (p
REFERENCES 1.
Austin CR, Perry JC, eds. Agents Affecting Boston, 1965.
2.
Lipner, H. Mechanism of mammalian ovulation. In: Knobil E, NeiU JD, eds. The Physiology of Reproduction. Raven Press, New York, 1988:447-88.
194
Fertility.
Little, Brown and Company,
FEBRUARY 1990 VOL. 41 NO. 2
CONTRACEPTION
3.
Alleva JL, Bonventre PF, Lamanna C. Inhibition of ovulation in hamsters by protein synthesis inhibitors Diphteria Toxin and cycloheximide. Proc. Sot. Exp. Biol. & Med. 1979; 162:170-4.
4.
Saidapur SK, Greenwald GS. The role oestradioL17fi in the pro-oestrous hamster.
5.
Norman RL. Behavorial es&us, ovarian histology and progesterone proestrous hamster. Ph.D. thesis, University of Kansas, 1971.
6.
Greenwald GS. Preovulatory changes Endocrinology 1971; 88:671-7.
7.
Conney AH. Pharmacological implications Pharmacol. Rev. 1967; 19:317-66.
8.
Brannstrom M, Boberg BM, Tomell J, Janson PO, Ahren K_ Effects of inhibitors of protein synthesis on the ovulatory process of the perfused rat ovary. J. Reprod. Fert. 1989; 85:451-9.
FEBRUARY
1990 VOL. 41 NO. 2
of protein synthesis in regulation J. Reprod. Fert. 1981; 62, 379-384.
in ovulating
hormone
of microsomal
secretion
of
in the
in the cyclic hamster.
enzyme
induction.
195
OVULATION BLOCKADE THROUGH CYCLOHEXIMIDE WITH ASSORTED Zeev Dickmann
SYNERGISM VCS
OF
and Paul F. Terranova*
Departments of Gynecology & Obstetrics and *Physiology, and the Ralph L. Smith Research Center University of Kansas Medical Center Kansas City, Kansas 66103, U.S.A.
ABSTRACI This report addresses the following question: Can ovulation be blocked through a combined action of two drugs, which individually have no effect on ovulation. Ovulation blockade in hamsters was achieved through synergism of two drugs that individually had no effect on ovulation. Treatments were given at 18:OO h on pro-estrus. The keystone finding was that cycloheximide (CX), in a dose too small to affect ovulation, can be made to block ovulation by pretreating hamsters 15 min earlier with pentobarbital. Ovulation blockade was also achieved with phenobarbital + CX, ketamine + CX, and ether + CX. When given alone, none of the four anesthetics interferred with ovulation. The mechanism by which the anesthetic/CX synergism works is not known. The pituitary can be excluded as a target, because it is not necessary for ovulation after 15:30 on pro-estrus. It is speculated that CX reduces the synthesis of ovarian proteins needed for follicle rupture, and the anesthetics augment this process through action on either the ovary, the brain, the liver, or a combination thereof. INTRODUCDON Drugs able to block ovulation have been known for several decades (1,2). Although investigators have continued to search for additional ovulation-blocking drugs, we are not aware of any publication that has raised the question, “Is it is possible to block ovulation through the combined action of two different types of drugs, which individually have no effect on ovulation.” This question is addressed in the present report. Treating pro-estrous hamsters with cycloheximide (CX), a protein synthesis inhibitor, caused ovulation blockade (3,4). An experiment in our laboratory revealed that a dose of CX too smalJ to affect ovulation, can be made to block ovulation by pretreating hamsters Since ovulation with sodium pentobarbital (PB) -- a general, short-acting anesthetic. blockade through synergism of two drugs has not been reported for any animal species, this synergistic phenomenon was further investigated.
Correspondence
to: Dr. Z. Dickmann
This work was supported Submitted Accepted
by NIH grant HD 20389.
for publication for publication
September 25, 1989 October 24, 1989
FEBRUARY 1990 VOL. 41 NO. 2
189
CONTRACEPTION
MATERIAL
AND METHODS
Adult female golden hamsters (Mesocricetus auratus), weighing loo-130 g, were maintained in a room at 71-75°F. with lights on from 0500 to 19:00 h. Under this li.aht regimen, most ova are ovulated between24:30 and 02:OOh on estrus (5). Only animals exhibiting at least 2-3 consecutive 4-day cycles were used. Estrus was determined by the characteristic postovulatory vaginal discharge. On pro-estrus at 18:00h, animals were injected with either saline (controls), CX, PB, or PB followed 15 min later by CX. The hamsters were killed the next morning (Day 1) between 09:OO and 10:OOh. Their oviducts were excised and flushed with saline (9 g NaCI/L) to recover the ovulated ova which were embedded in granulosa cells. To enable accurate counting of ova, the granulosa cells surrounding them were dispersed with hyaluronidase. CX was purchased from Sigma Chemical Co., St. Louis, Missouri. It was dissolved in saline and injected s.c., in a concentration of 10 mg/ml. The amounts injected are indicated in the Tables. [The reader should note that the potency of different batches of CX may vary. Thus, in experiments not included in this report, we used CX which had a decidedly higher potency than the CX (Lot 68F-0638) used in the present work.] PB (Veterinary Laboratories, Inc., Lenexa, Kansas) in a concentration of 65 mg/ml was injected i.p. at a dose of 8.8 mg/lOO g body weight (B.W.), which is an anesthetic dose. Lower doses of PB were used in two experiments (Table II). Three additional anesthetics were tested alone, and in combination with CX. They were: 1) Phenobarbital (PH) (Elkins-Sinn, Inc., Cherry Hill, New Jersey) - a general, longacting anesthetic, which, like PB, belongs to the barbiturate group, it was injected i.p.; 2) Ketamine (KT) (Bristol Laboratories, Syracuse, New York) - a dissociative anesthetic, injected i.p.; and 3) Ether (ET) (Fisher Scientific, Fair Lawn, New Jersey) - an inhalant type anesthetic. Like PB, PH and KT were injected at 18:OOh on pro-estrus. In the PH/CX, KT/CX, and ET/CX treatments, CX was injected at 18:15h. ET anesthesia was begun at 18:00 h, and was maintained for one hour. The data were anlyzed by the student’s t test. Differences were considered statistically significant at the level of p 5 0.05. RESULTS Table I shows that, compared to controls, neither 2 mg nor 3 mg CX had a significant effect on ovulation, whereas 4 mg significantly (p
190
FEBRUARY 1990 VOL. 41 NO. 2
2 3 4 5
5
5
6
9
1
2
3
4
N.s.: Not significantly different from control * Significantly lower than control, p < 0.001
_-_
6
Cx me/100
Control (saline injected)
Exo. No.
No. Animals
Table 1. The number of ova ovulated by hamsters (CX) at lS:OOh, on pro-oestrus (Day 4)
g B.W.
treated
Ova Ovulated
1.77 kO.73 *
000112237 13 , , 7>93
l
NS
2.50 f 1.31
9.80 20.73
10.20 -c 1.24 N.S.
0,0,0,3,4,8
7,10,10,11,11
6,9,11,12,13
11.8 20.6
MeankS.E.M.
or with various doses of cycloheximide
10,11,11,12,13,14
Per animal
with saline (controls),
6
5
6
6
6
7
8
9
N.S.:
l
3
3
3
2
___
Treatment Cx me/100 e B.W.
Not significantly different from control Significantly lower than control, p < 0.001
5
No. animals
5
Exp. No. PB me/100
2.2
4.4
8.8
8.8
8.8
g B.W.
Ova ovulated
of pentobarbital
124777 , , I , ,
002248 , 1, , ,
O,O,O,O, 1
000117 7?, , 9
8,14,14,14,14
Per animal
Table II. The number of ova ovulated by hamsters treated with various dose-combinations (CX) at 18:OOh on pro-oestrus (Day 4)
1.2
N.S.
S.E.M
4.66 2 1.11 *
2.66 +- 1.23 *
0.20 -e 0.20 ’
1.50 5 1.12 *
12.80 f
Meant
(PB) + cycloheximide
6
5
6
5
5
8
11
12
13
14
15
16
different
_--
__-
** Significantly
higher than control, p < 0.02
__-
__-
8.0
15.0
8.0
___
___
KT mg/lOOe B.W.
from control
___
___
___
8.8
8.8
PH me/lOOe B.W
* Significantly lower than control, p < 0.001
N.s.: Not significantly
5
No. animals
10
Exp. No.
Treatment
yes
yes
___
___
_--
___
__-
ET for 1 hr
3.0
_--
3.0
_--
___
3.0
_--
0,0,0,0,1,2,3,3
12,13,13,14,15
00000 , 77.
8,10,10,13,14,15
12,13,14,14,15
0.16 f 0.16
00000 , 3 9 , 11
1.1 t 0.39 *
13.4 lr 0.50 N.S.
0.0 *
11.6 4 1.11 N.S.
13.6 + 0.50 N.S.
l
14.4 t 0.50 **
13,14,14,15,16
Per animal
Mean2S.E.M.
(KT), ether (ET) alone,
Ova ovulated
(PH), ketamine
cx me/lOOe B.W.
Table III. The number of ova ovulated by hamsters treated with either phenobarbital or in combination with 3 mg cycloheximide (CX), at 18:OOh on pro-oestrus (Day 4)
CONTRACEPTION
was followed by a 3 mg CX injection, the ovulation rates dropped very significantly (p
REFERENCES 1.
Austin CR, Perry JC, eds. Agents Affecting Boston, 1965.
2.
Lipner, H. Mechanism of mammalian ovulation. In: Knobil E, NeiU JD, eds. The Physiology of Reproduction. Raven Press, New York, 1988:447-88.
194
Fertility.
Little, Brown and Company,
FEBRUARY 1990 VOL. 41 NO. 2
CONTRACEPTION
3.
Alleva JL, Bonventre PF, Lamanna C. Inhibition of ovulation in hamsters by protein synthesis inhibitors Diphteria Toxin and cycloheximide. Proc. Sot. Exp. Biol. & Med. 1979; 162:170-4.
4.
Saidapur SK, Greenwald GS. The role oestradioL17fi in the pro-oestrous hamster.
5.
Norman RL. Behavorial es&us, ovarian histology and progesterone proestrous hamster. Ph.D. thesis, University of Kansas, 1971.
6.
Greenwald GS. Preovulatory changes Endocrinology 1971; 88:671-7.
7.
Conney AH. Pharmacological implications Pharmacol. Rev. 1967; 19:317-66.
8.
Brannstrom M, Boberg BM, Tomell J, Janson PO, Ahren K_ Effects of inhibitors of protein synthesis on the ovulatory process of the perfused rat ovary. J. Reprod. Fert. 1989; 85:451-9.
FEBRUARY
1990 VOL. 41 NO. 2
of protein synthesis in regulation J. Reprod. Fert. 1981; 62, 379-384.
in ovulating
hormone
of microsomal
secretion
of
in the
in the cyclic hamster.
enzyme
induction.
195