Independence of temporal patterning of male mating behavior from the influence of androgen during the neonatal period

Physiology and Behavior, Vol. 14, pp. 251-254. Brain Research Publications Inc., 1975. Printed in the U.S.A.

Independence of Temporal Patterning of Male Mating Behavior from the Influence of Androgen During the Neonatal Period MONICA SCHOELCH-KRIEGER 1 AND RONALD J. BARFIELD

Department o f Biology, Livingston College, Rutgers University, New Brunswick, N J 08903

(Received 23 July 1974) SCHOELCH-KRIEGER, M. AND R. J. BARFIELD. Independence of temporal patterning of male mating behavior from the influence of androgen during the neonatal period. PHYSIOL. BEHAV. 14(3) 251-254, 1975.- The purpose of this experiment was to determine whether the presence of androgen neonatally influences the development of temporal patterning of male copulatory behavior in rats. Neonatally castrated males, neonatally androgenized females, and postpubertally castrated males and females were tested for male mating behavior following androgen treatment in adulthood. All groups exhibited a similar periodicity of male copulatory behavior (mount-bouts); however, postpubertally castrated males showed a higher frequency of intromission and ejaculatory patterns, while postpubertally castrated females exhibited fewer copulatory events per mount-bout than either neonatally castrated males or androgenized females. It was concluded that there is an identity of the temporal patterning of male mating behavior that is unaffected by either genetic sex or neonatal hormonal milieu; but, super-imposed upon this fundamental rhythm there are hormonally determined differences in copulatory performance. Androgen

Neonatal

Malecopulation

Temporal patterning

ADMINISTRATION of androgen to neonatal female rats results in an increase in the number of mounts [ 10,17] and intromission reflexes [20,21] shown by these animals as adults. Neonatal castration of male rats results in a decreased capacity to exhibit patterns of intromission and ejaculation [5, 8, 9, 11, 20]. Thus, androgen present neonatally affects the performance of male copulatory behavior exhibited by adults. Androgen present neonatally, however, also affects the potential for genital development [5,6]. This latter effect may account for the observed alterations in male copulatory behavior since the frequency of occurrence of the intromission and ejaculatory reflexes is generally well correlated with phallic size [4, 12, 18, 19]. To assess the influence of androgen in the neonate on the development of male mating behavior it would be desirable to analyze the resultant behavior (seen in the adult) in such a way as to avoid confounding performance of sexual behavior with genital development. Such an analysis can be carried out on the temporal patterning of copulatory activity since it is relatively independent of factors influencing the genitalia [ 15]. This should allow us to better differentiate the relative contribution of peripheral (genital) factors and hypothetical CNS factors in the development of male sexual behavior. It was the objective of the present study to determine whether androgen present neonatally plays a role in the development of temporal patterning of male copulatory behavior.

Mount-bouts

METHOD

Animals Seventy-three Long-Evans hooded rats born in this laboratory were used. They were weaned at 28 days and caged in unisexual groups of 2 - 3 of the same age and treatment group. Cages measured 30 × 18 x 18 cm. A constant supply of food and water was available to the animals. Lighting was maintained on a 1 2 - 1 2 reversed light-dark cycle, with lights off at 10 a. m. Treatment groups. Litters were sexed on the day of birth (Day 1). Each animal was assigned to one of the following groups according to sex and treatment received: (A) males untreated at birth; (B) males castrated on Day 1; (C) females untreated at birth; (D) females treated with testosterone propionate (TP) on Day 1.

Procedure E x p e r i m e n t a l treatments. For neonatal castration animals were placed under ether anesthesia; incisions and removal of testes were carried out with watchmakers forceps. After the operation the neonate was returned to the nursing mother. TP (500 ug) was administered to neonatal female rats by subcutaneous injection in 0.01 cc of oil. All females and intact males were castrated at 8 weeks of age. At 12 weeks all experimental animals had a 10 mg pellet of TP implanted subcutaneously.

Present address: Department of Biology, Mount Holyoke College, South Hadley, Mass. U.S.A. 251

Development

252

SCHOELCH-KRIGER AND BARFIELD

Test procedure. Testing began 4 weeks after the TP pellet was implanted. Animals were tested 2 to 4 times at intervals of 3 to 7 days. Tests were 15 min long and were carried out under red light between 1300 and 1800 hours. Ten gallon aquaria (40 x 26 × 29 cm) with cedar shaving covering the floor were used as observation cages. Each animal was allowed 5 min to adapt to the test situation prior to the introduction of a receptive female. Receptivity was induced in stimulus females by injection of 60 ug of estradiol benzoate 54 hr before the test and injection of 0.5 mg of progesterone 4 - 6 hr before testing. The following patterns of behavior were recorded on a push-button-actuated strip-chart event recorder: (a) Mount, with pelvic thrusts; (b) Intromission Pattern, mount with pelvic thrusts followed by a deep thrust and a rapid dismount; (c) Ejaculation Pattern, vigorous mounting with thrusting followed by a deep thrust with a slow dismount; (d) Termination of Mount-bout, a mount-bout has been defined as a sequence of mounts and/or intromissions, uninterrupted by any behavior (other than genital autogrooming) that is not oriented toward the female [ 15 ]. The termination is the point at which the copulating animal takes its attention away from the stimulus female or ceases to lick its own genitalia following a bout of copulatory activity. Tests were terminated if the animal did not mate for the first 10 min of the test or if the animal had intervals greater than 300 sec between any successive mount-bout (except postejaculatory intervals). Any animal which exhibited no positive tests was termed an unreliable mater and the data from those animals were excluded. Data analysis. Data from the first 12 min following the first copulatory response, or until the first ejaculation, were used in the analysis. This provided a comparable time base among all of the groups since ejaculation latency of the normal males was 10 to 12 min on the average. The standard measures of copulatory behavior obtained were as follows: mount latency (ML); mount frequency (MF); and intromission frequency (IF). Other measures based on the occurrence of mount bouts (MB) were also obtained. They were: mount-bout frequency (MBF); number of copulatory responses (mounts plus intromissions) per MB; mount-bout

period (MBP), the time from the first copulatory response of a MB to the first response of the next MB; and time out (TO), the time from the last copulatory response of a MB until the first response of the next MB. For each measure each animal received an average score based on its performance in all of the tests in which it met mating criteria (see test procedure). RESULTS A comparable percentage of all groups were found to satisfy the criteria for mating; a high percentage of all maters exhibited the intromission pattern, but only the n o r m a l males regularly demonstrated the ejaculatory pattern (See Table 1). In Table 2 it is shown that while ML does not significantly differ among groups, postpubertally castrated males have a significantly greater IF (p~<0.0005) and a smaller MF (p~<0.005) than the other 3 groups. When the individual mount-bouts are examined it is found that the neonatally castrated males and androgenized females showed more total responses per mount-bout than either postpubertally castrated males or females (p~<0.025). The temporal analysis of male mating behavior shows that the periodicity of mount-bouts is similar among all of the .groups. Analyses of variance indicate that although normal males have somewhat shorter MB periods and TO the difference does not approach statistical significance (p~0.15, see Table 3). DISCUSSION In the present study we have demonstrated that androgen present neonatally has no effect either on the percentage of animals that were reliable maters or on their ML (Table 1). Earlier, Pfaff and Zigmond [ 13] also found that similarly treated animals showed no differences in either ML or time spent sniffing the genitalia of the estrous female. We have further demonstrated that the temporal patterning of male copulatory behavior is also independent of androgen present during infancy. Previously we showed that the periodicity of male mating behavior exhibited by

TABLE 1 FREQUENCY OF EXHIBITION OF MASCULINE COPULATORY BEHAVIOR IN NORMAL MALES, NEONATALLY CASTRATED MALES, NORMAL FEMALES AND NEONATALLY ANDROGENIZED FEMALES

Normal Males

Neonatally Castrated Males

Normal Females

Neonatally Androgenized Females

Number Tested

18

11

19

25

Number Mating

12 66%

8 73%

11 58%

20 80%

% Maters Exhibiting Ejaculatory Reflex

100

0

0

10

% Maters Exhibiting Intromission Reflex

100

100

82

85

ANDROGEN AND MALE MATING BEHAVIOR

253 TABLE 2

COMPARISON OF THE COPULATORY BEHAVIOR OF NEONATALLY CASTRATED MALES, NEONATALLY ANDROGENIZED FEMALES, NORMAL FEMALES AND NORMAL MALES. (MEAN VALUES -+ S.E.M. ARE PRESENTED)

Normal Males

Castrated Males

Neonatally Normal Females

Androgenized Females

F*

56.0 -+ 30.0

51.0 -+ 23.0

53.0 -+ 24.0

102.0 -+ 30.0

1.77

Mount Frequency

8.9 -+ 2.1

22.9 -+ 5.2

17.3 -+ 2.0

21.9 -+ 1.9

4.925

Intromission Frequency

8.4 _+ 0.8

2.5 -+ 0.8

1.9 -+ 0.6

3.4 -+ 0.7

44.16§

Mount-bout Frequency

11.8 -+ 0.7

10.8 -+ 1.9

12.8 _+ 1.2

12.4 +- 1.0

0.71

Mounts + Intromissions per Mount-bout

1.4 -+ 0.1

2.0 -+ 0.3

1.5 -+ 0.1

1.9 -+ 0.1

3.8t

Mount Latency

Neonatally

*Analysis of variance, df = 3,47 tP<0.025 $p~0.005 §p~<0.0005 TABLE 3 COMPARISON OF THE TEMPORAL PATTERNING OF COPULATORY BEHAVIOR IN NEONATALLY CASTRATED MALES, NEONATALLY ANDROGENIZED FEMALES, NORMAL FEMALES AND NORMAL MALES. (MEAN VALUES -+ S.E.M. ARE PRESENTED)

Normal Males

NeonataUy Castrated Males

Normal Females

Neonatally Androgenized Females

F*

Mount-bout Period

52.2 -+ 5.2

66.1 -+ 3.8

64.4 +- 5.3

61.4 -+ 3.9

1.8 t

Time out

46.7 -+ 5.5

59.0 +- 5.8

59.6 -+ 4.8

50.6 -+ 3.9

1.5~:

*Analysis of variance, df = 3,47 tP~0.15 $p>~0.20

normal males, neonatally androgenized females and perinatally androgenized females was identical [ 1 6 ] . These findings taken t o g e t h e r indicate that androgen plays no role in the d e v e l o p m e n t o f the capacity to either initiate male c o p u l a t o r y behavior or, once initiated, to carry it forth with a typical periodicity of m o u n t - b o u t s . The p e r f o r m a n c e of sexual behavior was, however, affected by the presence of androgen in neonatal rats. N o r m a l males exhibited m o r e ejaculatory patterns and m o r e intromission patterns per test than any o t h e r group. If t h e i n d i v i d u a l m o u n t - b o u t s are dissected, neonatally c a s t r a t e d males and neonatally androgenized females showed m o r e c o p u l a t o r y events ( m o u n t s plus intromission patterns) per m o u n t - b o u t than did normal females and normal males. Thus, the differences in mating behavior are reflected not in the rate o f the m o u n t - b o u t s , but in their internal structure. The process by which male c o p u l a t o r y behavior develops appears to involve at least two groups o f c o m p o n e n t responses. The first group would include the initiation and the t e m p o r a l patterning of mating. These responses might

be considered indicative o f male c o p u l a t o r y m o t i v a t i o n ; they are not influenced by the presence o f androgen in infancy. The second group consists of intromission and ejaculatory patterns. These responses are highly d e p e n d e n t upon androgenic influences in early life in b o t h males and females [5, 9, 1 1, 20, 2 1 ] . Since the d e v e l o p m e n t of these responses is also correlated with increased phallic growth in response to androgen administered in adulthood [ 4 , 1 8 ] , it has been proposed that the principal action of androgen on the d e v e l o p m e n t of these responses is peripheral by virtue of a sensitization o f the phallus neonatally to stimulatory effects of androgen in a d u l t h o o d [4,19]. These two groups of responses are co-ordinated such that underlying the hormonally d e p e n d e n t differences in c o p u l a t o r y performance there are mechanisms controlling the initiation and timing o f male mating behavior which are i n d e p e n d e n t of neonatal h o r m o n a l influences. The finding that the t e m p o r a l patterning of male mating behavior is i n d e p e n d e n t o f the presence of androgen in the perinatal period leads to a m o r e general consideration of the pacing o f c o p u l a t o r y behavior. The t e m p o r a l patterning

254

S C H O E L C H - K R I E G E R AND B A R F I E L I )

of sexual behavior o f females has been s h o w n to be very similar to t h a t o f n o r m a l males. E s t r o u s females given the o p p o r t u n i t y t o a p p r o a c h or bar-press for a male did so at intervals w h i c h are virtually identical to the inter-intromission intervals s h o w n by males o f the same strain [ 7 , 1 4 ] . The fact that the male t i m e r is i n d e p e n d e n t o f b o t h h o r m o n a l c o n d i t i o n and genetic sex suggests that it could be one and the same as that controlling the pacing o f female sexual behavior. Perhaps, a unitary, sex indep e n d e n t , t i m e r can drive either the male or the female c o p u l a t o r y r h y t h m according to the behavioral and physiological d e t e r m i n a n t s prevailing. Such a m e c h a n i s m may be i m p o r t a n t in d e t e r m i n i n g the r e p r o d u c t i v e success o f the species. First, it w o u l d provide a basis for the s y n c h r o n y of

male and female c o p u l a t o r y r h y t h m s . S e c o n d , it may provide a basis for the c o r r e l a t i o n of the s t e r e o t y p e d c o p u l a t o r y behavior o f the male and its role in the facilitation o f i m p l a n t a t i o n in the female so clearly d e m o n s t r a t e d in rats [ 2 , 3 ] . W h e t h e r there are one, or t w o related, timers will d o u b t l e s s be the subject o f future research. ACKNOWLEDGEMENTS This research was supported by research grant HD 04484, from the USPHS, and by a Biomedical Science Support (}rant to Rutgers University from the USPHS. Hormones used in this study were generously donated by Dr. P. Perlman, Schering Corp., Bloomfield, New Jersey.

REFERENCES I. Adler, N. and G. Bermant. Sexual behavior in male rats; effects of reduced sensory feedback. J. comp. physiol. Psyehol. 61: 240-243, 1966. 2. Adler, N. T., J. A. Resko and R. W. Goy. The effect of copulatory behavior on hormonal change in the female rat prior to implantation. PhysioL Behav. 5: 1003-1008, 1970. 3. Adler, N. T. and S. R. Zoloth. Copulatory behavior can inhibit pregnancy in the female rat. Science 168: 1480-1482, 1970. 4. Beach, F. A. Hormonal factors controlling the differentiation, development and display of copulatory behavior in the ramstergig and related species. In: The Bio-psychology of Development, edited by E. Tobach, L. R. Aronson and E. Shaw. New York: Academic Press, 1971, pp. 249--296. 5. Beach, F. A. and A. M. Holz. Mating behavior in male rats castrated at various ages and injected with androgen. J. exp. ZooL 101:91-142, 1946. 6. Beach, F. A., R. G. Noble and R. K. Ordnoff. Effects of perinatal treatment on responses of male rats to gonadal hormones in adulthood. J. comp. physiol. PsychoL 68: 490-497, 1969. 7. Bermant, G. Response latencies of female rats during sexual intercourse. Science 133: 1771, 1961. 8. Gerall, A. A. S. E. Hendricks, L. L. Johnson and T. W. Bounds. Effects of early castration in male rats on adult sexual behavior. J. comp. physiol. Psychol. 64: 206-212, 1967. 9. Grady, K. L., C. H. Phoenix and W. C. Young. Role of the developing testis in differentiation of the neural tissues mediating mating behavior. J. comp. physiol. Psychol. 59: 176-182, 1965. 10. Harris, G. W. and S. Levine. Sexual differentiation of the brain and its experimental control. J. Physiol. 181: 379-400, 1965. 11. Larsson, K. Effects of neonatal castration upon the development of mating behavior of male rats. Z. Tierpsychol. 13: 867-873, 1966.

12. Nadler, R. D. Differentiation of the capacity for male sexual behavior in the rat. Hormones Behav. 1: 53 -63, 1969. 13. Pfaff, D. W. and R. E. Zigmond. Neonatal androgen effects on sexual and non-sexual behavior of adult rats tested under various hormone regimes. Neuroendoerinolog_v 7: 129.-145, 1971. 14. Pierce, J. T. and R. L. Nuttall. Self-paced sexual behavior in female rats. J. comp. physiol. Psychol. 54:310 313, 1961. 15. Sachs, B. D. and R. J. Barfield. Temporal patterning of sexual behavior in the male rat. J. comp. physiol. PsvchoL 73: 359 364, 1970. 16. Sachs, B. D., E. Pollak, M. S. Krieger and R. J. Barfield. Sexual behavior: Normal male patterning in androgenized females. Science 181: 770-772, 1973. 17. Sodersten, P. Increased mounting behavior in the female rat following a single neonatal injection of testosterone propionate. Hormones Behav. 4:1 - 17, 1973. 18. Ward, I. L. Differential effect of pre- and postnatal androgen on sexual behavior of intact and spayed female rats. Hormones Behav. 1: 25-36, 1969. 19. Whalen, R. E. Differentiation of the neural mechanisms which control gonadotropin secretion and sexual behavior. In: Perspective in Reproduction and Sexual Behavior, edited by M. Diamond. Bloomington, Indiana: Indiana University Press. 1968, pp. 303-340. 20. Whalen, R. E. and D. A. Edwards. Hormonal determinants of the development of masculine and feminine behavior in male and female rats. Anat. Rec. 157:173 180, 1967. 21. Whalen, R. E., D. A. Edwards, W. G. Luttge and R. T. Robertson. Early androgen treatment and male sexual behavior in female rats. Physiol. Behav. 4: 33-39, 1969.