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Differential effects of bromocriptine treatment on LH release and copulatory behavior in hyperprolactinemic male rats
HORMONES
AND
BEHAVIOR
15,
436-450 (1981)
Differential Effects of Bromocriptine Treatment on LH Release and Copulatory Behavior in Hyperprolactinemic Male Rats P. C. DOHERTY, A. BARTKE, AND M. S. SMITH* Departments of Anatomy and Obstetrics and Gynecology, The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78284, and *Department of Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261 Hyperprolactinemia (hyperPRL) induced by grafting four pituitary glands under the kidney capsule suppresses copulatory behavior in male rats and sexually naive male mice. In mice sexual experience attenuates the suppressive effects of hyperPRL on mating behavior, thus a comparison of the behavioral consequences of inducing hyperPRL in sexually naive and experienced mate rats was undertaken. Hyperprolactinemia had a significant suppressive effect on mating behavior in both groups of animals. Experienced animals showed deficits in all parameters studied except mount frequency and postejaculatory interval, while naive animals differed from respective controls only in mount latency, intromission latency, and intromission frequency. To determine if the inhibition of chronically elevated prolactin (PRL) levels would reverse the suppression of gonadotropin secretion and copulatory behavior in hyperprolactinemic animals, the effects of bromocriptine (CB-154) administration on plasma hormone levels and mating behavior were examined in pituitary-grafted and control rats. Bromocriptine treatment (1 mg/day for 14 days) led to increases in sexual activity in both the sham-operated and grafted animals. In the grafted animals, plasma PRL was reduced and plasma LH significantly increased in the CB-W-treated animals when compared to oil-treated controls. In sham-operated animals, CB-154 produced no significant changes in plasma LH or FSH despite the suppressed PRL levels. These results indicate that (1) hyperPRL induced by pituitary grafts can cause deficits in mating behavior in mate rats despite previous sexual experience, and (2) while CB-154 may be acting through other mechanisms to stimulate copulatory behavior, the reduction of chronically elevated PRL levels due to CB-154 treatment is responsible for reversal of the suppressive effects of hyperPRL on LH secretion.
In the human, pathologic elevation of plasma prolactin (PRL) levels, hyperprolactinemia (hyperPRL), is usually the result of a hypersecreting pituitary adenoma. In women, it is often associated with amenorrhea, anovulatory sterility, and, somewhat less frequently, galactorrhea (Ja436 0018-506x/81/040436-15$01.00/0 Copyright All rights
CD 1981 by Academic Press, Inc. of reproduction in any fom reserved.
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cobs, 1976). Several reports indicate that hyperPRL can also interfere with sexual function in men (Boyar, Kapen, Finkelstein, Perlow, Sassin, Fukushima, Weitzman, and Hellman, 1974; Tolis and Van Vliet, 1976; Thorner and Besser, 1977). Reduced gonadotropin levels, suppressed pituitary responsiveness to LHRH, and various degrees of hypogonadism are among the symptoms described; however, the most common complaints associated with hyperPRL in men are impotence and/or decreased libido. Recently a number of animal models have been described for the study of the effects of elevated circulating levels of PRL on the hypothalamo-pituitary-testicular axis. Male rats bearing tumors from PRL-producing cell lines have been reported to exhibit testicular atrophy (Fang, Refetoff, and Rosenfeld, 1974), or inhibition of the release of pituitary LH and hypothalamic LHRH associated with high serum PRL levels (Gil-Ad, Locatelli, Cocchi, Carminati, Arezzini, and Mtiller, 1978; Hodson, Simpkins, Pass, Aylsworth, Steger, and Meites, 1980). Male rats rendered hyperprolactinemic by transplantation of several isogeneic pituitary glands display reductions in plasma gonadotropin levels but normal plasma levels of testosterone (Bartke, Smith, Michael, Peron, and Dalterio, 1977; McNeilly, Sharpe, Davidson, and Fraser, 1978) and suppression of the postcastration increases in serum LH (Grandison, Hodson, Chen, Advis, Simpkins, and Meites, 1977). In addition, male rats with pituitary transplants exhibit impaired copulatory behavior patterns, including increased latencies to mount, intromit, and ejaculate, and fewer mounts, intromissions, and ejaculations during the test period (Svare, Bartke, Doherty, Mason, Michael, and Smith, 1979). Similar suppressive effects of elevated PRL levels on the copulatory behavior of male rabbits have also been reported (Hartmann, Endroczi, and Lissak, 1966). In male mice, the suppressive effects of hyperPRL on copulatory behavior attenuate with further sexual experience (Svare et al., 1979). Since previous studies in the rat utilized animals which were allowed sexual experience only after the induction of hyperPRL, one of the purposes of this investigation was to determine if sexual experience prior to surgery for pituitary transplantation can modify the effects of this treatment on copulatory behavior. Bromocriptine (CB-154), a dopamine agonist with prolactin-inhibiting capabilities, has been shown to be a potent agent in the relief of symptoms of pathologic hyperPRL (Thorner and Besser, 1978). Thus, bromocriptine treatment can result in decreased peripheral PRL levels and restored sexual function in hyperprolactinemic men (Ambrosi, Bara, Travaglini, Weber, Beck-Peccoz, Rondena, Elli, and Faglia, 1977; Franks, Jacobs, Martin, and Nabarro, 1978; Buvat, Asfour, Buvat-Herbaut, and Fossati, 1978). Bromocriptine treatment of rats bearing tumors of H&producing
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cell lines can reverse the reported inhibition of LHRH release from the hypothalamus (Gil-Ad ef al., 1978). The present study was undertaken to determine if bromocriptine treatment can reverse the suppressive effects of hyperprolactinemia on gonadotropin secretion and copulatory behavior in animals bearing pituitary grafts, and whether the effects of bromocriptine can be attributed to the suppression of chronically elevated PRL levels or a direct effect of bromocriptine itself. GENERAL METHODS Inbred CD-F rats purchased from Charles River Breeding Laboratories were maintained on a 14-hr light: IO-hr dark lighting schedule, with constant access to food and water. Male rats were rendered hyperprolactinemic by grafting whole pituitaries from four adult CD-F females under the kidney capsules (two pituitaries per kidney) or were sham operated. Animals were bled from the jugular vein under light ether anesthesia for determinations of plasma levels of PRL, LH, and FSH. Tests of copulatory behavior were performed between 3 and 6 hr after the onset of darkness under dim red illumination from a 25-W red light. Tests were conducted in neutral chambers consisting of IO-gal aquaria (25 x 47 x 29 cm) with sawdust on the floor. Each male was allowed an adaptation period of 30 min prior to introduction of a receptive female into the observation chamber. Adult ovariectomized females rendered behaviorally estrous by injections of estradiol benzoate (10 t&O.1 ml sesame oil) 48 hr prior to testing, and progesterone (500 t&O. 1 ml sesame oil) 4 hr prior to testing, served as stimulus partners. Each test lasted for a period of 1 hr. The following measures of copulatory behavior were recorded for each male: mount latency (ML), mounts with pelvic thrusting, intromission latency (IL), intromission responses, and ejaculation responses. In addition the following measures of masculine sexual behavior were calculated for the first copulatory series of each test: ejaculation latency (EL), time from the first intromission to ejaculation; postejaculatory interval (PEI), time from the first ejaculation to the first intromission of the next series; mount frequency (MF), the number of mounts prior to the first ejaculation; intromission frequency (IF), the number of intromissions prior to the first ejaculation; intromission ratio, the number of intromissions divided by the number of mounts prior to the first ejaculation; and the intromission rate (Urnin), the intromission frequency divided by the ejaculation latency. For the sexually experienced animals, test scores were evaluated using criteria adapted from Damassa, Smith, Tennent, and Davidson (1977). Thus, if IL > 15 min, EL > 30 min, or PEI > 15 min, the test was repeated. Animals were tested three times on alternate days, or until the
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first successful test occurred. Most animals achieved these criteria on the first test. If after the third test these criteria were not met, the test in which the best performance was achieved was recorded as the score for that animal. Test scores for the sexually naive animals, and those for Days 7 and 14 of bromocriptine treatment represent a single 1-hr test of copulatory behavior. Serum LH concentrations were assessed by the ovine-ovine radioimmunoassay system described previously (Niswender, Midgley, Monroe, and Reichert, 1968). The limit of detection of the assay was 5 q/ml, the intrassay variation, 6.2%, and the interassay variation, 7.4%. Plasma LH concentrations were expressed in terms of the NIAMDD rat LH-RP-1 standard (0.03 x NIH-LH-Sl). Follicle stimulating hormone concentrations were determined with the radioimmunoassay materials provided by the NIAMDD. Using the rat reference preparation (NIAMDD-RP-1, 2 IU/mg), the limit of detection of the assay was 100 r&ml. The coefficients of variation of this assay were 4.3% for intraassay variation and 5.8% for interassay variation. Plasma prolactin concentrations were determined by the NIAMDD rat prolactin radioimmunoassay and expressed in terms of the NIAMDD-RP-1 standard (11 IU/mg). The limit of detection of the assay was 3 r&ml, and the intraassay and interassay variations were 7.6 and 8.9%, respectively. Experiment Z To determine if the suppressive effects of hyperPRL on copulatory behavior of male rats are independent of previous sexual experience, 42 males were divided into four groups as follows: (a) 10 sexually naive males which received transplants of four whole pituitaries at the age of 90-95 days as previously described; (b) 11 sexually naive males which underwent sham surgery; (c) 10 sexually experienced males which received transplants of four whole pituitaries at the age of 100-105 days; and (d) 11 sexually experienced males which underwent sham surgery. When the males were 75 days of age, sexual experience was allowed by individually housing each of the animals with an intact cycling female until sperm was found in the vaginal smears and by testing for copulatory behavior. The results of the tests also served as the basis for assignment of individual animals to grafted and sham-operated groups to allow similar distribution of highly active copulators and less vigorous copulators. Prior to grafting, the experienced animals had ejaculated on three separate occasions as determined by the presence of sperm in vaginal smears or by direct observation in tests of copulatory behavior. On Day 24 after surgery, further tests of copulatory behavior were begun. Statistics. Statistical analysis was performed using data from only those animals which exhibited the specific behavioral parameter. No values were assigned to animals which did not perform. Comparisons
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were made only between the grafted and sham-operated animals in either the naive or the experienced groups, but not across all groups. Differences were considered significant at the 0.05 level using a two-tailed Mann-Whitney U test. Experiment
II
To determine if the suppression of chronically elevated PRL levels would be accompanied by improved copulatory behavior and increased plasma gonadotropin levels in the grafted rats, bromocriptine treatment was initiated. One month elapsed between Experiment I and Experiment II. During this time, male rats from the first experiment were allowed further sexual experience as described above. Animals were observed for copulatory behavior in a 1-hr test and assigned to one of four treatment groups with an equal number of the originally sexually naive and experienced animals assigned to each of the groups. The results of this test of copulatory behavior served as the pretreatment value. Bromocriptine treatment (1 mg/day in 0.1 ml of sesame oil) was administered for 15 days in a group of sham-operated animals (n = 10) and grafted animals (n = lo), while additional groups of sham-operated (n = 10) and grafted males (n = 10) received only vehicle. Tests of copulatory behavior were performed on Days 7 and 14 of treatment. Injections were given 12 hr prior to the time of testing. Plasma samples for determination of PRL, LH, and FSH were obtained from the jugular vein under light ether anesthesia 1 day prior to commencement of treatment, on Day 15 of treatment 12 hr after the last injection, and 2 weeks after the cessation of treatment. To decrease diurnal variability in responsiveness to drug treatment, copulatory behavior, and plasma hormone levels, the experimental procedures were staggered over S-day intervals (two animals from each treatment group/day), with all animals undergoing experimental manipulations at approximately the same time of day throughout the course of this experiment. In addition, to determine the degree of suppression achieved over the 24-hr period between injections, bromocriptine treatment was resumed for 3 days, as previously described, 1 month after the initial treatment period and the animals were bled 24 hr after the last injection. Siutistics. The effects of bromocriptine were assessed by comparing the behavior of the oil-treated vs the bromocriptine-treated animals within the grafted or sham-operated groups. Differences were considered significant at the 0.05 level using a two-tailed Mann-Whitney U test. For plasma hormone levels, significant treatment effects were determined by analysis of variance, while differences between the individual treatment groups were determined a posteriori using Duncan’s multiplerange test.
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RESULTS Experiment I Pituitary grafts exerted a significant suppressive effect on copulatory behavior in both the naive and experienced groups when tested between 3 and 4 weeks after surgery (Table 1). In the sexually naive group the grafted animals displayed longer latencies to mount (P < 0.01) and to intromit (P < 0.01) and fewer intromissions before the first ejaculation (P < 0.05) and during the test period (P < 0.05) than the sham-operated controls, The suppression of copulatory behavior was more pronounced in the animals which had had previous sexual experience. Thus grafted animals differed from the sham-operated controls not only in ML, IL, and total number of intromissions, but also in number of ejaculations and intromission rate. In Fig. 1, the Iatencies to mount, intromit, and ejaculate of the sexually experienced rats which received pituitary transplants or were sham operated are contrasted with their performance prior to surgery. While the sham-operated animals exhibited unchanged or decreased latencies, the grafted animals show large increases in the time to initiation of these behaviors over the preoperative values. Experiment
II
Fourteen days of treatment with bromocriptine resulted in increases in sexual activity in both the sham-operated and grafted animals. Specifically, the bromoctiptine-treated, sham-operated animals showed a reduction in the number of mounts without intromission prior to the first ejaculation (mount frequency) (P < 0.05), and an increase in the number of ejaculations which occurred during the 1-hr test period (P < 0.05; Table 2). In the grafted animals, only the PEI was significantly affected. The mean values of ML, IL, and EL, and the number of intromissions and ejaculations for each of the four treatment groups for the pretreatment, Day 7 and Day 14 tests of copulatory behavior, are shown in Figs. 2 and 3. It is evident that bromocriptine treatment resulted in a steady improvement in all these measures of copulatory behavior in the grafted animals. Furthermore, parallel changes in behavior may be seen between the bromocriptine-treated, sham, and grafted animals. Pretreatment hormone levels show the expected increase in plasma PRL and the decrease in plasma LH and FSH levels in the pituitary grafted animals (Table 3). Bromocriptine treatment led to reductions in plasma PRL levels in both the sham and grafted animals (Day 15 CB154, Table 3) far below the levels seen in the oil-treated sham-operated group. Additionally, plasma levels of LH in grafted males were significantly increased by CB-154. Two weeks after cessation of treatment, the
10/l 11.87 2 5.03 k 3.9 -c
Ejaculations Latency (mitt) PEI (mitt) Number
7110 14.63 f 1.67 6.50 f 1.44 2.6 2 0.4O.lO >o. 10
9110 5.94 rt 1.49 20.6 2 1.7 8.0 f 0.7 0.72 2 0.14 8/10 13.8 k 2.1 5.07 2 0.42 2.9 f 0.3
11111 1.36 2 0.25 29.6 rt 1.2 9.9 + 0.7 1.23 + 0.14 ll/ll 9.04 k 1.08 4.85 k 0.24 4.2 k 0.4
co.10 >O.lO co.05
co.02 co.02 BO.10 co.05
>O.lO
0.36 -c 0.05
0.40 ” 0.03
P co.05 >o. 10 20.10
Graft 9110 2.38 + 0.40 53.3 2 5.8 17.9 e 4.1
ll/ll 0.53 + 0.07 56.9 + 6.7 16.4 + 2.3
Sham
Experienced
a All values reported as mean 2 SEM. ’ The number of animals which exhibited the specified behavior (numerator) versus the total number of animals in the group (denominator). ’ Total number during the I-hr test period. * Number prior to the first ejaculation. ’ Intromission ratio = intromission frequency/mount frequency + intromission frequency.
1 1.51 0.41 0.4
10/l 2.87 k 33.5 + 12.0 k 1.08 2
Intromissions Latency (mitt) Number Frequency Rate
9110 10.98 f 2.27 20.6 e 3.0 9.4 + 0.6 0.68 -c 0.06
co. 10
0.29 2 0.04
0.43 f 0.05
1 0.42 3.5 1.1 0.14
O.lO >o. 10
9/10 6.87 2 1.39 66.4 rl: 4.5 25.9 f 4.0
P
lb 0.27 8.3 4.3
10/l 1.78 2 59.5 k 19.7 k
Graft
Mounts Latency (min) Number’ Frequent? Intromission ratio’
Sham
Naive
TABLE 1 Effects of Previous Sexual Experience on Copulatory Behavior of Male Rats Which Have Received Four Ectopic Pituitary Grafts (Graft) or Were Sham Operated (Sham)”
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Latency
IN HYPERPROLACTINEMIA Ejaculation Latew
Latency 6-
3-
16
s-
443
1
432 :,
OJ op Post.Op
4J PTLGS. op. Post-op.
PL-&S. op Post- op
FIG. 1. Latencies to mount, intromit, and ejaculate in sexually experienced male rats prior to or 3 weeks after surgery for pituitary transplantation (Graft) or sham surgery (Sham). Mean -C SEM.
PRL values had returned to the levels seen in the oil-treated animals, and there were no significant changes in plasma LH levels in the animals formerly treated with bromocriptine when compared to their respective oil-treated controls. In the animals which were injected for 3 days with 1 mg of CB-154 and bled 24 hr after the last injection, PRL levels were again suppressed in both groups which received bromocriptine below the level seen in the oil-treated sham-operated animals (Table 3). In addition, 3 days of treatment with bromocriptine induced a significant increase in plasma LH levels in the grafted males as compared to the oil-treated grafted animals. DISCUSSION Chronic elevation of plasma PRL levels suppresses copulatory behavior primarily by affecting the temporal pattern of behavioral responses. Typically, hyperprolactinemic animals exhibit longer ML, IL, and EL. However, no consistent effects on PEI could be demonstrated. These experiments also suggest a decreased level of performance, including difficulty in achieving intromission, particularly in the naive animals. It is possible, though, that decreased activity in the grafted males may be merely a reflection of changes in pacing. It should be noted that the hyperprolactinemic animals are capable of successful mating, and are no less fertile than their sham-operated controls (Ware et al., 1979). In sexual exhaustion studies, hyperprolactinemic male rats exhibit significantly longer ejaculation latencies, lower intromission to mount ratios, and other differences indicative of lowered sexual performance, when compared with sham-operated controls. (Quadagno, Herbert, and Bailey, personal communication). Thus, to exclude the possibility that disparate experiential factors may be playing a role in the expression of deficits
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x
AND SMITH
0000 -34-c
22%
dddd AhAh
ddd AVA
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in copulatory behavior, we have attempted to give control and experimental animals the same degree of sexual experience prior to grafting. The results demonstrate that the suppressive effects of pituitary grafts on copulatory behavior are even more readily demonstrable in the sexually experienced animals. The experienced grafted males differed from the controls in nearly all measures except number of mounts, MF, and PEI (Table I). Furthermore, these differences represent a change to a lower level of activity than that which occurred in the same animals prior to grafting (Fig. 1). The response of male rats to bromocriptine in terms of copulatory behavior differed from the effects of this treatment on gonadotropin secretion. Bromocriptine treatment proved to stimulate copulatory activity in both sham-operated and grafted animals (Table 2; Fig. 2 and 3). In the grafted animals only the PEI was significantly affected, but the Intromission Latency
Mount Latency
Ejaculation Latency 25
1T 0 Sham CB-154
oJ -
OJ I
I4
Intromissions
1
0’ I Pre Day7 Trtmnt.
Day14
Ejoculotions 7,
351
Graft CB-154 0 Sham CB-154 l
IO 1 0
Trtmnt.
FIGS. 2 AND 3. Latencies and ejaculations of sexually
Pre Day 7 Day 14 Trtmnt.
to mount, intromit, and ejaculate, and total number of mounts experienced pituitary grafted (Graft) and sham-operated (Sham) male rats prior to (Pretreatment) and on Days 7 and 14 of treatment with CB-154. Mean 2 SEM.
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TABLE 3 Plasma Levels of LH, FSH, and Prolactin (PRL) in Adult Male CD-F Rats Which Received Ectopic Pituitary Grafts (Graft) or Were Sham Operated (Sham) Prior to Initiation of Drug Treatment (Pretreatment), 2 Weeks after Initiation of Treatment with Bromocriptine (CB-154) or Vehicle (Oil) (Day I5 CB-154), and 2 Weeks after Cessation of Drug Treatment (Post-Treatment) Sham Oil
Graft CB-154
Oil
CB-154
LH FSH PRL
96.2 f 20.4 163.5 2 15.6 57.7 2 5.4
Pretreatment 82.2 + 17.1 45.9 4 5.96.= 196.2 -’ 19.9 152.6 4 9.2 53.6 z? 12.4 450.2 2 34.1”.’
39.4 + 6.2’.’ 156.1 2 8.w 411.5 2 42.2’*’
LH FSH PRL
40.3 ‘_ 4.1 205.8 t 15.8 74.2 -+ 17.pd,’
Day 15, CB-154 53.5 i 9.0 19.3 Y? 2.6b,cc 217.5 2 18.1 189.3 -f 14.8 4.8 rc_ C1.27~*~,’ 417.7 2 49.8*.‘,’
31.5 2 2.8’d 208,9 ? 16.2 7.8 2 0.8pd
LH FSH PRL
41.2 ” 6.8 227.4 + 12.9 72.1 2 4.1
Post-treatment 61.9 & 10.7 20.8 i 2.1*.’ 234.8 ? 17.6 201.8 z 20.2 64.1 t 9.4 247.7 * 14.6b.’
29.4 f 4.2 218.5 ? 10.9 2%.5 -t 37.8b.Csd
’ All values reported in terms of ng/ml of the NIAMDD reference expressed as mean 2 SEM. b Sig&icantly different from oil-treated sham animals. ’ Significantly different from CB-15Ctreated sham animals. d Significantly different from oil-treated grafted animals. ’ Significantly different from CB-154-treated grafted animals.
preparation
and
apparent, though not statistically significant, improvements which occurred in nearly all parameters of copulatory behavior examined in the CB-154-treated grafted animals are consistent with the pattern of change which occurred in the drug-treated controls. It is possible that treatment for a longer period may have been necessary for more definitive results in the grafted animals. The stimulatory effects of bromocriptine on copulatory behavior in the grafted rats cannot be attributed solely to the suppression of chronically elevated PRL levels. This does not imply that something other than PRL is responsible for the inhibition of sexual activity. In fact, deficits in the copulatory behavior of experienced male rats can also be produced by injecting ovine PRL (Doherty, Bartke, and Smith, 1980). Rather, increases in copulatory activity in the bromocriptine-treated sham-operated animals indicate the possibility of a direct stimulatory effect of this dopamine agonist on sexual behavior. This effect was not unexpected since both the dopamine precursor L-Dopa and agonist apomorphine are known to stimulate copulatory behavior (Tagliamonte, Fratta, Del Fiacco, and
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IN HYPERPROLACTINEMIA
Gessa, 1973; Malmnas, 1976). Furthermore, ergot alkaloids, including lisuride hydrogen maleate (Da Prada, Bonnetti, and Keller, 1977; Ahlenius, Larsson, and Svensson, 1980) and bromocriptine (Poggioli, Genedani, Castelli, and Bertolini, 1978) have been shown to stimulate various aspects of male sexual behavior. However, despite the suggestion that CB-154 may act directly to increase libido in men (Thomer and Besser, 1978), treatment with this drug has been reported to improve potency only in men with hyperPRL, but not in men with impotence of other etiology (Ambrosi et al., 1977; Buvat et al., 1978). Nor have any reports to date clearly demonstrated a stimulatory effect of bromocriptine on sexual activity in normal subjects. The effects of bromocriptine treatment on plasma gonadotropin levels in hyperprolactinemic rats suggest that the suppression of chronically elevated PRL levels was directly responsible for significant increases in plasma LH levels since they occurred only in the grafted animals (Tables 3,4). Hokfelt and Fuxe (1972) have reported similar effects of ergots on reversing the PRL-induced increase in dopamine fluorescence in the external palisade layer of the median eminence, which may be involved in inhibition of LHRH secretion and autoregulation of PRL release. However, a suggestive increase in plasma LH levels also occurred in the CB-15Ctreated shams with long-term treatment. Data from Gil-Ad et al. (1978) suggest that long-term, but not acute, treatment with bromocriptine may stimulate LHRH in both hyperprolactinemic tumor-bearing rats and intact controls. Thus long-term treatment with the ergot alkaloid may itself stimulate LH release. The apparent reduction in plasma PRL levels in pituitary grafted animals seen in the post-treatment period (Table 3) could not be correlated with any changes of behavior in these animals. While it is difficult to TABLE 4 Plasma Levels of LH, FSH, and Prolactin (PRL) in Adult Male CD-F Rats after Bromocriptine Treatment (3 days), 24 hr after Last Injection” Sham
LH FSH PRL
Graft
Oil
CB-154
52.2 e 6.4 213.8 2 12.6 75.2 * 9.Vd,’
59.7 f 8.3 244.9 + 23.0 5.4 f o.3b,“’
Oil 21.5 * 3.36.C.d 216.6 f 23.7 327.6 2 29.Sb.‘.”
CB-154 45.3 “_ 8.3 207.1 k 18.4 16.4 k 4.0bcc
’ All values reported in terms of rig/ml of the NIAMDD reference preparations expressed as mean r SEM. ’ Significantly different from oil-treated sham animals. ’ Significantly diierent from CB-W-treated sham animals. d Signilicantly different from CB-154-treated grafted animals. ’ Significantly diierent from oil-treated grafted animals.
and
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explain such a reduction in PRL output from the grafts, this effect has been reported previously in animals with anterior pituitary grafts, and was not associated with any changes in plasma LH or testosterone levels (Bartke et al., 1977). The lack of consistent suppressive effects of hyperPRL on FSH levels in the grafted animals may be a reflection of a differential responsiveness of LH and FSH to either the stimulatory effects of LHRH or the inhibitory effects of hyperPRL (Gil-Ad et al., 1978; Beck, Engelbart, Gelato, and Wuttke, 1977). These studies do not clarify a mechanism by which hyperPRL induces changes in copulatory activity. However, several possibilities seem apparent. Prolactin has been shown to increase dopaminergic activity in various regions of the brain including the median eminence (Hokfelt and Fuxe, 1972) and mesolimbic and nigrostriatal dopamine systems (Fuxe, Eneroth, Gustafsson, Loftstrom, and Skett, 1977; Wood, Cheney, and Costa, 1980). However, the relationship of hyperPRL and dopamine (DA) turnover to the suppression of copulatory behavior seems somewhat of a paradox, since it is thought that DA stimulates copulatory activity in male rats (Tagliamonte et al., 1973; Malmnas, 1976). Changes in the sensitivity of postsynaptic neurons to chronically increased DA release, or changes in dopaminergic activity due to neuronal feedback mechanisms or autoreceptor regulation could account for this discrepancy. Perhaps the differential responses to bromocriptine treatment observed in hyperprolactinemic and in normal males, in both rats and humans, is a reflection of this possible change in sensitivity. Alternately, increased DA turnover in the median eminence, where such neuronal feedback mechanisms do not appear to exist (Demarest and Moore, 1979), may chronically inhibit LHRH release to effect both decreased LH release and suppressed copulatory behavior (Moss, Dudley, Foreman, and McCann, 1975). ACKNOWLEDGMENTS We wish to thank Drs. D. M. Quadagno, J. Herbert, and D. Bailey for communicating their unpublished results to us. The surgical assistance of Arturo Moreno is also gratefully acknowledged. Bromocriptine was the gift of Dr. Richard Elton of Sandoz Pharmaceuticals, East Hanover, New Jersey; and material for radioimmunoassay of rat prolactin, LH, and FSH was generously supplied by the hormone distribution program of NIAMDD, and Dr. A. F. Parlow. We also wish to thank Ms. Lynn Rudloff and Ms. Gretta Small for their help in preparing this manuscript. This work was supported by NICHHD through Grant HD12671 and the Bioassay Core of Grant P30-HD10202.
REFERENCES Ahlenius, S., Larsson, K., and Svensson, L. (1980). Stimulating effects of lisuride on masculine sexual behavior of rats. Eur. J. Pharmacol. 64, 47-52. Ambrosi, B., Bara, R., Travaglini, P., Weber, G., Beck-Peccoz, P., Rondena, M., Elh, R., and Faglia, B. (1977). Study of the effects of bromocriptine on sexual impotence. Clin. Endocrinol. 7, 417-42 I.
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Bartke, A., Smith, M. S., Michael, S. D., Peron, F. G., and Dalterio, S. (1977). Effect of experimentally induced chronic hyperprolactinemia on testosterone and gonadotropin levels in male rats and mice. Endocrinology 100, 182-186. Beck, W., Engelbart, S., Gelato, M., and Wuttke, W. (1977). Antigonadotrophic effect of prolactin in adult castrated and in immature female rats. Acta Endocrinol. 84, 67-71. Boyar, R. M., Kapen, S. Finkelstein, J. W., Perlow, M., Sassin, J. F., Fukushima, D. K., Weitzman, E. D., and Hellman, L. (1974). Hypothalamic-pituitary function in diverse hyperprolactinemic states. J. C/in. Invest. 53, 1588-1598. Buvat, J., Asfour, M., Buvat-Herbaut, M., and Fossati, P. (1978). Prolactin and human sexual behavior. In C. Robyn and M. Hatter (Eds.), Progress in Pro&tin Physiology and Pathology, pp. 317-329. Elsevier, Amsterdam. Damassa, D. A., Smith, E. R., Tennent, B., and Davidson, J. M. (1977). The relationship between circulating testosterone levels and male sexual behavior in rats. Harm. Behav. 8, 275-286. Da Prada, M., Bonnetti, E. P., and Keller, H. H. (1977). Induction of mounting behavior in female and male rats by lisuride. Neurosci. Left. 6, 349-353. Demarest, K. T., and Moore, K. E. (1979). Comparison of dopamine synthesis regulation in the terminals of nigrostriatal, mesolimbic, tuberoinfundibular and tuberhypophyseal neurons. J. Neural. Transm. 46, 263-277. Doherty, P. C., Bartke, A., and Smith, M. S. (1980). The suppression of copulatory behavior in male rats with ectopic pituitary grafts: Role of prolactin. In Abstracts, ZOth Annual Meeting of the Society for Neuroscience, Vol. 6, p. 776. Fang, V. S., Refetoff, S., and Rosenfeld, R. L. (1974). Hypogonadism induced by a transplantable, prolactin-producing tumor in male rats: Hormonal and morphological studies. Endocrinology 95, 991-998. Franks, S., Jacobs, H. S., Martin, N., and Nabarro, J. D. N. (1978). Hyperprolactinemia and impotence. Clin. Endocrinol. 8, 277-287. Fuxe, K., Eneroth, P., Gustaffsson, J-A., Lofstrom, A., and Skett, P. (1977). Dopamine in the nucleus accumbens: Preferential increase of DA turnover by rat prolactin. Brain Res. 122, 177-182. Gil-Ad, I., Locatelli, V., Cocchi, D., Carminati, R., Arrezzini, C., and Mtlller, E. E. (1978). Effect of hyperprolactinemia and 2-BR-a-ergocryptine on neuroendocrine mechanism(s) for gonadotropin control. Life Sci. 23, 2245-2256. Grandison, L. J., Hodson, C. A., Chen, H. T., Advis, J., Simpkins, J., and Meites, J. (1977). Inhibition by prolactin of post-castration rise in LH. Nemoendocrinology 23, 312-322. Hartman, G., Endroczi, E., and Lissak, K. (1966). The effect of hypothalamic implantation of 17-B oestradiol and systemic administration of prolactin (LTH) on sexual behavior in male rabbits. Acta Physiol. Acad. Sci. Hung. 30, 53-59. Hodson, C. A., Simpkins, J. W., Pass, K. A., Aylsworth, C. F., Steger, R. W., and Meites, J. (1980). Effects of a prolactin-secreting pituitary tumor on hypothalamic, gonadotropic and testicular function in male rats. Neuroendocrinology 30, 7-10. Hokfelt, T., and Fuxe, K. (1972). Effects of prolactin and ergot alkaloids on the tuberoinfundibular dopamine system. Neuroendocrinology 9, 100-122. Jacobs, H. S. (1976). Prolactin and amenorrhea. N. Engl. J. Med. 295, 954-956. Malmnls, C. 0. (1976). The significance of dopamine, versus other catecholamines, for L-DOPA induced facilitation of sexual behavior in the male rat. Pharmacol. Riochem. Behav. 4, 521-526. McNeilly, A. S., Sharpe, R. M., Davidson, D. W., and Fraser, H. M. (1978). Inhibition of gonadotropin secretion by induced hyperprolactinemia in the male rat. J. Endocrinol. 79, 59-68. Moss, R. L., Dudley, C. A., Foreman, M. M., and McCann, S. M. (1975). Synthetic LRF:
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A potentiator of sexual behavior in the rat. In M. Motta, P. G. Crosignani, and L. Martini (Eds.), Hypothalamic Hormones, pp. 269-278. Academic Press, New York. Niswender, G. D., Midgley, A. R., Jr., Monroe, S. E., and Reichert, L. E., Jr. (1968). Radioimmunoassay for rat luteinizing hormone with antiovine LH serum and ovine LH-“‘I. Proc. Sot. Exp. Biol. Med. 128, 807-U 1. Poggioli, R., Genedani, S., Castelli, M., and Bertolini, A. (1978). Behavioral effects of 2Br-a-ergocryptine (Bromocriptine) in the male rat. Riv. Farmacol. Ter. 9, 213-220. Svare, B., Bartke, A., Doherty, P., Mason, I., Michael, S. D., and Smith, M. S. (1979). Hyperprolactinemia suppresses copulatory behavior in male rats and mice. Biol. Reprod. 21, 529-535. Tagliamonte, A., Fratta, W., Del Fiacco, M., and Gessa, G. L. (1973). Evidence that brain dopamine stimulates copulatory behavior in male rats. Riv. Farmacol. Ter. 4, 177-181. Thomer, M. O., and Besser, G. M. (1977). Hyperprolactinemia and gonadal function: Results of bromocriptine treatment. In P. G. Crosignani and C. Robyn (Eds.), Prolactin and Human Reproduction, pp. 285-301. Academic Press, New York. Thomer, M. O., and Besser, G. M. (1978). Bromocriptine treatment of hyperprolactinemic hypogonadism. Acta Endocrinol. 88, (Suppl. 216), 131-146. Tolis, G., and Van Vliet, S. (1976). Leydig cell function in hyperprolactinemia. Clin. Res. 24, 179A. Wood, P. L., Cheney, D. L., and Costa, E. (1980). A prolactin action on acetylcholine metabolism in striatum, hippocampus, and thalamus. .I. Neurochem. 34, 1053-1057.
AND
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436-450 (1981)
Differential Effects of Bromocriptine Treatment on LH Release and Copulatory Behavior in Hyperprolactinemic Male Rats P. C. DOHERTY, A. BARTKE, AND M. S. SMITH* Departments of Anatomy and Obstetrics and Gynecology, The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78284, and *Department of Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261 Hyperprolactinemia (hyperPRL) induced by grafting four pituitary glands under the kidney capsule suppresses copulatory behavior in male rats and sexually naive male mice. In mice sexual experience attenuates the suppressive effects of hyperPRL on mating behavior, thus a comparison of the behavioral consequences of inducing hyperPRL in sexually naive and experienced mate rats was undertaken. Hyperprolactinemia had a significant suppressive effect on mating behavior in both groups of animals. Experienced animals showed deficits in all parameters studied except mount frequency and postejaculatory interval, while naive animals differed from respective controls only in mount latency, intromission latency, and intromission frequency. To determine if the inhibition of chronically elevated prolactin (PRL) levels would reverse the suppression of gonadotropin secretion and copulatory behavior in hyperprolactinemic animals, the effects of bromocriptine (CB-154) administration on plasma hormone levels and mating behavior were examined in pituitary-grafted and control rats. Bromocriptine treatment (1 mg/day for 14 days) led to increases in sexual activity in both the sham-operated and grafted animals. In the grafted animals, plasma PRL was reduced and plasma LH significantly increased in the CB-W-treated animals when compared to oil-treated controls. In sham-operated animals, CB-154 produced no significant changes in plasma LH or FSH despite the suppressed PRL levels. These results indicate that (1) hyperPRL induced by pituitary grafts can cause deficits in mating behavior in mate rats despite previous sexual experience, and (2) while CB-154 may be acting through other mechanisms to stimulate copulatory behavior, the reduction of chronically elevated PRL levels due to CB-154 treatment is responsible for reversal of the suppressive effects of hyperPRL on LH secretion.
In the human, pathologic elevation of plasma prolactin (PRL) levels, hyperprolactinemia (hyperPRL), is usually the result of a hypersecreting pituitary adenoma. In women, it is often associated with amenorrhea, anovulatory sterility, and, somewhat less frequently, galactorrhea (Ja436 0018-506x/81/040436-15$01.00/0 Copyright All rights
CD 1981 by Academic Press, Inc. of reproduction in any fom reserved.
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cobs, 1976). Several reports indicate that hyperPRL can also interfere with sexual function in men (Boyar, Kapen, Finkelstein, Perlow, Sassin, Fukushima, Weitzman, and Hellman, 1974; Tolis and Van Vliet, 1976; Thorner and Besser, 1977). Reduced gonadotropin levels, suppressed pituitary responsiveness to LHRH, and various degrees of hypogonadism are among the symptoms described; however, the most common complaints associated with hyperPRL in men are impotence and/or decreased libido. Recently a number of animal models have been described for the study of the effects of elevated circulating levels of PRL on the hypothalamo-pituitary-testicular axis. Male rats bearing tumors from PRL-producing cell lines have been reported to exhibit testicular atrophy (Fang, Refetoff, and Rosenfeld, 1974), or inhibition of the release of pituitary LH and hypothalamic LHRH associated with high serum PRL levels (Gil-Ad, Locatelli, Cocchi, Carminati, Arezzini, and Mtiller, 1978; Hodson, Simpkins, Pass, Aylsworth, Steger, and Meites, 1980). Male rats rendered hyperprolactinemic by transplantation of several isogeneic pituitary glands display reductions in plasma gonadotropin levels but normal plasma levels of testosterone (Bartke, Smith, Michael, Peron, and Dalterio, 1977; McNeilly, Sharpe, Davidson, and Fraser, 1978) and suppression of the postcastration increases in serum LH (Grandison, Hodson, Chen, Advis, Simpkins, and Meites, 1977). In addition, male rats with pituitary transplants exhibit impaired copulatory behavior patterns, including increased latencies to mount, intromit, and ejaculate, and fewer mounts, intromissions, and ejaculations during the test period (Svare, Bartke, Doherty, Mason, Michael, and Smith, 1979). Similar suppressive effects of elevated PRL levels on the copulatory behavior of male rabbits have also been reported (Hartmann, Endroczi, and Lissak, 1966). In male mice, the suppressive effects of hyperPRL on copulatory behavior attenuate with further sexual experience (Svare et al., 1979). Since previous studies in the rat utilized animals which were allowed sexual experience only after the induction of hyperPRL, one of the purposes of this investigation was to determine if sexual experience prior to surgery for pituitary transplantation can modify the effects of this treatment on copulatory behavior. Bromocriptine (CB-154), a dopamine agonist with prolactin-inhibiting capabilities, has been shown to be a potent agent in the relief of symptoms of pathologic hyperPRL (Thorner and Besser, 1978). Thus, bromocriptine treatment can result in decreased peripheral PRL levels and restored sexual function in hyperprolactinemic men (Ambrosi, Bara, Travaglini, Weber, Beck-Peccoz, Rondena, Elli, and Faglia, 1977; Franks, Jacobs, Martin, and Nabarro, 1978; Buvat, Asfour, Buvat-Herbaut, and Fossati, 1978). Bromocriptine treatment of rats bearing tumors of H&producing
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cell lines can reverse the reported inhibition of LHRH release from the hypothalamus (Gil-Ad ef al., 1978). The present study was undertaken to determine if bromocriptine treatment can reverse the suppressive effects of hyperprolactinemia on gonadotropin secretion and copulatory behavior in animals bearing pituitary grafts, and whether the effects of bromocriptine can be attributed to the suppression of chronically elevated PRL levels or a direct effect of bromocriptine itself. GENERAL METHODS Inbred CD-F rats purchased from Charles River Breeding Laboratories were maintained on a 14-hr light: IO-hr dark lighting schedule, with constant access to food and water. Male rats were rendered hyperprolactinemic by grafting whole pituitaries from four adult CD-F females under the kidney capsules (two pituitaries per kidney) or were sham operated. Animals were bled from the jugular vein under light ether anesthesia for determinations of plasma levels of PRL, LH, and FSH. Tests of copulatory behavior were performed between 3 and 6 hr after the onset of darkness under dim red illumination from a 25-W red light. Tests were conducted in neutral chambers consisting of IO-gal aquaria (25 x 47 x 29 cm) with sawdust on the floor. Each male was allowed an adaptation period of 30 min prior to introduction of a receptive female into the observation chamber. Adult ovariectomized females rendered behaviorally estrous by injections of estradiol benzoate (10 t&O.1 ml sesame oil) 48 hr prior to testing, and progesterone (500 t&O. 1 ml sesame oil) 4 hr prior to testing, served as stimulus partners. Each test lasted for a period of 1 hr. The following measures of copulatory behavior were recorded for each male: mount latency (ML), mounts with pelvic thrusting, intromission latency (IL), intromission responses, and ejaculation responses. In addition the following measures of masculine sexual behavior were calculated for the first copulatory series of each test: ejaculation latency (EL), time from the first intromission to ejaculation; postejaculatory interval (PEI), time from the first ejaculation to the first intromission of the next series; mount frequency (MF), the number of mounts prior to the first ejaculation; intromission frequency (IF), the number of intromissions prior to the first ejaculation; intromission ratio, the number of intromissions divided by the number of mounts prior to the first ejaculation; and the intromission rate (Urnin), the intromission frequency divided by the ejaculation latency. For the sexually experienced animals, test scores were evaluated using criteria adapted from Damassa, Smith, Tennent, and Davidson (1977). Thus, if IL > 15 min, EL > 30 min, or PEI > 15 min, the test was repeated. Animals were tested three times on alternate days, or until the
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first successful test occurred. Most animals achieved these criteria on the first test. If after the third test these criteria were not met, the test in which the best performance was achieved was recorded as the score for that animal. Test scores for the sexually naive animals, and those for Days 7 and 14 of bromocriptine treatment represent a single 1-hr test of copulatory behavior. Serum LH concentrations were assessed by the ovine-ovine radioimmunoassay system described previously (Niswender, Midgley, Monroe, and Reichert, 1968). The limit of detection of the assay was 5 q/ml, the intrassay variation, 6.2%, and the interassay variation, 7.4%. Plasma LH concentrations were expressed in terms of the NIAMDD rat LH-RP-1 standard (0.03 x NIH-LH-Sl). Follicle stimulating hormone concentrations were determined with the radioimmunoassay materials provided by the NIAMDD. Using the rat reference preparation (NIAMDD-RP-1, 2 IU/mg), the limit of detection of the assay was 100 r&ml. The coefficients of variation of this assay were 4.3% for intraassay variation and 5.8% for interassay variation. Plasma prolactin concentrations were determined by the NIAMDD rat prolactin radioimmunoassay and expressed in terms of the NIAMDD-RP-1 standard (11 IU/mg). The limit of detection of the assay was 3 r&ml, and the intraassay and interassay variations were 7.6 and 8.9%, respectively. Experiment Z To determine if the suppressive effects of hyperPRL on copulatory behavior of male rats are independent of previous sexual experience, 42 males were divided into four groups as follows: (a) 10 sexually naive males which received transplants of four whole pituitaries at the age of 90-95 days as previously described; (b) 11 sexually naive males which underwent sham surgery; (c) 10 sexually experienced males which received transplants of four whole pituitaries at the age of 100-105 days; and (d) 11 sexually experienced males which underwent sham surgery. When the males were 75 days of age, sexual experience was allowed by individually housing each of the animals with an intact cycling female until sperm was found in the vaginal smears and by testing for copulatory behavior. The results of the tests also served as the basis for assignment of individual animals to grafted and sham-operated groups to allow similar distribution of highly active copulators and less vigorous copulators. Prior to grafting, the experienced animals had ejaculated on three separate occasions as determined by the presence of sperm in vaginal smears or by direct observation in tests of copulatory behavior. On Day 24 after surgery, further tests of copulatory behavior were begun. Statistics. Statistical analysis was performed using data from only those animals which exhibited the specific behavioral parameter. No values were assigned to animals which did not perform. Comparisons
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were made only between the grafted and sham-operated animals in either the naive or the experienced groups, but not across all groups. Differences were considered significant at the 0.05 level using a two-tailed Mann-Whitney U test. Experiment
II
To determine if the suppression of chronically elevated PRL levels would be accompanied by improved copulatory behavior and increased plasma gonadotropin levels in the grafted rats, bromocriptine treatment was initiated. One month elapsed between Experiment I and Experiment II. During this time, male rats from the first experiment were allowed further sexual experience as described above. Animals were observed for copulatory behavior in a 1-hr test and assigned to one of four treatment groups with an equal number of the originally sexually naive and experienced animals assigned to each of the groups. The results of this test of copulatory behavior served as the pretreatment value. Bromocriptine treatment (1 mg/day in 0.1 ml of sesame oil) was administered for 15 days in a group of sham-operated animals (n = 10) and grafted animals (n = lo), while additional groups of sham-operated (n = 10) and grafted males (n = 10) received only vehicle. Tests of copulatory behavior were performed on Days 7 and 14 of treatment. Injections were given 12 hr prior to the time of testing. Plasma samples for determination of PRL, LH, and FSH were obtained from the jugular vein under light ether anesthesia 1 day prior to commencement of treatment, on Day 15 of treatment 12 hr after the last injection, and 2 weeks after the cessation of treatment. To decrease diurnal variability in responsiveness to drug treatment, copulatory behavior, and plasma hormone levels, the experimental procedures were staggered over S-day intervals (two animals from each treatment group/day), with all animals undergoing experimental manipulations at approximately the same time of day throughout the course of this experiment. In addition, to determine the degree of suppression achieved over the 24-hr period between injections, bromocriptine treatment was resumed for 3 days, as previously described, 1 month after the initial treatment period and the animals were bled 24 hr after the last injection. Siutistics. The effects of bromocriptine were assessed by comparing the behavior of the oil-treated vs the bromocriptine-treated animals within the grafted or sham-operated groups. Differences were considered significant at the 0.05 level using a two-tailed Mann-Whitney U test. For plasma hormone levels, significant treatment effects were determined by analysis of variance, while differences between the individual treatment groups were determined a posteriori using Duncan’s multiplerange test.
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RESULTS Experiment I Pituitary grafts exerted a significant suppressive effect on copulatory behavior in both the naive and experienced groups when tested between 3 and 4 weeks after surgery (Table 1). In the sexually naive group the grafted animals displayed longer latencies to mount (P < 0.01) and to intromit (P < 0.01) and fewer intromissions before the first ejaculation (P < 0.05) and during the test period (P < 0.05) than the sham-operated controls, The suppression of copulatory behavior was more pronounced in the animals which had had previous sexual experience. Thus grafted animals differed from the sham-operated controls not only in ML, IL, and total number of intromissions, but also in number of ejaculations and intromission rate. In Fig. 1, the Iatencies to mount, intromit, and ejaculate of the sexually experienced rats which received pituitary transplants or were sham operated are contrasted with their performance prior to surgery. While the sham-operated animals exhibited unchanged or decreased latencies, the grafted animals show large increases in the time to initiation of these behaviors over the preoperative values. Experiment
II
Fourteen days of treatment with bromocriptine resulted in increases in sexual activity in both the sham-operated and grafted animals. Specifically, the bromoctiptine-treated, sham-operated animals showed a reduction in the number of mounts without intromission prior to the first ejaculation (mount frequency) (P < 0.05), and an increase in the number of ejaculations which occurred during the 1-hr test period (P < 0.05; Table 2). In the grafted animals, only the PEI was significantly affected. The mean values of ML, IL, and EL, and the number of intromissions and ejaculations for each of the four treatment groups for the pretreatment, Day 7 and Day 14 tests of copulatory behavior, are shown in Figs. 2 and 3. It is evident that bromocriptine treatment resulted in a steady improvement in all these measures of copulatory behavior in the grafted animals. Furthermore, parallel changes in behavior may be seen between the bromocriptine-treated, sham, and grafted animals. Pretreatment hormone levels show the expected increase in plasma PRL and the decrease in plasma LH and FSH levels in the pituitary grafted animals (Table 3). Bromocriptine treatment led to reductions in plasma PRL levels in both the sham and grafted animals (Day 15 CB154, Table 3) far below the levels seen in the oil-treated sham-operated group. Additionally, plasma levels of LH in grafted males were significantly increased by CB-154. Two weeks after cessation of treatment, the
10/l 11.87 2 5.03 k 3.9 -c
Ejaculations Latency (mitt) PEI (mitt) Number
7110 14.63 f 1.67 6.50 f 1.44 2.6 2 0.4
9110 5.94 rt 1.49 20.6 2 1.7 8.0 f 0.7 0.72 2 0.14 8/10 13.8 k 2.1 5.07 2 0.42 2.9 f 0.3
11111 1.36 2 0.25 29.6 rt 1.2 9.9 + 0.7 1.23 + 0.14 ll/ll 9.04 k 1.08 4.85 k 0.24 4.2 k 0.4
co.10 >O.lO co.05
co.02 co.02 BO.10 co.05
>O.lO
0.36 -c 0.05
0.40 ” 0.03
P co.05 >o. 10 20.10
Graft 9110 2.38 + 0.40 53.3 2 5.8 17.9 e 4.1
ll/ll 0.53 + 0.07 56.9 + 6.7 16.4 + 2.3
Sham
Experienced
a All values reported as mean 2 SEM. ’ The number of animals which exhibited the specified behavior (numerator) versus the total number of animals in the group (denominator). ’ Total number during the I-hr test period. * Number prior to the first ejaculation. ’ Intromission ratio = intromission frequency/mount frequency + intromission frequency.
1 1.51 0.41 0.4
10/l 2.87 k 33.5 + 12.0 k 1.08 2
Intromissions Latency (mitt) Number Frequency Rate
9110 10.98 f 2.27 20.6 e 3.0 9.4 + 0.6 0.68 -c 0.06
co. 10
0.29 2 0.04
0.43 f 0.05
1 0.42 3.5 1.1 0.14
9/10 6.87 2 1.39 66.4 rl: 4.5 25.9 f 4.0
P
lb 0.27 8.3 4.3
10/l 1.78 2 59.5 k 19.7 k
Graft
Mounts Latency (min) Number’ Frequent? Intromission ratio’
Sham
Naive
TABLE 1 Effects of Previous Sexual Experience on Copulatory Behavior of Male Rats Which Have Received Four Ectopic Pituitary Grafts (Graft) or Were Sham Operated (Sham)”
CB-154, LH, AND SEX BEHAVIOR
Latency
IN HYPERPROLACTINEMIA Ejaculation Latew
Latency 6-
3-
16
s-
443
1
432 :,
OJ op Post.Op
4J PTLGS. op. Post-op.
PL-&S. op Post- op
FIG. 1. Latencies to mount, intromit, and ejaculate in sexually experienced male rats prior to or 3 weeks after surgery for pituitary transplantation (Graft) or sham surgery (Sham). Mean -C SEM.
PRL values had returned to the levels seen in the oil-treated animals, and there were no significant changes in plasma LH levels in the animals formerly treated with bromocriptine when compared to their respective oil-treated controls. In the animals which were injected for 3 days with 1 mg of CB-154 and bled 24 hr after the last injection, PRL levels were again suppressed in both groups which received bromocriptine below the level seen in the oil-treated sham-operated animals (Table 3). In addition, 3 days of treatment with bromocriptine induced a significant increase in plasma LH levels in the grafted males as compared to the oil-treated grafted animals. DISCUSSION Chronic elevation of plasma PRL levels suppresses copulatory behavior primarily by affecting the temporal pattern of behavioral responses. Typically, hyperprolactinemic animals exhibit longer ML, IL, and EL. However, no consistent effects on PEI could be demonstrated. These experiments also suggest a decreased level of performance, including difficulty in achieving intromission, particularly in the naive animals. It is possible, though, that decreased activity in the grafted males may be merely a reflection of changes in pacing. It should be noted that the hyperprolactinemic animals are capable of successful mating, and are no less fertile than their sham-operated controls (Ware et al., 1979). In sexual exhaustion studies, hyperprolactinemic male rats exhibit significantly longer ejaculation latencies, lower intromission to mount ratios, and other differences indicative of lowered sexual performance, when compared with sham-operated controls. (Quadagno, Herbert, and Bailey, personal communication). Thus, to exclude the possibility that disparate experiential factors may be playing a role in the expression of deficits
444
DOHERTY,
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x
AND SMITH
0000 -34-c
22%
dddd AhAh
ddd AVA
CB-154, LH, AND SEX BEHAVIOR
IN HYPERPROLACTINEMIA
445
in copulatory behavior, we have attempted to give control and experimental animals the same degree of sexual experience prior to grafting. The results demonstrate that the suppressive effects of pituitary grafts on copulatory behavior are even more readily demonstrable in the sexually experienced animals. The experienced grafted males differed from the controls in nearly all measures except number of mounts, MF, and PEI (Table I). Furthermore, these differences represent a change to a lower level of activity than that which occurred in the same animals prior to grafting (Fig. 1). The response of male rats to bromocriptine in terms of copulatory behavior differed from the effects of this treatment on gonadotropin secretion. Bromocriptine treatment proved to stimulate copulatory activity in both sham-operated and grafted animals (Table 2; Fig. 2 and 3). In the grafted animals only the PEI was significantly affected, but the Intromission Latency
Mount Latency
Ejaculation Latency 25
1T 0 Sham CB-154
oJ -
OJ I
I4
Intromissions
1
0’ I Pre Day7 Trtmnt.
Day14
Ejoculotions 7,
351
Graft CB-154 0 Sham CB-154 l
IO 1 0
Trtmnt.
FIGS. 2 AND 3. Latencies and ejaculations of sexually
Pre Day 7 Day 14 Trtmnt.
to mount, intromit, and ejaculate, and total number of mounts experienced pituitary grafted (Graft) and sham-operated (Sham) male rats prior to (Pretreatment) and on Days 7 and 14 of treatment with CB-154. Mean 2 SEM.
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TABLE 3 Plasma Levels of LH, FSH, and Prolactin (PRL) in Adult Male CD-F Rats Which Received Ectopic Pituitary Grafts (Graft) or Were Sham Operated (Sham) Prior to Initiation of Drug Treatment (Pretreatment), 2 Weeks after Initiation of Treatment with Bromocriptine (CB-154) or Vehicle (Oil) (Day I5 CB-154), and 2 Weeks after Cessation of Drug Treatment (Post-Treatment) Sham Oil
Graft CB-154
Oil
CB-154
LH FSH PRL
96.2 f 20.4 163.5 2 15.6 57.7 2 5.4
Pretreatment 82.2 + 17.1 45.9 4 5.96.= 196.2 -’ 19.9 152.6 4 9.2 53.6 z? 12.4 450.2 2 34.1”.’
39.4 + 6.2’.’ 156.1 2 8.w 411.5 2 42.2’*’
LH FSH PRL
40.3 ‘_ 4.1 205.8 t 15.8 74.2 -+ 17.pd,’
Day 15, CB-154 53.5 i 9.0 19.3 Y? 2.6b,cc 217.5 2 18.1 189.3 -f 14.8 4.8 rc_ C1.27~*~,’ 417.7 2 49.8*.‘,’
31.5 2 2.8’d 208,9 ? 16.2 7.8 2 0.8pd
LH FSH PRL
41.2 ” 6.8 227.4 + 12.9 72.1 2 4.1
Post-treatment 61.9 & 10.7 20.8 i 2.1*.’ 234.8 ? 17.6 201.8 z 20.2 64.1 t 9.4 247.7 * 14.6b.’
29.4 f 4.2 218.5 ? 10.9 2%.5 -t 37.8b.Csd
’ All values reported in terms of ng/ml of the NIAMDD reference expressed as mean 2 SEM. b Sig&icantly different from oil-treated sham animals. ’ Significantly different from CB-15Ctreated sham animals. d Significantly different from oil-treated grafted animals. ’ Significantly different from CB-154-treated grafted animals.
preparation
and
apparent, though not statistically significant, improvements which occurred in nearly all parameters of copulatory behavior examined in the CB-154-treated grafted animals are consistent with the pattern of change which occurred in the drug-treated controls. It is possible that treatment for a longer period may have been necessary for more definitive results in the grafted animals. The stimulatory effects of bromocriptine on copulatory behavior in the grafted rats cannot be attributed solely to the suppression of chronically elevated PRL levels. This does not imply that something other than PRL is responsible for the inhibition of sexual activity. In fact, deficits in the copulatory behavior of experienced male rats can also be produced by injecting ovine PRL (Doherty, Bartke, and Smith, 1980). Rather, increases in copulatory activity in the bromocriptine-treated sham-operated animals indicate the possibility of a direct stimulatory effect of this dopamine agonist on sexual behavior. This effect was not unexpected since both the dopamine precursor L-Dopa and agonist apomorphine are known to stimulate copulatory behavior (Tagliamonte, Fratta, Del Fiacco, and
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Gessa, 1973; Malmnas, 1976). Furthermore, ergot alkaloids, including lisuride hydrogen maleate (Da Prada, Bonnetti, and Keller, 1977; Ahlenius, Larsson, and Svensson, 1980) and bromocriptine (Poggioli, Genedani, Castelli, and Bertolini, 1978) have been shown to stimulate various aspects of male sexual behavior. However, despite the suggestion that CB-154 may act directly to increase libido in men (Thomer and Besser, 1978), treatment with this drug has been reported to improve potency only in men with hyperPRL, but not in men with impotence of other etiology (Ambrosi et al., 1977; Buvat et al., 1978). Nor have any reports to date clearly demonstrated a stimulatory effect of bromocriptine on sexual activity in normal subjects. The effects of bromocriptine treatment on plasma gonadotropin levels in hyperprolactinemic rats suggest that the suppression of chronically elevated PRL levels was directly responsible for significant increases in plasma LH levels since they occurred only in the grafted animals (Tables 3,4). Hokfelt and Fuxe (1972) have reported similar effects of ergots on reversing the PRL-induced increase in dopamine fluorescence in the external palisade layer of the median eminence, which may be involved in inhibition of LHRH secretion and autoregulation of PRL release. However, a suggestive increase in plasma LH levels also occurred in the CB-15Ctreated shams with long-term treatment. Data from Gil-Ad et al. (1978) suggest that long-term, but not acute, treatment with bromocriptine may stimulate LHRH in both hyperprolactinemic tumor-bearing rats and intact controls. Thus long-term treatment with the ergot alkaloid may itself stimulate LH release. The apparent reduction in plasma PRL levels in pituitary grafted animals seen in the post-treatment period (Table 3) could not be correlated with any changes of behavior in these animals. While it is difficult to TABLE 4 Plasma Levels of LH, FSH, and Prolactin (PRL) in Adult Male CD-F Rats after Bromocriptine Treatment (3 days), 24 hr after Last Injection” Sham
LH FSH PRL
Graft
Oil
CB-154
52.2 e 6.4 213.8 2 12.6 75.2 * 9.Vd,’
59.7 f 8.3 244.9 + 23.0 5.4 f o.3b,“’
Oil 21.5 * 3.36.C.d 216.6 f 23.7 327.6 2 29.Sb.‘.”
CB-154 45.3 “_ 8.3 207.1 k 18.4 16.4 k 4.0bcc
’ All values reported in terms of rig/ml of the NIAMDD reference preparations expressed as mean r SEM. ’ Significantly different from oil-treated sham animals. ’ Significantly diierent from CB-W-treated sham animals. d Signilicantly different from CB-154-treated grafted animals. ’ Significantly diierent from oil-treated grafted animals.
and
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explain such a reduction in PRL output from the grafts, this effect has been reported previously in animals with anterior pituitary grafts, and was not associated with any changes in plasma LH or testosterone levels (Bartke et al., 1977). The lack of consistent suppressive effects of hyperPRL on FSH levels in the grafted animals may be a reflection of a differential responsiveness of LH and FSH to either the stimulatory effects of LHRH or the inhibitory effects of hyperPRL (Gil-Ad et al., 1978; Beck, Engelbart, Gelato, and Wuttke, 1977). These studies do not clarify a mechanism by which hyperPRL induces changes in copulatory activity. However, several possibilities seem apparent. Prolactin has been shown to increase dopaminergic activity in various regions of the brain including the median eminence (Hokfelt and Fuxe, 1972) and mesolimbic and nigrostriatal dopamine systems (Fuxe, Eneroth, Gustafsson, Loftstrom, and Skett, 1977; Wood, Cheney, and Costa, 1980). However, the relationship of hyperPRL and dopamine (DA) turnover to the suppression of copulatory behavior seems somewhat of a paradox, since it is thought that DA stimulates copulatory activity in male rats (Tagliamonte et al., 1973; Malmnas, 1976). Changes in the sensitivity of postsynaptic neurons to chronically increased DA release, or changes in dopaminergic activity due to neuronal feedback mechanisms or autoreceptor regulation could account for this discrepancy. Perhaps the differential responses to bromocriptine treatment observed in hyperprolactinemic and in normal males, in both rats and humans, is a reflection of this possible change in sensitivity. Alternately, increased DA turnover in the median eminence, where such neuronal feedback mechanisms do not appear to exist (Demarest and Moore, 1979), may chronically inhibit LHRH release to effect both decreased LH release and suppressed copulatory behavior (Moss, Dudley, Foreman, and McCann, 1975). ACKNOWLEDGMENTS We wish to thank Drs. D. M. Quadagno, J. Herbert, and D. Bailey for communicating their unpublished results to us. The surgical assistance of Arturo Moreno is also gratefully acknowledged. Bromocriptine was the gift of Dr. Richard Elton of Sandoz Pharmaceuticals, East Hanover, New Jersey; and material for radioimmunoassay of rat prolactin, LH, and FSH was generously supplied by the hormone distribution program of NIAMDD, and Dr. A. F. Parlow. We also wish to thank Ms. Lynn Rudloff and Ms. Gretta Small for their help in preparing this manuscript. This work was supported by NICHHD through Grant HD12671 and the Bioassay Core of Grant P30-HD10202.
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Bartke, A., Smith, M. S., Michael, S. D., Peron, F. G., and Dalterio, S. (1977). Effect of experimentally induced chronic hyperprolactinemia on testosterone and gonadotropin levels in male rats and mice. Endocrinology 100, 182-186. Beck, W., Engelbart, S., Gelato, M., and Wuttke, W. (1977). Antigonadotrophic effect of prolactin in adult castrated and in immature female rats. Acta Endocrinol. 84, 67-71. Boyar, R. M., Kapen, S. Finkelstein, J. W., Perlow, M., Sassin, J. F., Fukushima, D. K., Weitzman, E. D., and Hellman, L. (1974). Hypothalamic-pituitary function in diverse hyperprolactinemic states. J. C/in. Invest. 53, 1588-1598. Buvat, J., Asfour, M., Buvat-Herbaut, M., and Fossati, P. (1978). Prolactin and human sexual behavior. In C. Robyn and M. Hatter (Eds.), Progress in Pro&tin Physiology and Pathology, pp. 317-329. Elsevier, Amsterdam. Damassa, D. A., Smith, E. R., Tennent, B., and Davidson, J. M. (1977). The relationship between circulating testosterone levels and male sexual behavior in rats. Harm. Behav. 8, 275-286. Da Prada, M., Bonnetti, E. P., and Keller, H. H. (1977). Induction of mounting behavior in female and male rats by lisuride. Neurosci. Left. 6, 349-353. Demarest, K. T., and Moore, K. E. (1979). Comparison of dopamine synthesis regulation in the terminals of nigrostriatal, mesolimbic, tuberoinfundibular and tuberhypophyseal neurons. J. Neural. Transm. 46, 263-277. Doherty, P. C., Bartke, A., and Smith, M. S. (1980). The suppression of copulatory behavior in male rats with ectopic pituitary grafts: Role of prolactin. In Abstracts, ZOth Annual Meeting of the Society for Neuroscience, Vol. 6, p. 776. Fang, V. S., Refetoff, S., and Rosenfeld, R. L. (1974). Hypogonadism induced by a transplantable, prolactin-producing tumor in male rats: Hormonal and morphological studies. Endocrinology 95, 991-998. Franks, S., Jacobs, H. S., Martin, N., and Nabarro, J. D. N. (1978). Hyperprolactinemia and impotence. Clin. Endocrinol. 8, 277-287. Fuxe, K., Eneroth, P., Gustaffsson, J-A., Lofstrom, A., and Skett, P. (1977). Dopamine in the nucleus accumbens: Preferential increase of DA turnover by rat prolactin. Brain Res. 122, 177-182. Gil-Ad, I., Locatelli, V., Cocchi, D., Carminati, R., Arrezzini, C., and Mtlller, E. E. (1978). Effect of hyperprolactinemia and 2-BR-a-ergocryptine on neuroendocrine mechanism(s) for gonadotropin control. Life Sci. 23, 2245-2256. Grandison, L. J., Hodson, C. A., Chen, H. T., Advis, J., Simpkins, J., and Meites, J. (1977). Inhibition by prolactin of post-castration rise in LH. Nemoendocrinology 23, 312-322. Hartman, G., Endroczi, E., and Lissak, K. (1966). The effect of hypothalamic implantation of 17-B oestradiol and systemic administration of prolactin (LTH) on sexual behavior in male rabbits. Acta Physiol. Acad. Sci. Hung. 30, 53-59. Hodson, C. A., Simpkins, J. W., Pass, K. A., Aylsworth, C. F., Steger, R. W., and Meites, J. (1980). Effects of a prolactin-secreting pituitary tumor on hypothalamic, gonadotropic and testicular function in male rats. Neuroendocrinology 30, 7-10. Hokfelt, T., and Fuxe, K. (1972). Effects of prolactin and ergot alkaloids on the tuberoinfundibular dopamine system. Neuroendocrinology 9, 100-122. Jacobs, H. S. (1976). Prolactin and amenorrhea. N. Engl. J. Med. 295, 954-956. Malmnls, C. 0. (1976). The significance of dopamine, versus other catecholamines, for L-DOPA induced facilitation of sexual behavior in the male rat. Pharmacol. Riochem. Behav. 4, 521-526. McNeilly, A. S., Sharpe, R. M., Davidson, D. W., and Fraser, H. M. (1978). Inhibition of gonadotropin secretion by induced hyperprolactinemia in the male rat. J. Endocrinol. 79, 59-68. Moss, R. L., Dudley, C. A., Foreman, M. M., and McCann, S. M. (1975). Synthetic LRF:
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