HORMONES
AND BEHAVIOR,
6, l-g (1975)
Effects of Dihydrotestosterone and Estradiol Benzoate Pretreatment Upon Testosterone-Induced Sexual Behavior in the Castrated Male Rat
K. LARSSON,
G. PEREZPALACIOS,
G. MORALI,
and C. BEYER
Departamento de Investigacibn Gent&a, Institute Mexican0 de1 Seguro Social, Apartado PostaZ 73-032, M&co 73, D.F., M&co
Institute National de la Nutricibn, Viaduct0 Tlalpan y San Fernando, M&co 22, D.F., MPxico
Three groups of inexperienced castrated male rats were treated daily oil, e&radio1 benzoate (1 crp), or diiydrotestosterone (1 mg), and thereafter injected daily with testosterone (1 mg) for 21 days. Sexual behavior was tested every third day after the start of the pretreatment until day 36. Estradiol benzoate or dihydrotestosterone failed to elicit
for 15 days with
sexual behavior. Pretreatment with dihydrotestosterone, but not estradiol benzoate, significantly shortened the intervals to initiation of mounting and intromission in response to testosterone. The results suggest that fully developed genitals (penis and/or sexual accessories) facilitate initiation of copulatory behavior in response to testosterone administration.
Castration of inexperienced male rats prevents initiation of copulatory behavior (see Beach, 1948). Sexual behavior is initiated in the inexperienced castrated male rat by free testosterone (T) treatment but the full mating pattern occurs with a considerable latency (Beyer, Larsson, Perez-Palacios, and Morali, 1973). The cause for this delay has not been established but lack of testicular hormones results in atrophy of the penis and sexual accessories as well as a reduction in the concentration of enzymes (Singhal and Valadares, 1968; Singhal and Ling, 1969; Liao and Fang, 1969), cyclic nucleotides (Espinosa, Sayao, Beyer, and Perez-Palacios, 1973) and androphilic molecules (Liao, Tymoczko, Castaiieda, and Shao, 1973) in some androgen effecters. Therefore, the delay in the behavioral response to T could reflect the temporal course of events involved in restoring the normal functioning of these systems.
Copyright 01975 by Academic Press, Inc. AU rights of reproduction in any form reserved.
2
LARSSON ET AL.
The present study was aimed at investigating the effects of estradiol benzoate (EB) and dihydrotestosterone (DHT) pretreatments on the subsequent behavioral response to T administration. Neither DHT nor EB are effective in low dosages to activate mating behavior in castrated rats (Beyer, Larsson, Perez-Palacios, and Morali, 1973; Davidson, 1969). The rationale of this study was that pretreatment with DHT by developing sexual accessories or with EB by stimulating the synthesis of some molecules probably involved in androgen action (see Koide, 1969; Steeno, 1970; Pitot and Yatvin, 1973) would shorten the latency for the initiation of sexual behavior in response to T.
METHODS
Subjects Subjects were 56 sexually inexperienced young Sprague-Dawley male rats (av weight 222 gm at the start of treatment) from our colony. They were housed in individual cageswith food and water ad lib. and 10 light/14 hr dark cycles. The rats were castrated 25 days before initiation of the hormone treatment.
Hormone Treatment The rats were assigned randomly to three groups, (see Table 1 for number of animals) were injected SC once daily during 15 days with either sesame oil, EB (1 pg), or DHT (1 mg). These or larger doses of these steroids have been found ineffective in stimulating copulatory behavior in castrated male rats (Larsson, Sodersten, and Beyer, 1973; Beyer, Larsson, Perez-Palacios, and Morali, 1973). The volumes of injections were 0.02 ml (EB) or 0.04 ml (DHT and sesame oil). After the pretreatments all groups were injected once daily with T (1 mg in 0.1 ml sesameoil) for 21 days.
Testing Tests were begun 72 hr after the initiation of the pretreatment (Day 3) and were continued every third day until Day 36 during the dark phase of the light-dark cycle. The testing arena was a cylindrical observation cage (l/16 in. Plexiglas, 53 cm diam, 42 cm high). Females used as stimulus animals were brought into estrus with EB (3 pg/animal/day). After a 3-min adaptation period in the observation cage, the male was presented with a sexually receptive female. Every 5 min the female was exchanged for a new one, thus providing the male with optimal sexual stimulation.
STEROID HORMONESAND MATING
3
The following behavior variables were measured: (1) Mount, (2) mount latency, (3) intromission, (4) intromission latency, (5) ejaculation latency, and (6) postejaculatory interval. These behavioral variables have been previously defined (Beyer, Larsson, Perez-Palacios, and Morali, 1973). The term “ejaculation” will be used in this paper for the display of the ejaculatory pattern. The test was ended when one of the following conditions was fulfilled: (a) 15 min after the presentation of the female to the male, if at that time no intromission had taken place, (b) 30 min after the first intromission if no ejaculation had taken place, (c) 15 min after ejaculation, if no intromission had occurred; (d) after the first intromission after ejaculation. After the last test the animals were killed by overexposure to ether. Statistical Analysis Behavioral measures were analyzed by the Mann-Whitney U test or by the x2 test. Group differences were considered as significant when P = .05 was reached (two-tailed test). RESULTS The main results of this study are presented in Figs. 1 and 2 showing the average intervals in days for the first occurrence of mounting, intromission, and ejaculation in the three groups (Fig. l), as well as the proportion of responsive rats in each group during both the pretreatment (DHT; EB) and T treatment periods (Fig. 2). Rats pretreated with DHT showed mounts (P< .Ol), intromissions (P-C .OS), but not ejaculations earlier than did oiltreated rats (V Mann-Whitney test). Statistical comparison of the proportions of rats in the diverse groups showing the complete mating pattern at each test day showed that 9 days after the initiation of T treatment the DHT pretreated group showed a significantly larger proportion of responsive rats than the control group (5 of 19 pretreated rats mated vs 0 of 19 oil-treated rats mated, x2 = 5.76, P< 0.02). This suggests that DHT pretreatmenf accelerated the appearance of sexual behavior in response to T treatment. EB pretreatment had no significant effect on either the interval to the display of sexual activity or in the proportion of responsive rats to treatment. As can be seen in Table 1, the mating pattern did not show any group differences with the exception of the ejaculation latency which was prolonged in both experimental groups, i.e., those pretreated with EB or DHT. DISCUSSION Control rats displayed the ejaculatory pattern at least 14 days after the beginning of T treatment. This long delay in the behavioral response to free T
LARSSON ET AL. MOUNT
INTROMISSION
EJACULATION
16-
14-
2-
0-
an EB aL
an
a
aL
Fig. 1. Latency in days for the first appearance of mount, intromission, and ejaculation in the various treatment groups (M + SE).
agrees with previous observations on inexperienced castrated male rats from our colony (Beyer et al., 1973). Pretreatment with DHT, but not EB, shortened this delay. A similar result with DHT has been independently obtained by Sodersten (personal communication). The mechanism through which DHT exerted this effect is not clear, and several possible sites of action can be proposed for further analysis. DHT could have acted on the central nervous system either by “sensitizing” the brain substrate for sexual behavior to the subsequent action of T or by increasing the excitability of the spinal reflexes involved in mating. On the other hand, it is also possible that DHT exerted its effect by acting on the male genitalia (sexual accessories and penis). Data exist indicating that a fully developed penis is important, though not essential, for mating in the male rat (Beach and Holtz, 1946). Moreover, it appears that sensory inflow from the penis and/or genital region might have some role in sexual behavior since anesthetization of the penis (Carlsson and Larsson, 1964; Adler and Bermant, 1966) or sectioning of the dorsal penile nerve (Larsson and Sodersten, 1973) interferes with intromission and ejaculation. Although there is no evidence
STEROID HORMONES AND MATING
loo
5
_.a---
1
IJIQEB
-..I-..
I mg
I-PRETREATMENT-TESTOSTERONE
DHT
TREATMENT-
&PRETREATMENTI
TESTOSTERONE
TREATMENT
+
0 I2
I5
I8
24
30
36
(day*)
Fig. 2. Cumulative percentage of rats showing mounting, intromission, and ejaculation during various treatments. Note that pretreatment with DHT c---X--) clearly accelerated the display of sexual behavior in response to T administration.
19
Dihydrotestosterone
12
10
13
Mount
*p
AND BEHAVIOR,
6, l-g (1975)
Effects of Dihydrotestosterone and Estradiol Benzoate Pretreatment Upon Testosterone-Induced Sexual Behavior in the Castrated Male Rat
K. LARSSON,
G. PEREZPALACIOS,
G. MORALI,
and C. BEYER
Departamento de Investigacibn Gent&a, Institute Mexican0 de1 Seguro Social, Apartado PostaZ 73-032, M&co 73, D.F., M&co
Institute National de la Nutricibn, Viaduct0 Tlalpan y San Fernando, M&co 22, D.F., MPxico
Three groups of inexperienced castrated male rats were treated daily oil, e&radio1 benzoate (1 crp), or diiydrotestosterone (1 mg), and thereafter injected daily with testosterone (1 mg) for 21 days. Sexual behavior was tested every third day after the start of the pretreatment until day 36. Estradiol benzoate or dihydrotestosterone failed to elicit
for 15 days with
sexual behavior. Pretreatment with dihydrotestosterone, but not estradiol benzoate, significantly shortened the intervals to initiation of mounting and intromission in response to testosterone. The results suggest that fully developed genitals (penis and/or sexual accessories) facilitate initiation of copulatory behavior in response to testosterone administration.
Castration of inexperienced male rats prevents initiation of copulatory behavior (see Beach, 1948). Sexual behavior is initiated in the inexperienced castrated male rat by free testosterone (T) treatment but the full mating pattern occurs with a considerable latency (Beyer, Larsson, Perez-Palacios, and Morali, 1973). The cause for this delay has not been established but lack of testicular hormones results in atrophy of the penis and sexual accessories as well as a reduction in the concentration of enzymes (Singhal and Valadares, 1968; Singhal and Ling, 1969; Liao and Fang, 1969), cyclic nucleotides (Espinosa, Sayao, Beyer, and Perez-Palacios, 1973) and androphilic molecules (Liao, Tymoczko, Castaiieda, and Shao, 1973) in some androgen effecters. Therefore, the delay in the behavioral response to T could reflect the temporal course of events involved in restoring the normal functioning of these systems.
Copyright 01975 by Academic Press, Inc. AU rights of reproduction in any form reserved.
2
LARSSON ET AL.
The present study was aimed at investigating the effects of estradiol benzoate (EB) and dihydrotestosterone (DHT) pretreatments on the subsequent behavioral response to T administration. Neither DHT nor EB are effective in low dosages to activate mating behavior in castrated rats (Beyer, Larsson, Perez-Palacios, and Morali, 1973; Davidson, 1969). The rationale of this study was that pretreatment with DHT by developing sexual accessories or with EB by stimulating the synthesis of some molecules probably involved in androgen action (see Koide, 1969; Steeno, 1970; Pitot and Yatvin, 1973) would shorten the latency for the initiation of sexual behavior in response to T.
METHODS
Subjects Subjects were 56 sexually inexperienced young Sprague-Dawley male rats (av weight 222 gm at the start of treatment) from our colony. They were housed in individual cageswith food and water ad lib. and 10 light/14 hr dark cycles. The rats were castrated 25 days before initiation of the hormone treatment.
Hormone Treatment The rats were assigned randomly to three groups, (see Table 1 for number of animals) were injected SC once daily during 15 days with either sesame oil, EB (1 pg), or DHT (1 mg). These or larger doses of these steroids have been found ineffective in stimulating copulatory behavior in castrated male rats (Larsson, Sodersten, and Beyer, 1973; Beyer, Larsson, Perez-Palacios, and Morali, 1973). The volumes of injections were 0.02 ml (EB) or 0.04 ml (DHT and sesame oil). After the pretreatments all groups were injected once daily with T (1 mg in 0.1 ml sesameoil) for 21 days.
Testing Tests were begun 72 hr after the initiation of the pretreatment (Day 3) and were continued every third day until Day 36 during the dark phase of the light-dark cycle. The testing arena was a cylindrical observation cage (l/16 in. Plexiglas, 53 cm diam, 42 cm high). Females used as stimulus animals were brought into estrus with EB (3 pg/animal/day). After a 3-min adaptation period in the observation cage, the male was presented with a sexually receptive female. Every 5 min the female was exchanged for a new one, thus providing the male with optimal sexual stimulation.
STEROID HORMONESAND MATING
3
The following behavior variables were measured: (1) Mount, (2) mount latency, (3) intromission, (4) intromission latency, (5) ejaculation latency, and (6) postejaculatory interval. These behavioral variables have been previously defined (Beyer, Larsson, Perez-Palacios, and Morali, 1973). The term “ejaculation” will be used in this paper for the display of the ejaculatory pattern. The test was ended when one of the following conditions was fulfilled: (a) 15 min after the presentation of the female to the male, if at that time no intromission had taken place, (b) 30 min after the first intromission if no ejaculation had taken place, (c) 15 min after ejaculation, if no intromission had occurred; (d) after the first intromission after ejaculation. After the last test the animals were killed by overexposure to ether. Statistical Analysis Behavioral measures were analyzed by the Mann-Whitney U test or by the x2 test. Group differences were considered as significant when P = .05 was reached (two-tailed test). RESULTS The main results of this study are presented in Figs. 1 and 2 showing the average intervals in days for the first occurrence of mounting, intromission, and ejaculation in the three groups (Fig. l), as well as the proportion of responsive rats in each group during both the pretreatment (DHT; EB) and T treatment periods (Fig. 2). Rats pretreated with DHT showed mounts (P< .Ol), intromissions (P-C .OS), but not ejaculations earlier than did oiltreated rats (V Mann-Whitney test). Statistical comparison of the proportions of rats in the diverse groups showing the complete mating pattern at each test day showed that 9 days after the initiation of T treatment the DHT pretreated group showed a significantly larger proportion of responsive rats than the control group (5 of 19 pretreated rats mated vs 0 of 19 oil-treated rats mated, x2 = 5.76, P< 0.02). This suggests that DHT pretreatmenf accelerated the appearance of sexual behavior in response to T treatment. EB pretreatment had no significant effect on either the interval to the display of sexual activity or in the proportion of responsive rats to treatment. As can be seen in Table 1, the mating pattern did not show any group differences with the exception of the ejaculation latency which was prolonged in both experimental groups, i.e., those pretreated with EB or DHT. DISCUSSION Control rats displayed the ejaculatory pattern at least 14 days after the beginning of T treatment. This long delay in the behavioral response to free T
LARSSON ET AL. MOUNT
INTROMISSION
EJACULATION
16-
14-
2-
0-
an EB aL
an
a
aL
Fig. 1. Latency in days for the first appearance of mount, intromission, and ejaculation in the various treatment groups (M + SE).
agrees with previous observations on inexperienced castrated male rats from our colony (Beyer et al., 1973). Pretreatment with DHT, but not EB, shortened this delay. A similar result with DHT has been independently obtained by Sodersten (personal communication). The mechanism through which DHT exerted this effect is not clear, and several possible sites of action can be proposed for further analysis. DHT could have acted on the central nervous system either by “sensitizing” the brain substrate for sexual behavior to the subsequent action of T or by increasing the excitability of the spinal reflexes involved in mating. On the other hand, it is also possible that DHT exerted its effect by acting on the male genitalia (sexual accessories and penis). Data exist indicating that a fully developed penis is important, though not essential, for mating in the male rat (Beach and Holtz, 1946). Moreover, it appears that sensory inflow from the penis and/or genital region might have some role in sexual behavior since anesthetization of the penis (Carlsson and Larsson, 1964; Adler and Bermant, 1966) or sectioning of the dorsal penile nerve (Larsson and Sodersten, 1973) interferes with intromission and ejaculation. Although there is no evidence
STEROID HORMONES AND MATING
loo
5
_.a---
1
IJIQEB
-..I-..
I mg
I-PRETREATMENT-TESTOSTERONE
DHT
TREATMENT-
&PRETREATMENTI
TESTOSTERONE
TREATMENT
+
0 I2
I5
I8
24
30
36
(day*)
Fig. 2. Cumulative percentage of rats showing mounting, intromission, and ejaculation during various treatments. Note that pretreatment with DHT c---X--) clearly accelerated the display of sexual behavior in response to T administration.
19
Dihydrotestosterone
12
10
13
Mount
*p
