HORMONES
AND
Initiation
BEHAVIOR
13, 9-20 (1979)
of Copulatory Behavior in Castrated Rats Injected with Critically Adjusted Doses of Testosterone
Z. HLIGK’, Psychiatric
J. MADLAFOUSEK, Research
Institute,
Male
AND A. MOHAPELOVA
181 03 Prague 8, Czechoslovakia
In castrated male rats it was possible by means of administration of adequate testosterone propionate doses to induce a state in which the males did not initiate copulatory behavior with a stimulus female displaying no (Lordotic female) or weak (Presenting female) intensity of precopulatory behavior. On the other hand, such males started to copulate and finished copulatory series when a stimulus female exhibiting the complete pattern of precopulatory behavior (Darting female) was offered. The males “prepared” in this manner displayed regularly precopulatory behavior directed toward all the stimulus females. The requisite testosterone doses differed individually. However, in the majority of cases they were in the range of 500-700 /*g of testosterone injected once a week.
In the previous study (Madlafousek, Hlinak, and Beran, 1976) we showed that the declining ability of castrated male rats to initiate copulatory behavior is substantially dependent on the stimulus female used in testing the male. Females exhibiting low-intensity precopulatory behavior (presenting posture) were compared with those exhibiting complete precopulatory behavior (characterized by darting responses). Such females do not differ with respect to receptivity; their copulatory readiness, as expressed by the lordosis quotient, is practically 100%. Several weeks after castration, when no male was stimulated to mount or intromit a female which showed the presenting postures (Presenting female), males would initiate copulatory behavior with females showing the darting responses (Darting female). About 9 weeks after castration not even a Darting female was sufficient to compensate for the hormonal insufficiency of castrated males. It is well known that the loss of copulatory behavior can be prevented by the administration of reasonable testosterone doses (Beach and HolzTucker, 1949; Malmnas and Meyerson, 1971; Hamburger-Bar and Rigterh, 1977). There is a question whether in castrated male rats it is I To whom correspondence and request for reprints should be sent 9 0018-506X/79/040009-12$01.00/0 Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
10
., ,
HLINAK,
MADLAFOUSEK,
AND MOHAPELOVA
possible to induce by means of suboptimal testosterone doses a more or less permanent state in which the intensity of the female’s precopulatory behavior proves to be the decisive factor for whether the male shall or shall not initiate copulatory behavior. The positive answer would also have methodological importance since the male in such a “suboptimal” state could be used as an advantageous “preparation” for the effective analysis of the regulation of sexual behavior whose components seem to be redundant, mutually compensating, and therefore masking one another (Madlafousek, Freund, and Grofova, 1970; Madlafousek and Hlitiak, 1977). The present study describes an attempt to solve the outlined question through once-a-week testosterone administration. The results were in part published in a preliminary form (Hlii%k, Madlafousek, and Mohapelova, 1977; Hlii%k and Madlafousek, 1979). GENERAL
METHODS
Animals
Wistar rats of commercial origin (Velaz, Prague) were reared in a colony room with controlled temperature under a reversed 12/12 daynight cycle with lights off at 9%) AM. The young were weaned at the age of 28 days; thereafter, males and females were kept separately in groups of three in breeding cages. Commercial pellets and tap water were available ad libitum. Selection
of Subjects
When 83 days old, the males were subjected to the standardized adaptation procedure to the experimental conditions in four sessions. Details were published elsewhere (Madlafousek et al., 1976). The last adaptation session (5 min) was followed by the selection test of copulatory readiness (the males aged 92 days). Every male was tested by means of a “stimulus” female (see below). The test was terminated when the sexually inexperienced male: (a) achieved three complete mounts ended by intromission; (b) had not performed any copulatory mounts within 10 min after the female was placed in the experimental box; (c) had not achieved intromission within 10 min after the first incomplete mount. Stimulus
Females
In testing the copulatory readiness of males the following stimulus females were used: (a) Lordotic females, i.e., females displaying no precopulatory behavior, but reacting rather regularly to the male’s copulatory mounts with lordosis posture; (b) Presenting females, i.e., females whose precopulatory behavior is reduced only to presenting postures; (c) Darting females, i.e., females exhibiting predominantly
TESTOSTERONE
DOSE AND
MATING
11
ritualized dartings terminated by presenting posture-this is the complete pattern of precopulatory behavior. The females exhibiting any intensity of precopulatory behavior have full copulatory readiness as demonstrated in the previous study (Madlafousek et al., 1977) where sexual behavior of female rats and its intensity degrees are described in detail. The technique used to induce precopulatory behavior of varying intensity through estradiol alone or in combination with progesterone was presented in Madlafousek and Hlinak (1977) and Hlin”Lk (1975). Lordotic females after priming with estradiol were, moreover, immobilized with haloperidol (0.5 mg im 1 hr before the test). The precopulatory behavior of the stimulus female was continuously observed during the actual tests of the male and she was replaced when necessary. Testing of Critical Copulatory Readiness Mating behavior of male rats includes incomplete mounts (clasping, clasping with palpation, clasping with palpation and thrusts) and copulatory behavior in the proper sense: complete mounts ended by intromission and the ejaculatory pattern. While incomplete mounts are frequently interspersed among intromissions, it is a rare phenomenon that the male performs only incomplete mounts without being able to achieve intromission. Further, after the first intromission the male usually continues the copulatory series until the ejaculatory behavioral pattern. In this study, “copulatory readiness” is defined as the ability to initiate and to perform at least three complete mounts ended by intromission. “Critical copulatory readiness” was then defined, as follows, by the threshold value of a stimulus female: Presenting female versus Darting female. The testing was mostly carried out in weekly intervals. At the end of a 2-min adaptation period a Presenting female was placed in the experimental box. If the male did not start copulation with a Presenting female, he was exposed 2 hr later to the sexual stimuli of a Darting female. If the male exhibited copulatory behavior with a Presenting female, he was not tested with a Darting female; we have found (unpublished observation) that such a male is able to copulate also with a Darting female. The 2-hr interval between both tests was aimed at reducing the influence of the sexual excitement of the former test. Larsson (1958) showed in intact males that such a long enforced interval erases any influence of previous copulatory activity. According to the current experience in our laboratory the repeated testing of copulatory readiness of sexually naive adult males with a Presenting female has success only occasionally. The serial effect of repeated testing within the 2-hr interval can be even more justifiably neglected in castrated males. This presupposition was substantiated in a pilot study: In Weeks 18 and 20 a group of seven males did not initiate copulation with a Presenting female but 2 hr later they copulated
12
HLIfijAK,
MADLAFOUSEK,
AND MOI-IAPELOVA
with a Darting female. How much of the effectiveness of the Darting female was influenced by previous exposure of the male to a Presenting female was tested in the intermediate week (Week 19) when the males were confronted only with a Darting female: Only one male did not start to copulate under this condition. A test of critical copulatory readiness was identified as positive when the male was not able to initiate copulatory behavior with a Presenting female, but exhibited copulatory behavior with a Darting female. Such a male fulfilled the copulatory criterion termed shortly as P-D+. A test was identified as negative when the male: (a) displayed copulatory behavior with a Presenting female (P+D+) or (b) exhibited copulatory behavior neither with a Presenting female nor with a Darting one (P-D-). In the course of all the tests the following items of a male’s precopulatory behavior directed toward the female were recorded: sniffing, grooming, exploration of anogenital region, jumping over, touching the flanks, pursuing, standing on the female from behind, and various manipulations using the forelimbs. Testosterone Administration Starting on the day of castration the males were subjected to the hormonal treatment: Every male was injected im with testosterone propionate (Agovirin, Spofa) in oil solution (0.22 ml) once a week. The initial doses were 700 &animal. The subsequent doses were individually modified according to the results of the critical copulatory readiness tests. When the test was identified as positive, the testosterone dose was not changed. In case of a negative result the testosterone dose was either decreased or increased, or an additional dose was injected. The following individual doses were used during the hormonal treatment: 700,600, 500, 400, and 350 pg/animal. The additional dose-5OOpg-was applied in the middle of the weekly interval. EXPERIMENT
I
The first experiment was designed to elaborate the procedure which would make it possible to induce in castrated male rats a “critical” hormonal state in which the male does not start to copulate with a Presenting female, but is ultimately stimulated to copulate with a Darting female. In addition, a question was asked whether it is possible to chronically maintain such a hormonal state with individually modified testosterone doses. The preliminary study has shown that it is disadvantageous to start with low testosterone doses and to increase them gradually. Therefore, in this experiment we proceeded in the reverse order. Before castration and several weeks after castration (in the course of early hormonal treatment) the males were subjected to sufficient and controlled sexual experience.
TESTOSTERONE
DOSE AND
MATING
13
Methods From the initial number of 24 sexually inexperienced males 22 animals displayed copulatory behavior under the condition of the first interaction with a Darting female. These males were subjected to the following copulatory experience procedure: in 3-day intervals they received a chance to copulate with a Darting female, then with a Presenting female, and then again with a Darting one. In the experiment proper we selected 12 males fulfilling the criterion of the first intromission in the second copulatory series, in all the three sessions of copulatory experience procedure. The third day after the last session of copulatory experience the males were castrated under a short ether narcosis. Testosterone dose-700 pg once a week/animal-was the same for all the animals in the initial 5 weeks of the hormonal treatment. Individual modification of testosterone doses started from the sixth week. Testing of copulatory readiness as well as of critical readiness was performed once a week 48 hr after the testosterone administration. In the initial 4 weeks the males were tested only with a Darting female (postcastrational sexual experience); all the males copulated regularly with such a female. Tests of the critical copulatory readiness started in the 5th week after castration, i.e., in the 5th week of the hormonal treatment. In both cases, i.e., in the interaction with a Presenting as well as with a Darting female, the test was terminated: (a) when the male achieved the first intromission of the second copulatory series; (b) 10 min after the female was placed in the box, if the male did not start any copulatory behavior; or (c) when the male did not achieve intromission within 10 min after the first incomplete mount. In the 10th week the males were tested only with a Darting female to verify that the former exposition to a Presenting female has no critical effect on the successive test performed 2 hr later with a Darting female. Results The results of the first experiment are summarized in Fig. 1. The number of males fulfilling the desired copulatory criterion P-D+ increases with gradual individual modification of testosterone doses. Starting with Week 11 the majority of males achieved the criterion P-D+. In Week 10 when tests only with a Darting female for the purpose of controlling the residual effect of tests with a Presenting female were performed, all 12 males copulated. The critical hormonal state, varying in duration from 2 to 11 weeks, was induced in all 12 experimental males. Altogether, 63.5% of the tests, starting with the week when the male first fulfilled the criterion P-D+, were positive (73 out of 115). The conspicuous decrease in Weeks 13 and 14, when some males did not copulate at all, was compensated by in-
14
HLINAK,
MADLAFOUSEK,
AND MOHAPELOVA
IW-
SO-
P+D+ so-
OH \\
5
11
6 WEEK
AFTER
14
17
CASTRATION
FIG. 1. Initiation of copulatory behavior in castrated male rats in dependency on the testosterone propionate doses and on the intensity of female’s precopulatory behavior. Top: Percentage of males fulfilling the criterion, i.e., copulating with Darting but not with Presenting female (P-D+). Middle: Percentage of males not fulfilling the criterion, i.e., copulating with Darting as well as with Presenting female (P+D+) or copulating with neither female (P-D-). Bottom: The numerals indicate the number of males injected in the given week by pertinent testosterone doses. Up to the fifth week, all 12 males were injected with 700 pg of testosterone propionate weekly, starting the castration week. (From Week 15-18 only 9 males were tested.)
creased testosterone doses and/or by the additional testosterone dose (500 pg/animal) . The amount of testosterone administered differed individually in the range 350-1200 Fg. During the period of stabilized critical hormonal state, i.e., from Week 15 18, the most frequent dose used was 700 pg (in 5%); doses of 500 or 700 ,cLgwere injected in 85% of the cases.
TESTOSTERONE
DOSE AND
MATING
15
In all the tests, including those in which the males did not initiate copulatory behavior, we recorded clean-cut precopulatory behavior directed toward the female. Only in three cases out of 304 tests were incomplete mounts observed without any intromission and ejaculatory pattern. In 73 tests in which the criterion P-D+ was fulfilled, the males in all cases executed the ejaculatory behavioral pattern and with the exception of one male in one test started the second copulatory series. In comparison to the precastrational measurement, the males in the critical state P- D+ had longer latency to the first intromission (r = 7.033; N = 12; P < O.OOl),however, ejaculatory latency (time from the first intromission to the ejaculatory pattern) was shortened (t = 5,120; N = 12; P < 0,001) and frequency of intromissions was lower (t = 4,064; N = 12; P < 0,Ol). EXPERIMENT
II
Since the first experiment confirmed the validity of the experimental procedure, a second experiment was designed with the aim of simplifying the induction of the critical hormonal state in castrated males. The design eliminated the requirement of the male’s extensive sexual experience. Further, individual modification of testosterone doses in the early weeks after castration was studied. In addition, a stricter selection of males was performed in an effort to obtain a more compact experimental group: In this experiment only the males with a low threshold of copulatory readiness, i.e., the males already initiating copulatory behavior in the interaction with a female exhibiting weak precopulatory behavior (Presenting female), were used. The finding of Experiment I, that the majority of males fulfilled the desired effect under treatment with 700 pg testosterone, was employed in the second phase of Experiment II. The ability of males to initiate copulatory behavior only with a “strong” female was studied under the condition of a uniform testosterone dose; the difference between the stimulus females used was increased: Instead of a Presenting female, a Lordotic one was used. Methods
Only 14 out of 22 sexually inexperienced males initiated copulatory behavior when exposed to a Presenting female. These 14 males were subjected to minimal sexual experience consisting of three intromissions in each of two sessions: the first being the above mentioned test of copulatory readiness, the second a test performed 1 week later using a Darting female. Two days after the second test the males were castrated and the hormonal treatment was immediately started. In the first 3 weeks (Weeks O-2) the males were injected with a constant dose of 700 pg testosterone
16
HLIkkK,
MADLAFOUSEK,
AND MOHAPELOVli
propionate/animal/week. In the following weeks the doses were individually modified according to the criterion described above. The testing of Critical Copulatory Readiness was performed from the first to the seventh week after the castration and took place 48 hr after the hormone administration. The males were first tested with a Presenting female and 2 hr later with a Darting one. In all tests the male had a chance to achieve only three intromissions in the first copulatory series. Hormonal treatment continued throughout Weeks 8 to 20. However, starting with Week 15 the males were transferred to a constant weekly testosterone dose of 700 pg. In Weeks 9, 12, 13, and 16 the control test of copulatory readiness with a Darting female was performed 24 hr after testosterone administration. Critical copulatory readiness of males was tested from Week 17 to 20 in this manner: A Lordotic female was offered 24 hr after testosterone administration, and a Darting one was used 24 hr later. In one of the last 2 weeks, when the male fulfilled the criterion L-D+, he had an opportunity to finish three copulatory series. In other tests three intromissions were used as the criterion of copulatory readiness. Results In the first phase of the experiment (Weeks 4-7) the critical hormonal state P-D+ was induced in 12 animals out of the group of 14 males. Two males copulated also with a Presenting female, in spite of gradual decrease of testosterone dose. In all, 52% of positive tests (i.e., 29 out of 56) were obtained. The testosterone doses used were: 500 kg in 2%, 600 pg in 54%, and 700 ,ug in 13% of cases. All of the six males which fulfilled the criterion P-D+ in Week 7 copulated also in Week 9 when confronted only with a Darting female (i.e., without being exposed to a Presenting female 2 hr earlier). In the second phase of the experiment (Weeks 17-20) when a constant testosterone dose-700 pg-was injected, all 14 males met the copulatory criterion L-D+ . On the whole, 79% of the tests were positive (44 out of 56). Under these conditions, 14 males (100%) met the copulatory criterion for at least 2 weeks, 10 males (71%) for 3 weeks, and 6 males (43%) displayed permanent critical hormonal state for all 4 weeks. All 12 males fulfilling the criterion L-D+ in Week 17 had copulated in the week preceding that in which they were exposed to a Darting female 24 hr after testosterone administration. With the exception of one male which performed only the first copulatory series, all other males fulfilling the criterion L-D+ finished three copulatory series. Precopulatory behavior of the experimental males was observed, without exception, in all tests (in both phases of experiment), including those in which the males did not display any copulatory behavior.
TESTOSTERONE
DOSE AND
MATING
17
DISCUSSION
Overall, in 24 out of 26 castrated male rats we succeeded, by means of adjusted testosterone doses, in the induction of the state persisting for a shorter or longer time (even for several weeks), in which the males were not stimulated to copulate with a Presenting female but started copulatory behavior 2 hr later when tested with a Darting female. With regard to the experimental design it is not entirely possible to eliminate the possibility that the former test (with a Presenting female) influences the result of the latter test (with a Darting female). However, this serial effect could not play a decisive role: (a) When relatively large testosterone doses were administered (this holds, for example, for the majority of males in the fifth and sixth weeks in Experiment I), the males started to copulate with a Presenting female. When testosterone doses were relatively small (for example, in the 13th and 14th weeks in Experiment I), the males did not initiate copulations with a Darting female, in spite of being exposed to a Presenting female 2 hr before. (b) Males fulfilling the criterion P-D+ also initiated copulation with a Darting female in the control week in which they were not confronted with a Presenting female. (c) In the second phase of Experiment II, the interval between testing males with Lordotic female and with Darting female was 24 hr. It can be concluded that for the initiation of copulatory behavior the interaction between the male’s hormonal state and the intensity of the female’s precopulatory behavior is pronounced. The “critical hormonal state” P-D+ was achieved both in males having sexual experience and in males with minimal sexual experience. The selection of males in accordance with their threshold of copulatory readiness measured through precopulatory behavior of varied intensity of the stimulus female (HliSik and Madlafousek, 1972) was not necessary. However, it can be expected that this factor could have influenced the success of the initiation of the critical hormonal state. In the present study the technique of once-a-week testosterone propionate treatment was used. In the majority of cases the critical hormonal state P-D+ or L-D+ in the range 500-700 ,ug of testosterone was achieved. These testosterone doses converted to a daily dose (70- 100pg) are only a little larger than the daily maintenance doses determined by Beach and Holz-Tucker (1949) and recently used by Parrott (1974). For the achievement of the critical hormonal state and for its preservation it has shown to be more profitable to start with larger testosterone doses and decrease them gradually through individual modification. The critical hormonal state L-D+ (i.e., a larger range of the intensity of female precopulatory behavior) was achieved by means of a standard dose of 700 pg testosterone. A single testosterone administration (once a week/ animal) cannot ensure a longlasting and uniform activation of the central
18
HLItiAK,
MADLAFOUSEK,
AND MOHAPELOVbr
and peripheral target tissues. It seems that the use of testosterone-filled Silastic capsules (Kincl, Benagiano, and Angee, 1968; Damassa, Smith, Tennent, and Davidson, 1977) can contribute to the acquisition of more differentiated and more stabilized animal “preparations” in the future. This was suggested in the.preliminary experiments of our laboratory; in a certain phase after implantation of testosterone-filled Silastic capsules in castrated males the critical hormonal state was achieved-such males did not initiate copulations with a passive Lordotic female, but they started copulatory behavior with a Darting one. Comparing the copulatory performance of males in the critical hormonal state P-D+ (Hlirlak and Madlafousek, 1979) with the copulatory performance of males with implanted testosterone capsules (Damassa ef al., 1977) we surmise that weekly injections of 700 pg of testosterone propionate correspond to testosterone-filled capsules 2-5 mm long. Sexual behavior seems to be determined multilaterally and redundantly (Madlafousek et al., 1970, 1976). The absence or weakness of some determinant may result in no observable changes of sexual behavior, when remaining determinants are sufficient in number and quality. For example, sexually inexperienced adult intact male rats may initiate copulation, but only with females exhibiting appropriate intensity of precopulatory behavior (Hlin”ak and Madlafousek, 1972). After some sexual experience, the males copulate even with a Lordotic female exhibiting no precopulatory behavior (Madlafousek and Hlitiik, 1971). Sexual experience and specific sexual stimuli can mutually compensate for one another. Other determinants being present, one determinant can obscure or mask another. To test the role of suspected factors, it is desirable, if not necessary, to remove or weaken the known components of the determination of sexual behavior. For example, a lesion or stimulation of the relevant brain structures might have an observable effect only when the treated animal is in an adequate, sexually “critical” state. The reduction of the level of sexual hormones proved to be one of the suitable methods for weakening the determination of sexual behavior. The experimental males in this study are practically akin to such “preparations” applicable for further analysis of sexual behavior’s determinants. Their critical sexual state was defined through the dependency on the intensity of sexual stimuli on the part of the female. What are further characteristics of these experimental male “preparations” ? (a) The males exhibit precopulatory behavior, in the whole range of observed elements, even toward a stimulus female with which they are not stimulated to initiate copulatory behavior. A similar phenomenon was observed in inexperienced adult males (I-Ili%k and Madlafousek, 1972). (b) As soon as the males pass from precopulatory activities to the mating behavior they perform incomplete mounts as well as complete
TESTOSTERONE
DOSE AND MATING
19
mounts terminated by intromission patterns. In this respect they differ from the castrated males not hormonally treated. Such untreated animals pass through a temporary stage (in the course of the gradual decline of sexual behavior) in which they exhibit incomplete mounts without performing intromission and ejaculatory behavioral patterns (Madlafousek et al., 1976). (c) Although the intromission latency increases, the males terminate copulatory series with the ejaculatory pattern in a short time, performing a small number of intromissions. In this respect, these males are similar to untreated males following castration (Davidson, 1966). (d) The males are able to execute several (at least three) copulatory series.
REFERENCES Beach, F. A., and Holz-Tucker, M. A. (1949). Effects of different concentrations of androgen upon sexual behavior in castrated male rats. J. Camp. Physiol. Psycho/. 42, 433-453.
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. Horm. Behav. 8, 275-286.
Davidson, J. M. (1966). Characteristics of sex behavior in male rats following castration. Anim. Behav.
14, 266-272.
Hamburger-Bar, R., and Rigterh, H. (1977). Peripheral and central androgenic stimulation of sexual behavior of castrated male rats. Acta Endocrinol. 84, 813-828. HliiYak, Z. (1975). Strain-associated differences in the action of oestradiol and progesterone in inducing of precopulatory behaviour in ovariectomized rats. Physiol. Bohemoslov. 24, 381-383. Hliiiak, Z., and Madlafousek, J. (1972). The dependence of sexual behaviour of inexperienced males on the precopulatory behaviour of females in albino rat. Physiol. Bohemoslov. 21, 83-84.
Hlinak, Z., and Madlafousek, J. (1979). Copulatory behaviour of castrated male rats injected with suboptimal testosterone doses. Acriv. Nerv. Super. 21, 52-53. Hlinak, Z., Madlafousek, J., and Mohapelova, A. (1977). Initiation of copulatory activity in castrated male rats injected with submaximal testosterone doses. Physiol. Bohemoslov. 26, 448.
Kincl, F. A., Benagiano, G., and Angee, I. (1%8). Sustained release hormonal preparations. 1. Diffitssion of various steroids through polymer membranes. Steroids 11, 673-680. Larsson, K. (1958). Aftereffects of copulatory activity of the male rat: I. J. Comp. Physiol. Psycho/.
51, 325-327.
Madlafousek, J., Freund, K., and Grofova, I. (1970). Variables determining the effect of electrostimulation in the lateral preoptic area on the sexual behavior of male rats. J. Comp. Physiol.
Psychol.
72, 28-44.
Madlafousek, J., and Hlitiak, Z. (1971). The first copulations of adult males change their dependence on the female precopulatory behaviour (in rats). Is this a point of learning? ds. Psycho/. 15, 112-122. Madlafousek, J., and Hlitiak, Z. (1977). Sexual behaviour of the female laboratory rat: Inventory, patterning, and measurement. Behaviour 63, 129-174. Madlafousek, J., HlitW, Z., and Beran, J. (1976). Decline of sexual behavior in castrated male rats! Effects of female precopulatory behavior. Horm. Behav. 7, 245-252.
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AND MOHAPELOVA
Malmnls, C. O., and Meyerson, B. J. (1971). p-Chlorophenylalanine and copulatory behaviour in the male rats. Nature (London) 232, 398-400. Parrott, R. F. (1974). Effects of 17/3-hydro-4-androsten-19-ol-3-one (lPhydroxytestosterone) and Sa-androstan- 17@ol-3-one(dihydrotestosterone) on aspects of sexual behaviour in castrated male rats. J. Endocrinol. 61, 105-l 15.
AND
Initiation
BEHAVIOR
13, 9-20 (1979)
of Copulatory Behavior in Castrated Rats Injected with Critically Adjusted Doses of Testosterone
Z. HLIGK’, Psychiatric
J. MADLAFOUSEK, Research
Institute,
Male
AND A. MOHAPELOVA
181 03 Prague 8, Czechoslovakia
In castrated male rats it was possible by means of administration of adequate testosterone propionate doses to induce a state in which the males did not initiate copulatory behavior with a stimulus female displaying no (Lordotic female) or weak (Presenting female) intensity of precopulatory behavior. On the other hand, such males started to copulate and finished copulatory series when a stimulus female exhibiting the complete pattern of precopulatory behavior (Darting female) was offered. The males “prepared” in this manner displayed regularly precopulatory behavior directed toward all the stimulus females. The requisite testosterone doses differed individually. However, in the majority of cases they were in the range of 500-700 /*g of testosterone injected once a week.
In the previous study (Madlafousek, Hlinak, and Beran, 1976) we showed that the declining ability of castrated male rats to initiate copulatory behavior is substantially dependent on the stimulus female used in testing the male. Females exhibiting low-intensity precopulatory behavior (presenting posture) were compared with those exhibiting complete precopulatory behavior (characterized by darting responses). Such females do not differ with respect to receptivity; their copulatory readiness, as expressed by the lordosis quotient, is practically 100%. Several weeks after castration, when no male was stimulated to mount or intromit a female which showed the presenting postures (Presenting female), males would initiate copulatory behavior with females showing the darting responses (Darting female). About 9 weeks after castration not even a Darting female was sufficient to compensate for the hormonal insufficiency of castrated males. It is well known that the loss of copulatory behavior can be prevented by the administration of reasonable testosterone doses (Beach and HolzTucker, 1949; Malmnas and Meyerson, 1971; Hamburger-Bar and Rigterh, 1977). There is a question whether in castrated male rats it is I To whom correspondence and request for reprints should be sent 9 0018-506X/79/040009-12$01.00/0 Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
10
., ,
HLINAK,
MADLAFOUSEK,
AND MOHAPELOVA
possible to induce by means of suboptimal testosterone doses a more or less permanent state in which the intensity of the female’s precopulatory behavior proves to be the decisive factor for whether the male shall or shall not initiate copulatory behavior. The positive answer would also have methodological importance since the male in such a “suboptimal” state could be used as an advantageous “preparation” for the effective analysis of the regulation of sexual behavior whose components seem to be redundant, mutually compensating, and therefore masking one another (Madlafousek, Freund, and Grofova, 1970; Madlafousek and Hlitiak, 1977). The present study describes an attempt to solve the outlined question through once-a-week testosterone administration. The results were in part published in a preliminary form (Hlii%k, Madlafousek, and Mohapelova, 1977; Hlii%k and Madlafousek, 1979). GENERAL
METHODS
Animals
Wistar rats of commercial origin (Velaz, Prague) were reared in a colony room with controlled temperature under a reversed 12/12 daynight cycle with lights off at 9%) AM. The young were weaned at the age of 28 days; thereafter, males and females were kept separately in groups of three in breeding cages. Commercial pellets and tap water were available ad libitum. Selection
of Subjects
When 83 days old, the males were subjected to the standardized adaptation procedure to the experimental conditions in four sessions. Details were published elsewhere (Madlafousek et al., 1976). The last adaptation session (5 min) was followed by the selection test of copulatory readiness (the males aged 92 days). Every male was tested by means of a “stimulus” female (see below). The test was terminated when the sexually inexperienced male: (a) achieved three complete mounts ended by intromission; (b) had not performed any copulatory mounts within 10 min after the female was placed in the experimental box; (c) had not achieved intromission within 10 min after the first incomplete mount. Stimulus
Females
In testing the copulatory readiness of males the following stimulus females were used: (a) Lordotic females, i.e., females displaying no precopulatory behavior, but reacting rather regularly to the male’s copulatory mounts with lordosis posture; (b) Presenting females, i.e., females whose precopulatory behavior is reduced only to presenting postures; (c) Darting females, i.e., females exhibiting predominantly
TESTOSTERONE
DOSE AND
MATING
11
ritualized dartings terminated by presenting posture-this is the complete pattern of precopulatory behavior. The females exhibiting any intensity of precopulatory behavior have full copulatory readiness as demonstrated in the previous study (Madlafousek et al., 1977) where sexual behavior of female rats and its intensity degrees are described in detail. The technique used to induce precopulatory behavior of varying intensity through estradiol alone or in combination with progesterone was presented in Madlafousek and Hlinak (1977) and Hlin”Lk (1975). Lordotic females after priming with estradiol were, moreover, immobilized with haloperidol (0.5 mg im 1 hr before the test). The precopulatory behavior of the stimulus female was continuously observed during the actual tests of the male and she was replaced when necessary. Testing of Critical Copulatory Readiness Mating behavior of male rats includes incomplete mounts (clasping, clasping with palpation, clasping with palpation and thrusts) and copulatory behavior in the proper sense: complete mounts ended by intromission and the ejaculatory pattern. While incomplete mounts are frequently interspersed among intromissions, it is a rare phenomenon that the male performs only incomplete mounts without being able to achieve intromission. Further, after the first intromission the male usually continues the copulatory series until the ejaculatory behavioral pattern. In this study, “copulatory readiness” is defined as the ability to initiate and to perform at least three complete mounts ended by intromission. “Critical copulatory readiness” was then defined, as follows, by the threshold value of a stimulus female: Presenting female versus Darting female. The testing was mostly carried out in weekly intervals. At the end of a 2-min adaptation period a Presenting female was placed in the experimental box. If the male did not start copulation with a Presenting female, he was exposed 2 hr later to the sexual stimuli of a Darting female. If the male exhibited copulatory behavior with a Presenting female, he was not tested with a Darting female; we have found (unpublished observation) that such a male is able to copulate also with a Darting female. The 2-hr interval between both tests was aimed at reducing the influence of the sexual excitement of the former test. Larsson (1958) showed in intact males that such a long enforced interval erases any influence of previous copulatory activity. According to the current experience in our laboratory the repeated testing of copulatory readiness of sexually naive adult males with a Presenting female has success only occasionally. The serial effect of repeated testing within the 2-hr interval can be even more justifiably neglected in castrated males. This presupposition was substantiated in a pilot study: In Weeks 18 and 20 a group of seven males did not initiate copulation with a Presenting female but 2 hr later they copulated
12
HLIfijAK,
MADLAFOUSEK,
AND MOI-IAPELOVA
with a Darting female. How much of the effectiveness of the Darting female was influenced by previous exposure of the male to a Presenting female was tested in the intermediate week (Week 19) when the males were confronted only with a Darting female: Only one male did not start to copulate under this condition. A test of critical copulatory readiness was identified as positive when the male was not able to initiate copulatory behavior with a Presenting female, but exhibited copulatory behavior with a Darting female. Such a male fulfilled the copulatory criterion termed shortly as P-D+. A test was identified as negative when the male: (a) displayed copulatory behavior with a Presenting female (P+D+) or (b) exhibited copulatory behavior neither with a Presenting female nor with a Darting one (P-D-). In the course of all the tests the following items of a male’s precopulatory behavior directed toward the female were recorded: sniffing, grooming, exploration of anogenital region, jumping over, touching the flanks, pursuing, standing on the female from behind, and various manipulations using the forelimbs. Testosterone Administration Starting on the day of castration the males were subjected to the hormonal treatment: Every male was injected im with testosterone propionate (Agovirin, Spofa) in oil solution (0.22 ml) once a week. The initial doses were 700 &animal. The subsequent doses were individually modified according to the results of the critical copulatory readiness tests. When the test was identified as positive, the testosterone dose was not changed. In case of a negative result the testosterone dose was either decreased or increased, or an additional dose was injected. The following individual doses were used during the hormonal treatment: 700,600, 500, 400, and 350 pg/animal. The additional dose-5OOpg-was applied in the middle of the weekly interval. EXPERIMENT
I
The first experiment was designed to elaborate the procedure which would make it possible to induce in castrated male rats a “critical” hormonal state in which the male does not start to copulate with a Presenting female, but is ultimately stimulated to copulate with a Darting female. In addition, a question was asked whether it is possible to chronically maintain such a hormonal state with individually modified testosterone doses. The preliminary study has shown that it is disadvantageous to start with low testosterone doses and to increase them gradually. Therefore, in this experiment we proceeded in the reverse order. Before castration and several weeks after castration (in the course of early hormonal treatment) the males were subjected to sufficient and controlled sexual experience.
TESTOSTERONE
DOSE AND
MATING
13
Methods From the initial number of 24 sexually inexperienced males 22 animals displayed copulatory behavior under the condition of the first interaction with a Darting female. These males were subjected to the following copulatory experience procedure: in 3-day intervals they received a chance to copulate with a Darting female, then with a Presenting female, and then again with a Darting one. In the experiment proper we selected 12 males fulfilling the criterion of the first intromission in the second copulatory series, in all the three sessions of copulatory experience procedure. The third day after the last session of copulatory experience the males were castrated under a short ether narcosis. Testosterone dose-700 pg once a week/animal-was the same for all the animals in the initial 5 weeks of the hormonal treatment. Individual modification of testosterone doses started from the sixth week. Testing of copulatory readiness as well as of critical readiness was performed once a week 48 hr after the testosterone administration. In the initial 4 weeks the males were tested only with a Darting female (postcastrational sexual experience); all the males copulated regularly with such a female. Tests of the critical copulatory readiness started in the 5th week after castration, i.e., in the 5th week of the hormonal treatment. In both cases, i.e., in the interaction with a Presenting as well as with a Darting female, the test was terminated: (a) when the male achieved the first intromission of the second copulatory series; (b) 10 min after the female was placed in the box, if the male did not start any copulatory behavior; or (c) when the male did not achieve intromission within 10 min after the first incomplete mount. In the 10th week the males were tested only with a Darting female to verify that the former exposition to a Presenting female has no critical effect on the successive test performed 2 hr later with a Darting female. Results The results of the first experiment are summarized in Fig. 1. The number of males fulfilling the desired copulatory criterion P-D+ increases with gradual individual modification of testosterone doses. Starting with Week 11 the majority of males achieved the criterion P-D+. In Week 10 when tests only with a Darting female for the purpose of controlling the residual effect of tests with a Presenting female were performed, all 12 males copulated. The critical hormonal state, varying in duration from 2 to 11 weeks, was induced in all 12 experimental males. Altogether, 63.5% of the tests, starting with the week when the male first fulfilled the criterion P-D+, were positive (73 out of 115). The conspicuous decrease in Weeks 13 and 14, when some males did not copulate at all, was compensated by in-
14
HLINAK,
MADLAFOUSEK,
AND MOHAPELOVA
IW-
SO-
P+D+ so-
OH \\
5
11
6 WEEK
AFTER
14
17
CASTRATION
FIG. 1. Initiation of copulatory behavior in castrated male rats in dependency on the testosterone propionate doses and on the intensity of female’s precopulatory behavior. Top: Percentage of males fulfilling the criterion, i.e., copulating with Darting but not with Presenting female (P-D+). Middle: Percentage of males not fulfilling the criterion, i.e., copulating with Darting as well as with Presenting female (P+D+) or copulating with neither female (P-D-). Bottom: The numerals indicate the number of males injected in the given week by pertinent testosterone doses. Up to the fifth week, all 12 males were injected with 700 pg of testosterone propionate weekly, starting the castration week. (From Week 15-18 only 9 males were tested.)
creased testosterone doses and/or by the additional testosterone dose (500 pg/animal) . The amount of testosterone administered differed individually in the range 350-1200 Fg. During the period of stabilized critical hormonal state, i.e., from Week 15 18, the most frequent dose used was 700 pg (in 5%); doses of 500 or 700 ,cLgwere injected in 85% of the cases.
TESTOSTERONE
DOSE AND
MATING
15
In all the tests, including those in which the males did not initiate copulatory behavior, we recorded clean-cut precopulatory behavior directed toward the female. Only in three cases out of 304 tests were incomplete mounts observed without any intromission and ejaculatory pattern. In 73 tests in which the criterion P-D+ was fulfilled, the males in all cases executed the ejaculatory behavioral pattern and with the exception of one male in one test started the second copulatory series. In comparison to the precastrational measurement, the males in the critical state P- D+ had longer latency to the first intromission (r = 7.033; N = 12; P < O.OOl),however, ejaculatory latency (time from the first intromission to the ejaculatory pattern) was shortened (t = 5,120; N = 12; P < 0,001) and frequency of intromissions was lower (t = 4,064; N = 12; P < 0,Ol). EXPERIMENT
II
Since the first experiment confirmed the validity of the experimental procedure, a second experiment was designed with the aim of simplifying the induction of the critical hormonal state in castrated males. The design eliminated the requirement of the male’s extensive sexual experience. Further, individual modification of testosterone doses in the early weeks after castration was studied. In addition, a stricter selection of males was performed in an effort to obtain a more compact experimental group: In this experiment only the males with a low threshold of copulatory readiness, i.e., the males already initiating copulatory behavior in the interaction with a female exhibiting weak precopulatory behavior (Presenting female), were used. The finding of Experiment I, that the majority of males fulfilled the desired effect under treatment with 700 pg testosterone, was employed in the second phase of Experiment II. The ability of males to initiate copulatory behavior only with a “strong” female was studied under the condition of a uniform testosterone dose; the difference between the stimulus females used was increased: Instead of a Presenting female, a Lordotic one was used. Methods
Only 14 out of 22 sexually inexperienced males initiated copulatory behavior when exposed to a Presenting female. These 14 males were subjected to minimal sexual experience consisting of three intromissions in each of two sessions: the first being the above mentioned test of copulatory readiness, the second a test performed 1 week later using a Darting female. Two days after the second test the males were castrated and the hormonal treatment was immediately started. In the first 3 weeks (Weeks O-2) the males were injected with a constant dose of 700 pg testosterone
16
HLIkkK,
MADLAFOUSEK,
AND MOHAPELOVli
propionate/animal/week. In the following weeks the doses were individually modified according to the criterion described above. The testing of Critical Copulatory Readiness was performed from the first to the seventh week after the castration and took place 48 hr after the hormone administration. The males were first tested with a Presenting female and 2 hr later with a Darting one. In all tests the male had a chance to achieve only three intromissions in the first copulatory series. Hormonal treatment continued throughout Weeks 8 to 20. However, starting with Week 15 the males were transferred to a constant weekly testosterone dose of 700 pg. In Weeks 9, 12, 13, and 16 the control test of copulatory readiness with a Darting female was performed 24 hr after testosterone administration. Critical copulatory readiness of males was tested from Week 17 to 20 in this manner: A Lordotic female was offered 24 hr after testosterone administration, and a Darting one was used 24 hr later. In one of the last 2 weeks, when the male fulfilled the criterion L-D+, he had an opportunity to finish three copulatory series. In other tests three intromissions were used as the criterion of copulatory readiness. Results In the first phase of the experiment (Weeks 4-7) the critical hormonal state P-D+ was induced in 12 animals out of the group of 14 males. Two males copulated also with a Presenting female, in spite of gradual decrease of testosterone dose. In all, 52% of positive tests (i.e., 29 out of 56) were obtained. The testosterone doses used were: 500 kg in 2%, 600 pg in 54%, and 700 ,ug in 13% of cases. All of the six males which fulfilled the criterion P-D+ in Week 7 copulated also in Week 9 when confronted only with a Darting female (i.e., without being exposed to a Presenting female 2 hr earlier). In the second phase of the experiment (Weeks 17-20) when a constant testosterone dose-700 pg-was injected, all 14 males met the copulatory criterion L-D+ . On the whole, 79% of the tests were positive (44 out of 56). Under these conditions, 14 males (100%) met the copulatory criterion for at least 2 weeks, 10 males (71%) for 3 weeks, and 6 males (43%) displayed permanent critical hormonal state for all 4 weeks. All 12 males fulfilling the criterion L-D+ in Week 17 had copulated in the week preceding that in which they were exposed to a Darting female 24 hr after testosterone administration. With the exception of one male which performed only the first copulatory series, all other males fulfilling the criterion L-D+ finished three copulatory series. Precopulatory behavior of the experimental males was observed, without exception, in all tests (in both phases of experiment), including those in which the males did not display any copulatory behavior.
TESTOSTERONE
DOSE AND
MATING
17
DISCUSSION
Overall, in 24 out of 26 castrated male rats we succeeded, by means of adjusted testosterone doses, in the induction of the state persisting for a shorter or longer time (even for several weeks), in which the males were not stimulated to copulate with a Presenting female but started copulatory behavior 2 hr later when tested with a Darting female. With regard to the experimental design it is not entirely possible to eliminate the possibility that the former test (with a Presenting female) influences the result of the latter test (with a Darting female). However, this serial effect could not play a decisive role: (a) When relatively large testosterone doses were administered (this holds, for example, for the majority of males in the fifth and sixth weeks in Experiment I), the males started to copulate with a Presenting female. When testosterone doses were relatively small (for example, in the 13th and 14th weeks in Experiment I), the males did not initiate copulations with a Darting female, in spite of being exposed to a Presenting female 2 hr before. (b) Males fulfilling the criterion P-D+ also initiated copulation with a Darting female in the control week in which they were not confronted with a Presenting female. (c) In the second phase of Experiment II, the interval between testing males with Lordotic female and with Darting female was 24 hr. It can be concluded that for the initiation of copulatory behavior the interaction between the male’s hormonal state and the intensity of the female’s precopulatory behavior is pronounced. The “critical hormonal state” P-D+ was achieved both in males having sexual experience and in males with minimal sexual experience. The selection of males in accordance with their threshold of copulatory readiness measured through precopulatory behavior of varied intensity of the stimulus female (HliSik and Madlafousek, 1972) was not necessary. However, it can be expected that this factor could have influenced the success of the initiation of the critical hormonal state. In the present study the technique of once-a-week testosterone propionate treatment was used. In the majority of cases the critical hormonal state P-D+ or L-D+ in the range 500-700 ,ug of testosterone was achieved. These testosterone doses converted to a daily dose (70- 100pg) are only a little larger than the daily maintenance doses determined by Beach and Holz-Tucker (1949) and recently used by Parrott (1974). For the achievement of the critical hormonal state and for its preservation it has shown to be more profitable to start with larger testosterone doses and decrease them gradually through individual modification. The critical hormonal state L-D+ (i.e., a larger range of the intensity of female precopulatory behavior) was achieved by means of a standard dose of 700 pg testosterone. A single testosterone administration (once a week/ animal) cannot ensure a longlasting and uniform activation of the central
18
HLItiAK,
MADLAFOUSEK,
AND MOHAPELOVbr
and peripheral target tissues. It seems that the use of testosterone-filled Silastic capsules (Kincl, Benagiano, and Angee, 1968; Damassa, Smith, Tennent, and Davidson, 1977) can contribute to the acquisition of more differentiated and more stabilized animal “preparations” in the future. This was suggested in the.preliminary experiments of our laboratory; in a certain phase after implantation of testosterone-filled Silastic capsules in castrated males the critical hormonal state was achieved-such males did not initiate copulations with a passive Lordotic female, but they started copulatory behavior with a Darting one. Comparing the copulatory performance of males in the critical hormonal state P-D+ (Hlirlak and Madlafousek, 1979) with the copulatory performance of males with implanted testosterone capsules (Damassa ef al., 1977) we surmise that weekly injections of 700 pg of testosterone propionate correspond to testosterone-filled capsules 2-5 mm long. Sexual behavior seems to be determined multilaterally and redundantly (Madlafousek et al., 1970, 1976). The absence or weakness of some determinant may result in no observable changes of sexual behavior, when remaining determinants are sufficient in number and quality. For example, sexually inexperienced adult intact male rats may initiate copulation, but only with females exhibiting appropriate intensity of precopulatory behavior (Hlin”ak and Madlafousek, 1972). After some sexual experience, the males copulate even with a Lordotic female exhibiting no precopulatory behavior (Madlafousek and Hlitiik, 1971). Sexual experience and specific sexual stimuli can mutually compensate for one another. Other determinants being present, one determinant can obscure or mask another. To test the role of suspected factors, it is desirable, if not necessary, to remove or weaken the known components of the determination of sexual behavior. For example, a lesion or stimulation of the relevant brain structures might have an observable effect only when the treated animal is in an adequate, sexually “critical” state. The reduction of the level of sexual hormones proved to be one of the suitable methods for weakening the determination of sexual behavior. The experimental males in this study are practically akin to such “preparations” applicable for further analysis of sexual behavior’s determinants. Their critical sexual state was defined through the dependency on the intensity of sexual stimuli on the part of the female. What are further characteristics of these experimental male “preparations” ? (a) The males exhibit precopulatory behavior, in the whole range of observed elements, even toward a stimulus female with which they are not stimulated to initiate copulatory behavior. A similar phenomenon was observed in inexperienced adult males (I-Ili%k and Madlafousek, 1972). (b) As soon as the males pass from precopulatory activities to the mating behavior they perform incomplete mounts as well as complete
TESTOSTERONE
DOSE AND MATING
19
mounts terminated by intromission patterns. In this respect they differ from the castrated males not hormonally treated. Such untreated animals pass through a temporary stage (in the course of the gradual decline of sexual behavior) in which they exhibit incomplete mounts without performing intromission and ejaculatory behavioral patterns (Madlafousek et al., 1976). (c) Although the intromission latency increases, the males terminate copulatory series with the ejaculatory pattern in a short time, performing a small number of intromissions. In this respect, these males are similar to untreated males following castration (Davidson, 1966). (d) The males are able to execute several (at least three) copulatory series.
REFERENCES Beach, F. A., and Holz-Tucker, M. A. (1949). Effects of different concentrations of androgen upon sexual behavior in castrated male rats. J. Camp. Physiol. Psycho/. 42, 433-453.
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. Horm. Behav. 8, 275-286.
Davidson, J. M. (1966). Characteristics of sex behavior in male rats following castration. Anim. Behav.
14, 266-272.
Hamburger-Bar, R., and Rigterh, H. (1977). Peripheral and central androgenic stimulation of sexual behavior of castrated male rats. Acta Endocrinol. 84, 813-828. HliiYak, Z. (1975). Strain-associated differences in the action of oestradiol and progesterone in inducing of precopulatory behaviour in ovariectomized rats. Physiol. Bohemoslov. 24, 381-383. Hliiiak, Z., and Madlafousek, J. (1972). The dependence of sexual behaviour of inexperienced males on the precopulatory behaviour of females in albino rat. Physiol. Bohemoslov. 21, 83-84.
Hlinak, Z., and Madlafousek, J. (1979). Copulatory behaviour of castrated male rats injected with suboptimal testosterone doses. Acriv. Nerv. Super. 21, 52-53. Hlinak, Z., Madlafousek, J., and Mohapelova, A. (1977). Initiation of copulatory activity in castrated male rats injected with submaximal testosterone doses. Physiol. Bohemoslov. 26, 448.
Kincl, F. A., Benagiano, G., and Angee, I. (1%8). Sustained release hormonal preparations. 1. Diffitssion of various steroids through polymer membranes. Steroids 11, 673-680. Larsson, K. (1958). Aftereffects of copulatory activity of the male rat: I. J. Comp. Physiol. Psycho/.
51, 325-327.
Madlafousek, J., Freund, K., and Grofova, I. (1970). Variables determining the effect of electrostimulation in the lateral preoptic area on the sexual behavior of male rats. J. Comp. Physiol.
Psychol.
72, 28-44.
Madlafousek, J., and Hlitiak, Z. (1971). The first copulations of adult males change their dependence on the female precopulatory behaviour (in rats). Is this a point of learning? ds. Psycho/. 15, 112-122. Madlafousek, J., and Hlitiak, Z. (1977). Sexual behaviour of the female laboratory rat: Inventory, patterning, and measurement. Behaviour 63, 129-174. Madlafousek, J., HlitW, Z., and Beran, J. (1976). Decline of sexual behavior in castrated male rats! Effects of female precopulatory behavior. Horm. Behav. 7, 245-252.
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., ,
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AND MOHAPELOVA
Malmnls, C. O., and Meyerson, B. J. (1971). p-Chlorophenylalanine and copulatory behaviour in the male rats. Nature (London) 232, 398-400. Parrott, R. F. (1974). Effects of 17/3-hydro-4-androsten-19-ol-3-one (lPhydroxytestosterone) and Sa-androstan- 17@ol-3-one(dihydrotestosterone) on aspects of sexual behaviour in castrated male rats. J. Endocrinol. 61, 105-l 15.
