ENDOCRINE RESEARCH COMMUNICATIONS, 4 (1), 35-44
(1977)
SUBCUTANEOUS MELATONIN IMPLANTS INHIBIT REPRODUCTIVE ATROPHY IN MALE HAMSTERS INDUCED BY DAILY MELATONIN INJECTIONS Russel J. Reiter, P. Kevin Rudeen, Jeffrey W. Sackman, Mary K. Vaughan, Linda Y. Johnson, and John C. Little Department of Anatomy The University of Texas Health Science Center at San Antonio 7703 Floyd Curl Drive San Antonio, Texas 78284
Endocr Res Downloaded from informahealthcare.com by Nyu Medical Center on 10/04/13 For personal use only.
Abstract Daily afternoon (at 7 p.rn.1 injections of melatonin ( 2 5 pg in oil) into adult male hamsters for 50 days led to atrophy of the testes and accessory sex organs (seminal vesicles and coagulating glands) and in a significant depression in pituitary LH and prolactin content and concentration. These actions of melatonin were prevented if the animals had been pinealectomized before the daily melatonin injections were begun. Likewise, if hamsters received a weekly subcutaneous implant of melatonin in beeswax (1 mg melatonin in 24 mg beeswax) the daily melatonin injections failed to inhibit the growth of the reproductive organs and to depress pituitary LH and prolactin levels. Beeswax by itself had no such effect. Introduction Light deprivation in male hamsters is accompanied by involution of the pituitary-gonadal axis.
If the pineal gland is removed
from these animals, darkness can no longer induce the gonads to atrophy !l,
2).
Likewise, when melatonin is chronically administered
to light-deprived hamsters by means of a subcutaneous depot,
35 Copyright 0 1977 hy Marcel Dekker, Inc All Rights Reserved. Neither this work nor any p ~ r t may be reproduced or transmitted in a n y f o r m or by any meahs. electronlc or mechanical. includmp photocopying, microfilming, and recording, o r by a n y information storage and retrieval system, without permission in writing f r o m t h e publisher.
REITER ET AL.
36
darkness cannot cause involution of the sexual apparatus (2, 3 ) . That is, chronic melatonin treatment mimics the effects of pinealectomy; both procedures prevent darkness from inhibiting the function of the reproductive system. This has been referred to as the conntar antigonadotrophic action of melatonin. This is in marked contrast to the influence of acute melatonin administration on the reproductive organs of male hamsters kept in long daily photoperiods. The daily injection of melatonin into hamsters kept in long photoperiods (LD 14:lO) induces reproductive
Endocr Res Downloaded from informahealthcare.com by Nyu Medical Center on 10/04/13 For personal use only.
involution much like exposure to darkness.
However, for melatonin
to be effective in inhibiting reproduction it must be administered near the end of the light phase of the LD cycle (4, 5 ) . Interestingly, this antigonadotrophic action of acute melatonin injections is prevented by surgical removal of the pineal gland (4, 5 ) .
Since both pinealectomy and chronic melatonin treatment
(via subcutaneous deposits) prevent darkness from inducing gonadal regression and since pinealectomy also prevents the antigonadotrophic effects of acute melatonin injections, the obvious question that arises is, would chronic melatonin adminstration (via subcutaneous pellets) prevent the inhibitory action of acute melatonin injections given late in the day? The purpose of the following experiment was to examine this possibility. Materials and Methods Fifty young adult male hamsters (60-80gms) were used for this investigation. The animals were housed 4-6 per plastic cage in a room automatically illuminated for 14 hours daily (LD 14:lO).
37
MELATONIN AND REPRODUCTION Lights were on from 6 a.m. to 8 p-m. Food and water were continuously available. The animals were divided into the following 5 groups: l), control hamsters (8 each) injected subcutaneously with 0.1 cc peanut oil daily at 7 p.m.;
2).
hamsters (10 each) that received
daily injections of melatonin (25 ug in 0.1 cc oil) at 7 p.m.; 3 ) , hamsters (12 each) that received daily melatonin injections and were pinealectomized; 4 ) , hamsters (8 each) that received daily injections of melatonin and a weekly subcutaneous implant of a
Endocr Res Downloaded from informahealthcare.com by Nyu Medical Center on 10/04/13 For personal use only.
beeswax pellet; 5), hamsters (12 each) that received daily injections of melatonin and a weekly subcutaneous implant of a melatoninbeeswax pellet. Pinealectomies were done on hamsters anesthetized with ether 1 week before the beginning of the treatment period.
Melatonin for
the injections was mixed with peanut oil to a final concentration of 250 pg/cc.
This solution was prepared fresh every 2 or 3 days.
The injections were given daily on the dorsum of the back at 7 p-m. The preparation of the melatonin-beeswax pellets has been described elswhere
(2).
Each pellet contained 1 mg melatonin in 24 mg beeswax.
The beeswax pellets weighed approximately 25 mg.
Pellets were
implanted subcutaneously once per week while the animals were anesthetized with ether. All animals were treated for 50 consecutive days after which they were necropsied.
Following decapitation, trunk blood samples
were collected in heparinized tubes.
Anterior pituitary, testicular
and accessory organ (seminal vesicles and coagulating glands)
38
REITER ET AL.
weights were recorded.
Pituitaries were homogenized by sonication
and these, along with the plasma samples, were stored frozen for later hormone analysis.
Pituitary and plasma luteinizing hormone
(LH) and prolactin levels were estimated by double antibody
radioimmunoassays using published techniques (6, 7). Reagents for the assays were provided by the Rat Hormone Distribution Program, NIAMDD.
Because of lack of parallelism between rat and hamster
prolactin inhibition curves, hamster prolactin values are expressed in terms of a pool of standard hamster anterior pituitaries
Endocr Res Downloaded from informahealthcare.com by Nyu Medical Center on 10/04/13 For personal use only.
(SHAP).
Statistical analyses were carried out using an ANOVA and
a Student t test for differences between means. Results The body weights of the hamsters were not altered significantly by any of the experimental treatments (Table I).
Daily melatonin
injections at 7 p.m. caused a highly significant reduction in the weights of both the testes and the accessory sex organs (Fig. 1). The inhibitory effect of daily melatonin injections on the gonads and adnexa were prevented by removal of the pineal gland.
The
weekly implantation of a beeswax pellet into hamsters did not interfere with the antigonadotrophic activity of daily melatonin injections. However, animals that were treated chronically with melatonin by means of a melatonin-beeswax pellet had gonads and accessory organs equivalent in size to those of oil-treated controls even though they had received daily melatonin injections at 7 p.m. Stated in anoeher way, chronic melatonin treatment prevented the
MELATONIN AND REPRODUCTION
39 TABLE I
Endocr Res Downloaded from informahealthcare.com by Nyu Medical Center on 10/04/13 For personal use only.
Effect of various treatments on body weights (BW), absolute (mg) and relative (mg/lOOg BW) pituitary gland weights and plasma LH and prolactin levels in adult male hamsters.
Treatment (N)
BW 4
Ant. Pit. mg (mg/lOOg BW)
Plasma Hormones LH Prolactin ng/ml ygSHAP/ml
Oil-treated controls (8)
13324
2.942.17 (2.212.13)
5.46T.22
1.7Ot.18
Melatonin injections (10)
14528
2-84?.12 (1.962.09*)
2.11?31**
0.40+10**
Melatonin injections; pinealec tomy (12)
130f7
3.06f.10 (2.35f.08)
4.14t.36
0.81+.08**
Melatonin injections; beeswax pellet (8)
14427
2.872.18 (1.99*.11*)
3.05t.43
0.27+_.05**
Melatonin injections; 146k3 beeswax-melatonin pellet (12)
3.13f.15 (2.142-08)
3.98k.24
0.54?.05**
*p
(1977)
SUBCUTANEOUS MELATONIN IMPLANTS INHIBIT REPRODUCTIVE ATROPHY IN MALE HAMSTERS INDUCED BY DAILY MELATONIN INJECTIONS Russel J. Reiter, P. Kevin Rudeen, Jeffrey W. Sackman, Mary K. Vaughan, Linda Y. Johnson, and John C. Little Department of Anatomy The University of Texas Health Science Center at San Antonio 7703 Floyd Curl Drive San Antonio, Texas 78284
Endocr Res Downloaded from informahealthcare.com by Nyu Medical Center on 10/04/13 For personal use only.
Abstract Daily afternoon (at 7 p.rn.1 injections of melatonin ( 2 5 pg in oil) into adult male hamsters for 50 days led to atrophy of the testes and accessory sex organs (seminal vesicles and coagulating glands) and in a significant depression in pituitary LH and prolactin content and concentration. These actions of melatonin were prevented if the animals had been pinealectomized before the daily melatonin injections were begun. Likewise, if hamsters received a weekly subcutaneous implant of melatonin in beeswax (1 mg melatonin in 24 mg beeswax) the daily melatonin injections failed to inhibit the growth of the reproductive organs and to depress pituitary LH and prolactin levels. Beeswax by itself had no such effect. Introduction Light deprivation in male hamsters is accompanied by involution of the pituitary-gonadal axis.
If the pineal gland is removed
from these animals, darkness can no longer induce the gonads to atrophy !l,
2).
Likewise, when melatonin is chronically administered
to light-deprived hamsters by means of a subcutaneous depot,
35 Copyright 0 1977 hy Marcel Dekker, Inc All Rights Reserved. Neither this work nor any p ~ r t may be reproduced or transmitted in a n y f o r m or by any meahs. electronlc or mechanical. includmp photocopying, microfilming, and recording, o r by a n y information storage and retrieval system, without permission in writing f r o m t h e publisher.
REITER ET AL.
36
darkness cannot cause involution of the sexual apparatus (2, 3 ) . That is, chronic melatonin treatment mimics the effects of pinealectomy; both procedures prevent darkness from inhibiting the function of the reproductive system. This has been referred to as the conntar antigonadotrophic action of melatonin. This is in marked contrast to the influence of acute melatonin administration on the reproductive organs of male hamsters kept in long daily photoperiods. The daily injection of melatonin into hamsters kept in long photoperiods (LD 14:lO) induces reproductive
Endocr Res Downloaded from informahealthcare.com by Nyu Medical Center on 10/04/13 For personal use only.
involution much like exposure to darkness.
However, for melatonin
to be effective in inhibiting reproduction it must be administered near the end of the light phase of the LD cycle (4, 5 ) . Interestingly, this antigonadotrophic action of acute melatonin injections is prevented by surgical removal of the pineal gland (4, 5 ) .
Since both pinealectomy and chronic melatonin treatment
(via subcutaneous deposits) prevent darkness from inducing gonadal regression and since pinealectomy also prevents the antigonadotrophic effects of acute melatonin injections, the obvious question that arises is, would chronic melatonin adminstration (via subcutaneous pellets) prevent the inhibitory action of acute melatonin injections given late in the day? The purpose of the following experiment was to examine this possibility. Materials and Methods Fifty young adult male hamsters (60-80gms) were used for this investigation. The animals were housed 4-6 per plastic cage in a room automatically illuminated for 14 hours daily (LD 14:lO).
37
MELATONIN AND REPRODUCTION Lights were on from 6 a.m. to 8 p-m. Food and water were continuously available. The animals were divided into the following 5 groups: l), control hamsters (8 each) injected subcutaneously with 0.1 cc peanut oil daily at 7 p.m.;
2).
hamsters (10 each) that received
daily injections of melatonin (25 ug in 0.1 cc oil) at 7 p.m.; 3 ) , hamsters (12 each) that received daily melatonin injections and were pinealectomized; 4 ) , hamsters (8 each) that received daily injections of melatonin and a weekly subcutaneous implant of a
Endocr Res Downloaded from informahealthcare.com by Nyu Medical Center on 10/04/13 For personal use only.
beeswax pellet; 5), hamsters (12 each) that received daily injections of melatonin and a weekly subcutaneous implant of a melatoninbeeswax pellet. Pinealectomies were done on hamsters anesthetized with ether 1 week before the beginning of the treatment period.
Melatonin for
the injections was mixed with peanut oil to a final concentration of 250 pg/cc.
This solution was prepared fresh every 2 or 3 days.
The injections were given daily on the dorsum of the back at 7 p-m. The preparation of the melatonin-beeswax pellets has been described elswhere
(2).
Each pellet contained 1 mg melatonin in 24 mg beeswax.
The beeswax pellets weighed approximately 25 mg.
Pellets were
implanted subcutaneously once per week while the animals were anesthetized with ether. All animals were treated for 50 consecutive days after which they were necropsied.
Following decapitation, trunk blood samples
were collected in heparinized tubes.
Anterior pituitary, testicular
and accessory organ (seminal vesicles and coagulating glands)
38
REITER ET AL.
weights were recorded.
Pituitaries were homogenized by sonication
and these, along with the plasma samples, were stored frozen for later hormone analysis.
Pituitary and plasma luteinizing hormone
(LH) and prolactin levels were estimated by double antibody
radioimmunoassays using published techniques (6, 7). Reagents for the assays were provided by the Rat Hormone Distribution Program, NIAMDD.
Because of lack of parallelism between rat and hamster
prolactin inhibition curves, hamster prolactin values are expressed in terms of a pool of standard hamster anterior pituitaries
Endocr Res Downloaded from informahealthcare.com by Nyu Medical Center on 10/04/13 For personal use only.
(SHAP).
Statistical analyses were carried out using an ANOVA and
a Student t test for differences between means. Results The body weights of the hamsters were not altered significantly by any of the experimental treatments (Table I).
Daily melatonin
injections at 7 p.m. caused a highly significant reduction in the weights of both the testes and the accessory sex organs (Fig. 1). The inhibitory effect of daily melatonin injections on the gonads and adnexa were prevented by removal of the pineal gland.
The
weekly implantation of a beeswax pellet into hamsters did not interfere with the antigonadotrophic activity of daily melatonin injections. However, animals that were treated chronically with melatonin by means of a melatonin-beeswax pellet had gonads and accessory organs equivalent in size to those of oil-treated controls even though they had received daily melatonin injections at 7 p.m. Stated in anoeher way, chronic melatonin treatment prevented the
MELATONIN AND REPRODUCTION
39 TABLE I
Endocr Res Downloaded from informahealthcare.com by Nyu Medical Center on 10/04/13 For personal use only.
Effect of various treatments on body weights (BW), absolute (mg) and relative (mg/lOOg BW) pituitary gland weights and plasma LH and prolactin levels in adult male hamsters.
Treatment (N)
BW 4
Ant. Pit. mg (mg/lOOg BW)
Plasma Hormones LH Prolactin ng/ml ygSHAP/ml
Oil-treated controls (8)
13324
2.942.17 (2.212.13)
5.46T.22
1.7Ot.18
Melatonin injections (10)
14528
2-84?.12 (1.962.09*)
2.11?31**
0.40+10**
Melatonin injections; pinealec tomy (12)
130f7
3.06f.10 (2.35f.08)
4.14t.36
0.81+.08**
Melatonin injections; beeswax pellet (8)
14427
2.872.18 (1.99*.11*)
3.05t.43
0.27+_.05**
Melatonin injections; 146k3 beeswax-melatonin pellet (12)
3.13f.15 (2.142-08)
3.98k.24
0.54?.05**
*p
