THE EFFECT OF SOME B-ADRENERGIC BLOCKING AGENTS OVULATION IN RABBITS*
ON
M.S. Foda, N.A. Darwlsh and A.A. Shafeek Obstetrics and Gynecology Department E.M. Sharaf and M.A. Zakl Pharmacelogy Deportment Faculty of Medicine, Cairo University
ABSTRACT The effect of two B-adrenerglc blocking agents, propanolol hydrochloride and oxprenolol hydrochlorlde, on ovulation in rabbits is studied. The first drug prevented receptivity to male and suppressed ovulation after forced mating. Oxprenolol hydrochlorlde showed a similar effect as regards receptivity to male but did not prevent ovulation after forced mating. Both drugs did not interfere wlth ovulation induced by large doses of exogenous gonadotrophin. The possible made of action of these drugs is discussed.
*Supported by
a
W H O grant.
Accepted for publ£cation May 8, 1975
JULY 1975
VOL. 12 NO. 1
53
CONTRACEPTION
I NTRO DUCTION The existence of smooth muscle fibres in the ovarian stroma has been documented in a number of mammalian species (1,2,3,4). Ruorescent hlstochemlcal techniques have shown the presence of adrenergic terminal fibres in the ovarian stromata of cat, man, rabbit and monkey (3,5,6). The smooth muscle in the ovary end its apparent adrenergic innervatlan suggests that, at least in some species, neurogenlc contraction of the muscle may cause or facilitate ovum release (7). In the rabbit it was found that the 13-adrenergic blocker, dlbenzylene, administered locally to the ovary in conjunction with artificially induced ovulation caused a reduction in the number of ruptured follicles with a reciprocal increase in the number of mature follicles (8). It was proposed that dibenzylene inhibited ovarian smooth muscle cantractlon necessary for ovum ejection. The effect of g-adrenergic blockers in this regard has not yet been explored. The present work was undertaken to study: a) The effect of two ~]-adrenerglc blocking agents, namely, propanolol hydrochloride (Indral, ICI)and oxprenolol hydrochlorlde (Tronslcor, Ciba), on the process of ovulation in mature rabbits; and b) The effect of the same compounds an chorlanic gonodotrophln induced ovulation in immature rabbits. MATERIAL AND METHODS Effect of prapanolol hydrochlorlde and oxprenolol hydrochlorlde on mating induced ovulation in mature rabbits The study was conducted an mature female rabbits of the Boscat strain which had been isolated individually for 3 to 4 weeks to prevent the occurrence of spontaneous ovulation or pseudopregnancy. The animals were not less than one year old and the average weight was 2.5 kg. The animals were divided into 3 groups of 6 members each. The rabbits of the first group were injected intravenously through the marginal ear vein with propanolol hydrochlorlde at a dose of 1.5 mg/kg body weight per day for 7 consecutive days, so as to give enough time for the drug to act through different parameters. The members of the 2nd group were injected with oxprenolol hydrochlorlde by the same route and at the same dose level for the same period. The 3rd group was injected for 7 consecutive days wlth physiological saline as controls. On the 8th day, and for four consecutive days, the animals of the three graupswere exposed to male partners 15 minutes after the daily injection which was continued until day 11. Vaginal smears were obtained from the animals which accepted mating to demonstrate sperms in the vagina. Laparatamy was performed an the 14th day from
54
JULY 1975 VOL. 12 NO. 1
CONTRACEPTION
the start of the experiment. The ovaries ware inspected for follicles and corpora lutea and the uterine homs for early sites of implantation. The abdomen was then closed and each animal was returned to its cage. A second laparotomy was performed on the 23rd day to differentiate between true and pseudopregnancy and to obtain ovarian biopsies (Figure). Intravenous Pentobarbltal (45 mg/~)was used for anaesthesia. mating I
I
I
2 3 4
!
1
I
I
5 6 7 8 9
daily injection Figure.
I
I
I
I
10 11 I
12
|
I
I
13 14 ,
I
I
I
I
!
II
I
I
15 16 17 18 19 20 21 22 23
I
|st laparotomy
I
J
2nd laparo amy
A graphic representation of the study protocol.
Effect of 13-adrenerglc blockers on chorionlc gonadotrophln induced ovulation In immature female rabbits This study was conducted on immature female rabbits of the white Giza strain. Their ages ranged between 5 and 6 months, and the average weight was 1.5 kg. The animals were divided into 3 groups, each of 8 animals. The 1st group was iniected with physiological saline to be considered as the control group. The members of the 2nd and 3rd groups were injected through the marginal ear vein with 1.5 mg per kg body weight of propanolol hydrochlorlde or oxpmnolol hydrochlorlde, respectively. After 15 minutes, each member of the 3 groups was injected by the same route with 200 I.U. chorlonlc gonodotrophin. Lapamtomy was performed on the 3rd day and ovaries were inspected for haemorrhaglc follicles and ovarian biopsies were obtained. Effect of 13-adrenerg|c blockers on forced mating induced ovulation in mature female rabbits ~ A new batch of 18 mature female rabbits cf the Boscot strain was used for the study. Their ages were above one year and the average weight was 2.5 kg. After individual isolation of the female rabbits for 2 weeks, they were divided into 3 groups of 6 members each. The 1st group was used as the control; physiological saline was injected I.V. daily for 7 consecutive days before exposure to forced mating. On the 8th day, 15 minutes after the injection, forced mating was practiced. This procedure was repeated on the 9th, 10th and 11th days. The 2nd and 3rd groups were treated similarly but using the 2 test drugs instead of physiological saline at a dose of 1.5 mg/kg body ~elght. Twelve days elapsed between the last day of mating and laparotomy. During the surgery, the ovaries were inspected for the presence of mature follicles and corpora lutea, the uterine hams for implantation sites. Biopsies were obtained for further microscopic evaluation.
JULY 1975
VOL. 12 NO. I
55
CONTRACEPTION
RESULT Effect of propanolol hydrochloride and oxprenolol hydrochlorlde on matinginduced-ovulatlon Control group: the 6 members of this group accepted mating successfully. Examination at the 1st laparatomy revealed the presence of haemarrhagic Grafian follicles and corpora lutea in 5 out of the 6 cases. The 2nd laparotamy (after 12 days) shewed presence of implantation sites in 4 of the 6 cases and the average number of implantations was 3 in each horn and was mare or less parallel to the number of corpora lutea. In one animal no implantation site was detected and the ovaries contained lutelnized follicles as proved by microscopic examination. Propanolol hydrachlorlde treated group: all the female rabbits did not accept mating in all the exposure days after treatment with the drug. Laparotomies on the 14th and 23rd days proved absence of haemorrhagic follicles, corpora lutea and implantation sites in all the animals. Microscopic examination of the ovaries showed follicles in various stages of development but none had reached full maturity. No corpora lutea or atretlc follicles were demonstrated. The oxprenolol hydrochlorlde treated group: five animals out of the 6 refused mating in all the exposure days. One female accepted natural mating and deposition of sperms was proved by vaginal smear. Laparotomles in the 5 cases which refused mating revealed no evidence of ovulation or pregnancy. In the female which accepted mating, no implantation sites were detected and the ovaries contained no corpora lutea. Effect of prot0anolol hydrochlorlde andoxprenolol hydrochlorlde on chorlonlc gonadatrophln-induced-ovulatlon In this experiment injection of 200 I.U. of chorlonic gonadotrophln proved to be successful in inducing ovulation in all the female immature rabbits whether premedicated with physiological saline, oxprenolol hydrochloride or propanolol hydrochlorlde. This was demonstrated by laparatomy and ovarian biopsies in all the cases. Many developing follicles, several corpora lutea, and distended lutelnized follicles were found. Effect of propanolol hydrochlorlde and oxprenolol hydrochlorlde on ovulation induced by forced mating The technique of forced mating was effective in inducing ovulation and subsequent pregnancies in 5 out of the 6 members of the control group. The number of corpora lutea averaged 10 in each animal and the number of implantation sites 6 in each.
56
JULY 1975 VOL. 12 NO. 1
CONTRACEPTION
In the propanolol hydrochlorlde treated animals, all 6 female rabbits failed to ovulate or become pregnant as proved by the absence of corpora lutea and implantation sites during laporotomy. The number of implantation sites was markedly less than the number of corpora lutea in 2 animals of the oxprenolol group. In one of them, 6 corpora lutea were detected and only one Implantation slte, and In the other, 8 corpora lutea were found wlth 2 Implantation sites. DISCUSSION During these experiments, ovulation was induced by natural mating, forced mating and exogenous chorlonlc ganadetrophin. All 3 methods were effective in the control groups which makes deviation in drug treated rabbits significant. Propanolol hydrachlorlde in a dose of 1.5 mg/kg body weight prevented natural mating; the females were not receptive to male. The same dose was found to suppress ovulation in female rabbits exposed to forced mating. However, the drug did not neutralize the effect of a large d~e of exogenous ganadotrophln in inducing ovulation which excludes a direct effect on the ovaries at this dose level. It could however still interfere wlth the effect of a physiological level of LH. Prevention of ovulation after forced mating is probably due to inhibition of pituitary gonadotrophin release. This inhibition might be due to a direct effect on the pituitary or to interference wlth cortical or hypothalamlc control on the anterior pituitary. Oxprenolol hydrochlorlde at a dose of 1.5 mg/kg body weight did not prevent ovulation induced by forced mating or exogenous ganadotrophln. Therefore, the drug dld not interfere wlth Iiberatlon of endogenous gonadotraphln or neutrallze the exogenous gonadotrophln effect. On the other hand, oxprenolol prevented receptivity to male rabbits in 5 out of 6. Thls could be explained by the sedative and tranquillzlng effect of the drug which was observed in the animals used. On thls basis, its use was reported for various psychiatric indications (9). An interesting observation, which necessitates further investigation, was the fact that in 2 of the oxprenolol treated animals which became pregnant followlng forced mat|ng, the number of implantation sites were markedly less than corresponding corpora lutea. If thls observation is reproducible in a larger number of animals, it might be explalned by the effect of oxprenolol on ovum transport and implantatlon.
JULY 1975 VOL. 12 NO. 1
57
CONTRACEPTION REFERENCES 1.
Brambell, F.W.R.: Ovarian changes. In Marshall's Physiology of Reproduction, Vol. 1, Part 1, ed. by A.S.Parkes, pp. 397, 642. Longmans, London (1956).
2.
Fumagall, Z., Motta, P. and Calvieri, S.: The presence of smooth muscle cells in the ovary of several mammals as seen under the electron microscope. Experientla 27:682-683 (1971).
3.
Jacobowltz, D. and Wallach, E.E.: Histochemlcal and chemical studies of the autonomic innervatlan of the ovary. Endocrinology 81:1132-1139 (1967).
4.
O'Shea, J.D.: An ultrastruatural study of smooth muscle-llke cells in the theca extema of ovarian follicles in the rat. Anat. Rec. 167: 127-139 (1970).
5.
Owman, C., Rosengren, E. and Sioberg, N.O.: Origin of the admnerglc innervatian to the female genital tract of the mbblt." Life Scl. 5:1389-1396 (1966).
6.
Owman, C., Rosengmn, E. and Sjoberg, N.O.: Adrenerglc innervation of the human female reproductive organs: a hlstochemlcal and chemical investigation. Obstet. Gynec. 30:763-773 (1967).
7.
Rocemto, T., Jacobowltz, D. and Wallach, E.E.: Observations of spontaneous contmctlons of the cat ovary in vitro. Endocrinology 84:1336-1341 (1969).
8.
Virutamesen, P., Hlckok, R.L. and Wallach, E.E.: Local ovarian effects on human chorionlc gonadotmphln-lnduced-ovulatlon in the mbblt. Fert. Steril. 22:235-243 (1971).
9.
Volk, W., Bier, W., Braun, J.P., Gruter, W. and Spiegelberg, U.: Behandlung van erregten psychosen mlt einem Beta-Rezeptomn-Blocker (oxprenolol) in hoher Daslerung. Nervenarzt (D) 43, 9:491-492
(i~2).
58
JULY 1975 VOL. 12 NO. 1
ON
M.S. Foda, N.A. Darwlsh and A.A. Shafeek Obstetrics and Gynecology Department E.M. Sharaf and M.A. Zakl Pharmacelogy Deportment Faculty of Medicine, Cairo University
ABSTRACT The effect of two B-adrenerglc blocking agents, propanolol hydrochloride and oxprenolol hydrochlorlde, on ovulation in rabbits is studied. The first drug prevented receptivity to male and suppressed ovulation after forced mating. Oxprenolol hydrochlorlde showed a similar effect as regards receptivity to male but did not prevent ovulation after forced mating. Both drugs did not interfere wlth ovulation induced by large doses of exogenous gonadotrophin. The possible made of action of these drugs is discussed.
*Supported by
a
W H O grant.
Accepted for publ£cation May 8, 1975
JULY 1975
VOL. 12 NO. 1
53
CONTRACEPTION
I NTRO DUCTION The existence of smooth muscle fibres in the ovarian stroma has been documented in a number of mammalian species (1,2,3,4). Ruorescent hlstochemlcal techniques have shown the presence of adrenergic terminal fibres in the ovarian stromata of cat, man, rabbit and monkey (3,5,6). The smooth muscle in the ovary end its apparent adrenergic innervatlan suggests that, at least in some species, neurogenlc contraction of the muscle may cause or facilitate ovum release (7). In the rabbit it was found that the 13-adrenergic blocker, dlbenzylene, administered locally to the ovary in conjunction with artificially induced ovulation caused a reduction in the number of ruptured follicles with a reciprocal increase in the number of mature follicles (8). It was proposed that dibenzylene inhibited ovarian smooth muscle cantractlon necessary for ovum ejection. The effect of g-adrenergic blockers in this regard has not yet been explored. The present work was undertaken to study: a) The effect of two ~]-adrenerglc blocking agents, namely, propanolol hydrochloride (Indral, ICI)and oxprenolol hydrochlorlde (Tronslcor, Ciba), on the process of ovulation in mature rabbits; and b) The effect of the same compounds an chorlanic gonodotrophln induced ovulation in immature rabbits. MATERIAL AND METHODS Effect of prapanolol hydrochlorlde and oxprenolol hydrochlorlde on mating induced ovulation in mature rabbits The study was conducted an mature female rabbits of the Boscat strain which had been isolated individually for 3 to 4 weeks to prevent the occurrence of spontaneous ovulation or pseudopregnancy. The animals were not less than one year old and the average weight was 2.5 kg. The animals were divided into 3 groups of 6 members each. The rabbits of the first group were injected intravenously through the marginal ear vein with propanolol hydrochlorlde at a dose of 1.5 mg/kg body weight per day for 7 consecutive days, so as to give enough time for the drug to act through different parameters. The members of the 2nd group were injected with oxprenolol hydrochlorlde by the same route and at the same dose level for the same period. The 3rd group was injected for 7 consecutive days wlth physiological saline as controls. On the 8th day, and for four consecutive days, the animals of the three graupswere exposed to male partners 15 minutes after the daily injection which was continued until day 11. Vaginal smears were obtained from the animals which accepted mating to demonstrate sperms in the vagina. Laparatamy was performed an the 14th day from
54
JULY 1975 VOL. 12 NO. 1
CONTRACEPTION
the start of the experiment. The ovaries ware inspected for follicles and corpora lutea and the uterine homs for early sites of implantation. The abdomen was then closed and each animal was returned to its cage. A second laparotomy was performed on the 23rd day to differentiate between true and pseudopregnancy and to obtain ovarian biopsies (Figure). Intravenous Pentobarbltal (45 mg/~)was used for anaesthesia. mating I
I
I
2 3 4
!
1
I
I
5 6 7 8 9
daily injection Figure.
I
I
I
I
10 11 I
12
|
I
I
13 14 ,
I
I
I
I
!
II
I
I
15 16 17 18 19 20 21 22 23
I
|st laparotomy
I
J
2nd laparo amy
A graphic representation of the study protocol.
Effect of 13-adrenerglc blockers on chorionlc gonadotrophln induced ovulation In immature female rabbits This study was conducted on immature female rabbits of the white Giza strain. Their ages ranged between 5 and 6 months, and the average weight was 1.5 kg. The animals were divided into 3 groups, each of 8 animals. The 1st group was iniected with physiological saline to be considered as the control group. The members of the 2nd and 3rd groups were injected through the marginal ear vein with 1.5 mg per kg body weight of propanolol hydrochlorlde or oxpmnolol hydrochlorlde, respectively. After 15 minutes, each member of the 3 groups was injected by the same route with 200 I.U. chorlonlc gonodotrophin. Lapamtomy was performed on the 3rd day and ovaries were inspected for haemorrhaglc follicles and ovarian biopsies were obtained. Effect of 13-adrenerg|c blockers on forced mating induced ovulation in mature female rabbits ~ A new batch of 18 mature female rabbits cf the Boscot strain was used for the study. Their ages were above one year and the average weight was 2.5 kg. After individual isolation of the female rabbits for 2 weeks, they were divided into 3 groups of 6 members each. The 1st group was used as the control; physiological saline was injected I.V. daily for 7 consecutive days before exposure to forced mating. On the 8th day, 15 minutes after the injection, forced mating was practiced. This procedure was repeated on the 9th, 10th and 11th days. The 2nd and 3rd groups were treated similarly but using the 2 test drugs instead of physiological saline at a dose of 1.5 mg/kg body ~elght. Twelve days elapsed between the last day of mating and laparotomy. During the surgery, the ovaries were inspected for the presence of mature follicles and corpora lutea, the uterine hams for implantation sites. Biopsies were obtained for further microscopic evaluation.
JULY 1975
VOL. 12 NO. I
55
CONTRACEPTION
RESULT Effect of propanolol hydrochloride and oxprenolol hydrochlorlde on matinginduced-ovulatlon Control group: the 6 members of this group accepted mating successfully. Examination at the 1st laparatomy revealed the presence of haemarrhagic Grafian follicles and corpora lutea in 5 out of the 6 cases. The 2nd laparotamy (after 12 days) shewed presence of implantation sites in 4 of the 6 cases and the average number of implantations was 3 in each horn and was mare or less parallel to the number of corpora lutea. In one animal no implantation site was detected and the ovaries contained lutelnized follicles as proved by microscopic examination. Propanolol hydrachlorlde treated group: all the female rabbits did not accept mating in all the exposure days after treatment with the drug. Laparotomies on the 14th and 23rd days proved absence of haemorrhagic follicles, corpora lutea and implantation sites in all the animals. Microscopic examination of the ovaries showed follicles in various stages of development but none had reached full maturity. No corpora lutea or atretlc follicles were demonstrated. The oxprenolol hydrochlorlde treated group: five animals out of the 6 refused mating in all the exposure days. One female accepted natural mating and deposition of sperms was proved by vaginal smear. Laparotomles in the 5 cases which refused mating revealed no evidence of ovulation or pregnancy. In the female which accepted mating, no implantation sites were detected and the ovaries contained no corpora lutea. Effect of prot0anolol hydrochlorlde andoxprenolol hydrochlorlde on chorlonlc gonadatrophln-induced-ovulatlon In this experiment injection of 200 I.U. of chorlonic gonadotrophln proved to be successful in inducing ovulation in all the female immature rabbits whether premedicated with physiological saline, oxprenolol hydrochloride or propanolol hydrochlorlde. This was demonstrated by laparatomy and ovarian biopsies in all the cases. Many developing follicles, several corpora lutea, and distended lutelnized follicles were found. Effect of propanolol hydrochlorlde and oxprenolol hydrochlorlde on ovulation induced by forced mating The technique of forced mating was effective in inducing ovulation and subsequent pregnancies in 5 out of the 6 members of the control group. The number of corpora lutea averaged 10 in each animal and the number of implantation sites 6 in each.
56
JULY 1975 VOL. 12 NO. 1
CONTRACEPTION
In the propanolol hydrochlorlde treated animals, all 6 female rabbits failed to ovulate or become pregnant as proved by the absence of corpora lutea and implantation sites during laporotomy. The number of implantation sites was markedly less than the number of corpora lutea in 2 animals of the oxprenolol group. In one of them, 6 corpora lutea were detected and only one Implantation slte, and In the other, 8 corpora lutea were found wlth 2 Implantation sites. DISCUSSION During these experiments, ovulation was induced by natural mating, forced mating and exogenous chorlonlc ganadetrophin. All 3 methods were effective in the control groups which makes deviation in drug treated rabbits significant. Propanolol hydrachlorlde in a dose of 1.5 mg/kg body weight prevented natural mating; the females were not receptive to male. The same dose was found to suppress ovulation in female rabbits exposed to forced mating. However, the drug did not neutralize the effect of a large d~e of exogenous ganadotrophln in inducing ovulation which excludes a direct effect on the ovaries at this dose level. It could however still interfere wlth the effect of a physiological level of LH. Prevention of ovulation after forced mating is probably due to inhibition of pituitary gonadotrophin release. This inhibition might be due to a direct effect on the pituitary or to interference wlth cortical or hypothalamlc control on the anterior pituitary. Oxprenolol hydrochlorlde at a dose of 1.5 mg/kg body weight did not prevent ovulation induced by forced mating or exogenous ganadotrophln. Therefore, the drug dld not interfere wlth Iiberatlon of endogenous gonadotraphln or neutrallze the exogenous gonadotrophln effect. On the other hand, oxprenolol prevented receptivity to male rabbits in 5 out of 6. Thls could be explained by the sedative and tranquillzlng effect of the drug which was observed in the animals used. On thls basis, its use was reported for various psychiatric indications (9). An interesting observation, which necessitates further investigation, was the fact that in 2 of the oxprenolol treated animals which became pregnant followlng forced mat|ng, the number of implantation sites were markedly less than corresponding corpora lutea. If thls observation is reproducible in a larger number of animals, it might be explalned by the effect of oxprenolol on ovum transport and implantatlon.
JULY 1975 VOL. 12 NO. 1
57
CONTRACEPTION REFERENCES 1.
Brambell, F.W.R.: Ovarian changes. In Marshall's Physiology of Reproduction, Vol. 1, Part 1, ed. by A.S.Parkes, pp. 397, 642. Longmans, London (1956).
2.
Fumagall, Z., Motta, P. and Calvieri, S.: The presence of smooth muscle cells in the ovary of several mammals as seen under the electron microscope. Experientla 27:682-683 (1971).
3.
Jacobowltz, D. and Wallach, E.E.: Histochemlcal and chemical studies of the autonomic innervatlan of the ovary. Endocrinology 81:1132-1139 (1967).
4.
O'Shea, J.D.: An ultrastruatural study of smooth muscle-llke cells in the theca extema of ovarian follicles in the rat. Anat. Rec. 167: 127-139 (1970).
5.
Owman, C., Rosengren, E. and Sioberg, N.O.: Origin of the admnerglc innervatian to the female genital tract of the mbblt." Life Scl. 5:1389-1396 (1966).
6.
Owman, C., Rosengmn, E. and Sjoberg, N.O.: Adrenerglc innervation of the human female reproductive organs: a hlstochemlcal and chemical investigation. Obstet. Gynec. 30:763-773 (1967).
7.
Rocemto, T., Jacobowltz, D. and Wallach, E.E.: Observations of spontaneous contmctlons of the cat ovary in vitro. Endocrinology 84:1336-1341 (1969).
8.
Virutamesen, P., Hlckok, R.L. and Wallach, E.E.: Local ovarian effects on human chorionlc gonadotmphln-lnduced-ovulatlon in the mbblt. Fert. Steril. 22:235-243 (1971).
9.
Volk, W., Bier, W., Braun, J.P., Gruter, W. and Spiegelberg, U.: Behandlung van erregten psychosen mlt einem Beta-Rezeptomn-Blocker (oxprenolol) in hoher Daslerung. Nervenarzt (D) 43, 9:491-492
(i~2).
58
JULY 1975 VOL. 12 NO. 1
