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Testosterone induced mounting behavior in adult female rats born in litters of different female to male ratios
Physiology & Behavior, Voi. 28, pp. 1007-1010. Pergamon Press and Brain Research Publ., 1982. Printed in the U.S.A.
Testosterone Induced Mounting Behavior in Adult Female Rats Born in Litters of Different Female to Male Ratios A. KOOS S L O B A N D P I E T E R V A N D E R S C H O O T
Department of Endocrinology, Growth and Reproduction Faculty o f Medicine, Erasmus University, P.O. Box 1738, Rotterdam, The Netherlands Received 2 D e c e m b e r 1981 SLOB, A. K. AND P. VAN DER SCHOOT. Testosterone induced mounting behavior in adult female rats born in litters o f different female to male ratios. PHYSIOL. BEHAV. 28(6) 1007-1010, 1982.--Female rats can show mounting behavior towards isosexual or heterosexual conspecifics. The present experiments were designed to study whether the prenatal presence of male fetuses would affect display of this mounting behavior in adulthood. Therefore mounting behavior, after gonadectomy and during continuous treatment with testosterone propionate (TP) was studied in female rats which were born in litters without male siblings ("all female" litters) and in litters with a variable number of male siblings. Litter composition at birth did not affect display of adult mounting behavior during protracted tests (a total of 6 tests during 8 weeks of treatment with TP). The data indicate that TP induced mounting behavior in adulthood occurs independent of the prenatal presence of male fetuses, ff mounting behavior in adulthood has to be "organized" by prenatally present androgen (the current way of thinking), then the present data indicate that female rat fetuses provide themselves ~ith these hormones. It would then seem inappropriate to judge adult mounting behavior as a sign of "masculinization" of the rat brain. Mounting behavior
Litter composition at birth
Female rats
ADULT female rats have been reported to display mounting and intromission behavior without receiving exogenous hormones perinatally or in adulthood (e.g., [2, 3, 20, 27]). Such observations were initially interpreted as suggesting that neural mechanisms regulating "masculine" sexual behavior develop in the absence of either pre- or postnatal androgenic stimulation (see review in [1]). However, other evidence suggested that female rats were normally exposed prenatally to a certain amount of androgenic stimulation, and that this exposure may partially masculimze all females of this species (see also [14]). The first investigators to suggest an endogenous prenatal androgenic modification of the nervous system ("masculinization") of female rats were Ward and Renz [28]. Their finding that prenatal exposure to cyproterone acetate (a synthetic anti-androgen) "decreased markedly the amount and quality of male behavior below that of control females suggests that the low level of male behavior typically elicited in normal females by adult androgen treatment (Gerall & Ward, 1966; Ward & Sperr, 1969) results, at least in part, from prenatal stimulation by endogenous androgen" ([28], p. 354). They speculated that this androgen was of adrenal origin. Another line of supportive evidence for this (endogenous) hypothesis of prenatal androgen masculinization of female rats came from the work of Clemens and collaborators. They were the first to suggest a prenatal morphological and behavioral "masculinization" of female fetuses through exposure to androgen from the testes of male fetuses [ 7, 8, 9]. These
investigators reported that female fetuses next to male fetuses in one uterine horn had a larger ano-genital distance at birth and displayed more mounting behavior in response to administration of testosterone in adult life than did female fetuses that had not been in such close proximity to fetal male rats. Also in mice it was reported that the positions of the female fetuses in the uterus relative to the male fetuses determined the extent to which the female mouse was masculinized in terms of morphology (ano-genital distance at birth) and behavior (testosterone-induced fighting in adulthood) [10], as well as reproductively important characteristics during adulthood [23, 24, 25, 26]. Clemens and coworkers [9] as well as Vom Saal and co-workers [23] suggested that fetal male androgens reach the female fetus by way of direct diffusion across the amniotic membranes late in gestation. For this latter hypothesis, however, Slob, Ooms and Vreeburg [18] were not able to find supportive evidence. They found equivalent endogenous serum levels of testosterone in female rat fetuses on Day 20 of gestation, regardless of the in utero (one uterine horn) ratio of male to female fetuses. If the prenatal presence and/or proximity of male fetuses affects adult reproductive behavior in female rats, then clear behavioral differences should be apparent between females stemming from all female litters and females born in litters with an abundant number of male siblings. The present experiment was designed to test this hypothesis.
Copyright © 1982 Brain Research Publications Inc.--0031-9384/82/0601007-04503.00/0
1008
SLOB AND VAN DER SCHOOT TABLE 1 NUMBER OF FEMALES IN EACH OF THE 4 EXPERIMENTAL CONDITIONS AS WELL AS THE NUMBER OF LITTERS AND LITTERSIZE AT BIRTH
Litter Composition " S e x Ratio"
Number of Litters
Littersize at Birth Mean Median Range
Number of Females Used in Experiment
? 9 Q9 (all female) 9 Q9 d (predominantly female) 9 Qd d (fifty-fifty)
5
4.6
4
1-9
23
5
6.6
6
5-9
9
8
6.0
6
2-12
8
$ddd
4
6.8
7
4-9
4
(predominantly male)
METHOD
During November and December 1980 all litters born in a large breeding colony (Wistar, Amsterdam-R strain; Department of Anatomy, Faculty of Medicine, Erasmus University, Rotterdam) were checked immediately after delivery. Four "types" of litters were assigned to this experiment: (1) all female litters (n=5); (2) one male and 4 to 8 females (n=5); (3) equal number of female and male siblings (n=8); (4) one female and 3 to 8 males (n=4). Initially the animals were kept at 22-23°C and exposed to 14 hr light:10 hr darkness/day (lights on 05.00-19.00 hr). The animals grew up with their mothers in the litter composition at birth, were weaned at 22 days of age and then housed 3 or 4 of same sex to a cage. From litter-types 1 and 4 all females were used, from litter-types 2 and 3 one or two females were randomly selected from each litter at weaning and subsequently used in the experiment (see Table 1). The animals were allowed free access to pellet food and tap water. At the age of about 2 months all animals were transferred to an other animal quarter and exposed to a reversed 14 hr light: 10 hr darkness/day (lights on 21.30-7.30 hr). Beginning at the age of about 21/2 months all females were daily tested (in the middle of the dark period) for the occurrence of earwiggiing, hopping and darting (proceptivity, [5]) by introducing a sexually active male in the cage for a few minutes. This procedure was continued for 40 days and revealed information about the occurrence of behavioral estrus. Between the ages of approximately 31/2 to 4 months all females were first tested for mounting behavior during behavioral estrus (15 min with an estrogen/progesterone primed ovariectomized stimulus female), and then for proceptive and receptive behaviors during the next behavioral estrus (15 rain with a testosterone injected sexually active castrated male). These tests were carried out between 10.30 and 12.30 hr, during the dark period. Within one week following the last behavioral test at estrus, all females were operated under ether anesthesia and both ovaries were removed through bilateral incisions. From then on all females were injected SC with testosterone propionate (TP; 0.2 mg TP on Monday and Wednesday, 0.3 mg on Friday, to inject the equivalent of 0.1 mg TP/day). The females were tested for mounting and intromission behavior by bringing them together in a semi-circular arena with an
estrogen/progesterone primed ovariectomized stimulus female (15 rain/test). The first period of testing was 1-3 weeks after the onset of TP injections. The second period of testing was 7-8 weeks after the onset of TP treatment. Behaviors that were scored by two observers included: mount, mount with pelvic thrusting, intromission, abortive mount. For the analysis of the data only mounts with pelvic thrusts and intromittive mounts are considered and will be referred to as mounting behavior. RESULTS
Behavioral estrus occurred very regularly, predominantly at 4-day intervals, and no differences between females of the four experimental groups were found. Mounting behavior during behavioral estrus, when tested with a stimulus female, was displayed at very low frequencies with no differences between the females. Proceptive and receptive behaviors, when tested with an active male during behavioral estrus, were displayed at very high frequencies with lordosis quotients of 100. Again, no differences between the females of the four experimental groups were noted. For the initial behavior tests during TP treatment the data are not presented, because a considerable number of animals in each group did not yet show mounting behavior (50, 19 and 20%, respectively during 1st, 2nd and 3rd test). Nevertheless, there was an overall increase in mounting behavior of those animals displaying the behavior: 7.5, 14.6, and 18.1 mean frequencies for 1st, 2nd and 3rd test, respectively. During the 3rd test there were no significant differences in percentage of animals and mean (-+SEM) mount frequencies between the females stemming from "all female" (82%; 21.6___1.9), from "predominantly female" (88%; 22.0_5.6), and from "predominately male" litters (75%; 17.8---6.7). Only the females from "fifty-fifty" litters showed significantly lower mount frequencies than all other groups (3.8_+ 1.8), 1-way ANOVA, F(3/40)=3.45, p<0.05, although the percentage of animals showing the behavior (62%) was not significantly different. The results of the last three behavior tests (4, 5 and 6) during the 7th and 8th week of TP injections, are shown in Fig. 1. During tests 4, 5 and 6, respectively 98, 93 and 100% of the females displayed mounting behavior (only some "fifty-fifty" females did not mount). Two-way ANOVA
LITTER COMPOSITION AND FEMALE MOUNTING
60
t
[] qth test test [] 5th 6th test
S0
I10
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i ~
l I T
II
I~
II
II
/I
~l il li li
il II ii li
II I ~ I ~ I ~
II ii
II •
I~t
30
=
10
t
0
II I1 I1 Ii
'all female'
li Ii II
I x II I"
I/ i1 II II ii
'onemale,
p r e d o m , female ~
I/ ~" II I " II
'fifty-fifty'
font
female, p r e d o m , male'
LITTERCOMPOSITIONAT B I R T H FIG. 1. Frequency of TP-induced mounting behavior (mounts with or without accompanying "intromission" behavior towards hormortally primed estrous female rats) of female rats during 15 min tests: effect of litter composition at birth. There were no consistent significant differences in mounting behavior of female rats from "all female litters" and those from litters with predominantly male siblings.
(split-plot p.q. design, [15]) revealed a significant effect for groups, F(3/40)=4.67, p<0.05, as well as for groups x tests interaction, F(6/80)= 10.12, p<0.01. Subsequent analysis of this interaction (simple main effects method, [15]) showed significant F-values during test 4, F(3/120)=3.09, p<0.05, and test 5, F(3/120)=17.27, p<0.01. Further analysis indicated that during the 4th test only the "fifty-fifty" females differed significantly from all other females. During the 5th test "predominantly male" females and "fifty-fifty" females were significantly different, higher and lower, respectively, from all other females.
DISCUSSION The present experiment clearly demonstrates that the prenatal presence of male fetuses is not a prerequisite for TP-induced mounting behavior in adult female rats. These data corroborate recent findings by van de Poll et al. [22]. The present data are not in line with the results reported by Clemens and co-workers [7-9]. There is no easy explanation for this discrepancy. However, it might be of importance that in the experiments of Clemens the procedure was quite different from ours, and that their animals might have been exposed to considerable prenatal and postnatal stress. First of all "between Day 10 and 13 of gestation one horn of the uterus was r e m o v e d . . . " , and secondly "cesarian sections were performed 21 days after m a t i n g . . . " ([9], p. 41). Furthermore, the possible problems with cesarian sections became apparent through the following statement: "In the present experiment (Experiment 3; [9]), animals were not
1009 delivered by cesarian section and this alleviated their susceptibility to respiratory infection" ([9], p. 45) might indicate a substantial morbidity among the experimental females. Recently, some support for the hypothesis that fetal female rats are masculinized by male littermates was published by Meisel and Ward [16]. These authors found that TP-induced mounting behavior in adult female rats was related only to the presence of males on the caudal side of the females in the same uterine horn. However, if the experimental females (delivered by cesarian section) were grouped according to fetal male contiguity, as Clemens and coworkers did [7,9], no significant effects for mounting were found. The Meisel and Ward [16] experiment differed from the present investigation in that the percentage of females displaying mounting behavior (about 55%) was rather low. This difference might be explained by the method of TP administration and subsequent behavioral testing: 0.25 mg TP/day for 14 days. In the present investigation the equivalent of 0.1 mg TP/day was injected for 8 weeks, which resulted in virtually all of the females displaying mounting behavior. The results of the present investigation are in line with the idea that female rat fetuses are normally exposed to endogenous androgens (see also [19,30]). The origin of the relatively high levels of testosterone in female fetal plasma could very well be the placenta rather than male fetal siblings (e.g., [12,21]). In vitro studies have shown that the placenta is capable of converting pregnenolone and progesterone to androstenedione and testosterone [6,17]. Recent findings of high levels of androsterone (a metabolite of testosterone and androstenedione) in placentae of female rat fetuses (Day 20 of gestation) further support this idea (J. T. M. Vreeburg, personal communication). The hypothesis that female rats produce androgens at the end of their prenatal life, which couM affect CNS development, is consistent with the present findings. However, the consistency as such is no proof that androgens are required for development of CNS systems involved in the display of mounting behavior in adulthood. Such statement would require the analysis of behavior in animals in which prenatal androgen production and/or exposure is diminished or inhibfled. Present work addresses the latter question. The general picture which emerges from this study is, that female rats show mounting behavior in adulthood independent of litter composition at birth. There was only one group out of 4 ("predominantly male") that showed increased numbers of mounts in only one out of 3 tests. Thus in one test situation out of 12 would the results appear to support Clemens' hypothesis. As can be expected when several groups of rats are tested on a number of occasions, every once in a while an individual testresult may differ from the general picture. The consistent finding of large numbers of mounts in most of the tests with females from "all female" litters provides in fact the best evidence that the presence of males cannot be a large contribution to the development of CNS systems involved in adult mounting behavior. Finally, it seems inappropriate to talk about prenatal "masculinization" since organization of the nervous system of female rat fetuses through endogenous androgens seems to be a naturally occurring phenomenon. The finding that normal female mammals of many species will mount female and male partners (including pelvic thrusting and sometimes intromission and "orgasm" behavior) [3, 4, 13] indicate that this so-called "masculine" behavior is an intrinsic female behavioral characteristic.
1010
SLOB AND VAN DER SCHOOT ACKNOWLEDGEMENT
Special thanks are due to G. H. Ketting, Department of Anatomy, Faculty of Medicine, Erasmus University Rotterdam, for careful selection and taking care of the new born litters. We thank P. E.
Schenck for statistical advice and Professor J. J. van der Werff ten Bosch, and A. Bot for critical reading of the manuscript. This experiment was carded out in part with the able assistance of medical students during a course in endocrinology.
REFERENCES 1. Baum, M. J. Differentiation of coital behavior in mammals: a comparative analysis. Neurosci. Biobehav. Rev. 3: 265-284, 1979. 2. Beach, F. A. Sex reversals in the mating pattern of the rat. J. genet. Psychol. 53: 329-334, 1938. 3. Beach, F. A. Factors involved in the control of mounting behavior by female mammals. In: Perspectives in Reproduction and Sexual Behavior, edited by M. Diamond. Bloomington, IN: Indiana University Press, 1968, pp. 83-131. 4. Beach, F. A. Hormonal modification of sexually dimorphic behavior. Psychoneuroendocrinology 1: 3-23, 1975. 5. Beach, F. A. Sexual attractivity, proceptivity, and receptivity in female mammals. Hormones Behav. 7: 105-138, 1976. 6. Chan, S. W. C. and J. H. Leathern. Placental steroidogenesis in the rat: progesterone production by tissue of the basal zone. Endocrinology 96: 298-303, 1975. 7. Clemens, L. G. Neurohormonal control of male sexual behavior. In: Reproductive Behavior, edited by W. Montagna and W. A. Sadler. New York: Plenum Press, 1974, pp. 23-53. 8. Clemens, L. G. and L. Conigiio. Influence of prenatal litter composition on mounting behavior of female rats. Am. Zool. 11: 617-618, 1971. 9. Clemens, L. G., B. A. Gladue and L. P. Conigiio. Prenatal endogenous androgenic influences on masculine sexual behavior and genital morphology in male and female rats. Hormones Behav. 10: 40-53, 1978. 10. Gandelman, R., F. S. vom Saal and J. M. Reinisch. Contiguity to male foetuses affects morphology and behaviour of female mice. Nature 266: 722-724, 1977. 11. Gerall, A. A. and I. L. Ward. Effects of prenatal exogenous androgen on the sexual behavior of the female albino rat. J. comp. physiol. Psychol. 62: 370--375, 1966. 12. Gibori, G. and R. Sridaran. Sites of androgen and estradiol production in the second half of pregnancy in the rat. Biol. Reprod. 24: 249-256, 1981. 13. Goldfoot, D. A., H. Westerborg-van Loon, W. Groeneveld and A. Koos Slob. Behavioral and physiological evidence of sexual climax in the female stamp-tailed macaque (Macaca Arctoides). Science 208: 1477-1479, 1980. 14. Goy, R. W. and B. S. McEwen. Sexual Differentiation of the Brain. Cambridge, MA: The MIT Press, 1980. 15. Kirk, R. E. Experimental Design: Procedures for the Behavioral Sciences. Belmont, CA: Brooks/Cole, 1968. 16. Meisel, R. L. and I. L. Ward. Fetal female rats are masculinized by male littermates located caudally in the uterus. Science 213: 239-242, 1981.
17. Rembiesa, R., M. Marchut and A. Warchoi. Ovarian-placental dependency in rat. Part I. Biotransformation of C~ steroids to androgens by rat placenta in vitro. Steroids 19" 65-84, 1972. 18. Slob, A. K., M. P. Ooms and J. T. M. Vreeburg. Sex ratio in utero and the plasma concentration of testosterone in male and female rat foetuses. J. Endocr. 79: 395-396, 1978. 19. Slob, A. K., M. P. Ooms and J. T. M. Vreeburg. Prenatal and early postnatal sex differences in plasma and gonadal testosterone and plasma luteinizing hormone in female and male rats. J. Endocr. 87: 81-87, 1980. 20. Srdersten, P. Mounting behavior in the female rat during the estrous cycle, after ovariectomy, and after estrogen or testosterone administration. Hormones Behav. 3: 307-320, 1972. 21. Sridaran, R., R. Basuray and G. Gibori. Source and regulation of testosterone secretion in pregnant and pseudo-pregnant rats. Endocrinology 108: 855-861, 1981. 22. van de Poll, N. E., S. M. van der Zwan, H. G. van Oyen and J. H. Pater. Sexual behavior in female rats born in all female litters. Behav. Brain Res. 4: 103-109, 1982. 23. vom Saal, F. S. and F. H. Bronson. In utero proximity of female mouse fetuses to males: effect on reproductive performance during later life. Biol. Reprod. 19: 842--853, 1978. 24. vom Saal, F. S. and F. H. Bronson. Sexual characteristics of adult female mice are correlated with their blood testosterone levels during prenatal development. Science 208: 597-599, 1980. 25. vom Saal, F. S. and F. H. Bronson. Variation in length of the estrous cycle in mice due to former intrauterine proximity to male fetuses. Biol. Reprod. 22: 777-780, 1980. 26. vom Saal, F. S., S. Pryor and F. H. Bronson. Effects of prior intrauterine position and housing on oestrous cycle length in adolescent mice. J. Reprod. Fert. 62: 33-37, 1981. 27. Ward, I. L. Differential effect of pre- and postnatal androgen on the sexual behavior of intact and spayed female rats. Hormones Behav. 1: 25-36, 1969. 28. Ward, I. L. and F. J. Renz. Consequences ofperinatal hormone manipulation on the adult sexual behavior of female rats. J. comp. physiol. Psychol. 78: 349-355, 1972. 29. Ward, I. L. and E. V. Sperr. Effects of prolonged androgen on the sexual behavior of intact and spayed female rats. Physiol. Behav. 4: 765-768, 1969. 30. Weisz, J. and I. L. Ward. Plasma testosterone and progesterone titers of pregnant rats, their male and female fetuses and neonatal offspring. Endocrinology 106: 306--316, 1980.
Testosterone Induced Mounting Behavior in Adult Female Rats Born in Litters of Different Female to Male Ratios A. KOOS S L O B A N D P I E T E R V A N D E R S C H O O T
Department of Endocrinology, Growth and Reproduction Faculty o f Medicine, Erasmus University, P.O. Box 1738, Rotterdam, The Netherlands Received 2 D e c e m b e r 1981 SLOB, A. K. AND P. VAN DER SCHOOT. Testosterone induced mounting behavior in adult female rats born in litters o f different female to male ratios. PHYSIOL. BEHAV. 28(6) 1007-1010, 1982.--Female rats can show mounting behavior towards isosexual or heterosexual conspecifics. The present experiments were designed to study whether the prenatal presence of male fetuses would affect display of this mounting behavior in adulthood. Therefore mounting behavior, after gonadectomy and during continuous treatment with testosterone propionate (TP) was studied in female rats which were born in litters without male siblings ("all female" litters) and in litters with a variable number of male siblings. Litter composition at birth did not affect display of adult mounting behavior during protracted tests (a total of 6 tests during 8 weeks of treatment with TP). The data indicate that TP induced mounting behavior in adulthood occurs independent of the prenatal presence of male fetuses, ff mounting behavior in adulthood has to be "organized" by prenatally present androgen (the current way of thinking), then the present data indicate that female rat fetuses provide themselves ~ith these hormones. It would then seem inappropriate to judge adult mounting behavior as a sign of "masculinization" of the rat brain. Mounting behavior
Litter composition at birth
Female rats
ADULT female rats have been reported to display mounting and intromission behavior without receiving exogenous hormones perinatally or in adulthood (e.g., [2, 3, 20, 27]). Such observations were initially interpreted as suggesting that neural mechanisms regulating "masculine" sexual behavior develop in the absence of either pre- or postnatal androgenic stimulation (see review in [1]). However, other evidence suggested that female rats were normally exposed prenatally to a certain amount of androgenic stimulation, and that this exposure may partially masculimze all females of this species (see also [14]). The first investigators to suggest an endogenous prenatal androgenic modification of the nervous system ("masculinization") of female rats were Ward and Renz [28]. Their finding that prenatal exposure to cyproterone acetate (a synthetic anti-androgen) "decreased markedly the amount and quality of male behavior below that of control females suggests that the low level of male behavior typically elicited in normal females by adult androgen treatment (Gerall & Ward, 1966; Ward & Sperr, 1969) results, at least in part, from prenatal stimulation by endogenous androgen" ([28], p. 354). They speculated that this androgen was of adrenal origin. Another line of supportive evidence for this (endogenous) hypothesis of prenatal androgen masculinization of female rats came from the work of Clemens and collaborators. They were the first to suggest a prenatal morphological and behavioral "masculinization" of female fetuses through exposure to androgen from the testes of male fetuses [ 7, 8, 9]. These
investigators reported that female fetuses next to male fetuses in one uterine horn had a larger ano-genital distance at birth and displayed more mounting behavior in response to administration of testosterone in adult life than did female fetuses that had not been in such close proximity to fetal male rats. Also in mice it was reported that the positions of the female fetuses in the uterus relative to the male fetuses determined the extent to which the female mouse was masculinized in terms of morphology (ano-genital distance at birth) and behavior (testosterone-induced fighting in adulthood) [10], as well as reproductively important characteristics during adulthood [23, 24, 25, 26]. Clemens and coworkers [9] as well as Vom Saal and co-workers [23] suggested that fetal male androgens reach the female fetus by way of direct diffusion across the amniotic membranes late in gestation. For this latter hypothesis, however, Slob, Ooms and Vreeburg [18] were not able to find supportive evidence. They found equivalent endogenous serum levels of testosterone in female rat fetuses on Day 20 of gestation, regardless of the in utero (one uterine horn) ratio of male to female fetuses. If the prenatal presence and/or proximity of male fetuses affects adult reproductive behavior in female rats, then clear behavioral differences should be apparent between females stemming from all female litters and females born in litters with an abundant number of male siblings. The present experiment was designed to test this hypothesis.
Copyright © 1982 Brain Research Publications Inc.--0031-9384/82/0601007-04503.00/0
1008
SLOB AND VAN DER SCHOOT TABLE 1 NUMBER OF FEMALES IN EACH OF THE 4 EXPERIMENTAL CONDITIONS AS WELL AS THE NUMBER OF LITTERS AND LITTERSIZE AT BIRTH
Litter Composition " S e x Ratio"
Number of Litters
Littersize at Birth Mean Median Range
Number of Females Used in Experiment
? 9 Q9 (all female) 9 Q9 d (predominantly female) 9 Qd d (fifty-fifty)
5
4.6
4
1-9
23
5
6.6
6
5-9
9
8
6.0
6
2-12
8
$ddd
4
6.8
7
4-9
4
(predominantly male)
METHOD
During November and December 1980 all litters born in a large breeding colony (Wistar, Amsterdam-R strain; Department of Anatomy, Faculty of Medicine, Erasmus University, Rotterdam) were checked immediately after delivery. Four "types" of litters were assigned to this experiment: (1) all female litters (n=5); (2) one male and 4 to 8 females (n=5); (3) equal number of female and male siblings (n=8); (4) one female and 3 to 8 males (n=4). Initially the animals were kept at 22-23°C and exposed to 14 hr light:10 hr darkness/day (lights on 05.00-19.00 hr). The animals grew up with their mothers in the litter composition at birth, were weaned at 22 days of age and then housed 3 or 4 of same sex to a cage. From litter-types 1 and 4 all females were used, from litter-types 2 and 3 one or two females were randomly selected from each litter at weaning and subsequently used in the experiment (see Table 1). The animals were allowed free access to pellet food and tap water. At the age of about 2 months all animals were transferred to an other animal quarter and exposed to a reversed 14 hr light: 10 hr darkness/day (lights on 21.30-7.30 hr). Beginning at the age of about 21/2 months all females were daily tested (in the middle of the dark period) for the occurrence of earwiggiing, hopping and darting (proceptivity, [5]) by introducing a sexually active male in the cage for a few minutes. This procedure was continued for 40 days and revealed information about the occurrence of behavioral estrus. Between the ages of approximately 31/2 to 4 months all females were first tested for mounting behavior during behavioral estrus (15 min with an estrogen/progesterone primed ovariectomized stimulus female), and then for proceptive and receptive behaviors during the next behavioral estrus (15 rain with a testosterone injected sexually active castrated male). These tests were carried out between 10.30 and 12.30 hr, during the dark period. Within one week following the last behavioral test at estrus, all females were operated under ether anesthesia and both ovaries were removed through bilateral incisions. From then on all females were injected SC with testosterone propionate (TP; 0.2 mg TP on Monday and Wednesday, 0.3 mg on Friday, to inject the equivalent of 0.1 mg TP/day). The females were tested for mounting and intromission behavior by bringing them together in a semi-circular arena with an
estrogen/progesterone primed ovariectomized stimulus female (15 rain/test). The first period of testing was 1-3 weeks after the onset of TP injections. The second period of testing was 7-8 weeks after the onset of TP treatment. Behaviors that were scored by two observers included: mount, mount with pelvic thrusting, intromission, abortive mount. For the analysis of the data only mounts with pelvic thrusts and intromittive mounts are considered and will be referred to as mounting behavior. RESULTS
Behavioral estrus occurred very regularly, predominantly at 4-day intervals, and no differences between females of the four experimental groups were found. Mounting behavior during behavioral estrus, when tested with a stimulus female, was displayed at very low frequencies with no differences between the females. Proceptive and receptive behaviors, when tested with an active male during behavioral estrus, were displayed at very high frequencies with lordosis quotients of 100. Again, no differences between the females of the four experimental groups were noted. For the initial behavior tests during TP treatment the data are not presented, because a considerable number of animals in each group did not yet show mounting behavior (50, 19 and 20%, respectively during 1st, 2nd and 3rd test). Nevertheless, there was an overall increase in mounting behavior of those animals displaying the behavior: 7.5, 14.6, and 18.1 mean frequencies for 1st, 2nd and 3rd test, respectively. During the 3rd test there were no significant differences in percentage of animals and mean (-+SEM) mount frequencies between the females stemming from "all female" (82%; 21.6___1.9), from "predominantly female" (88%; 22.0_5.6), and from "predominately male" litters (75%; 17.8---6.7). Only the females from "fifty-fifty" litters showed significantly lower mount frequencies than all other groups (3.8_+ 1.8), 1-way ANOVA, F(3/40)=3.45, p<0.05, although the percentage of animals showing the behavior (62%) was not significantly different. The results of the last three behavior tests (4, 5 and 6) during the 7th and 8th week of TP injections, are shown in Fig. 1. During tests 4, 5 and 6, respectively 98, 93 and 100% of the females displayed mounting behavior (only some "fifty-fifty" females did not mount). Two-way ANOVA
LITTER COMPOSITION AND FEMALE MOUNTING
60
t
[] qth test test [] 5th 6th test
S0
I10
r~'
i ~
l I T
II
I~
II
II
/I
~l il li li
il II ii li
II I ~ I ~ I ~
II ii
II •
I~t
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=
10
t
0
II I1 I1 Ii
'all female'
li Ii II
I x II I"
I/ i1 II II ii
'onemale,
p r e d o m , female ~
I/ ~" II I " II
'fifty-fifty'
font
female, p r e d o m , male'
LITTERCOMPOSITIONAT B I R T H FIG. 1. Frequency of TP-induced mounting behavior (mounts with or without accompanying "intromission" behavior towards hormortally primed estrous female rats) of female rats during 15 min tests: effect of litter composition at birth. There were no consistent significant differences in mounting behavior of female rats from "all female litters" and those from litters with predominantly male siblings.
(split-plot p.q. design, [15]) revealed a significant effect for groups, F(3/40)=4.67, p<0.05, as well as for groups x tests interaction, F(6/80)= 10.12, p<0.01. Subsequent analysis of this interaction (simple main effects method, [15]) showed significant F-values during test 4, F(3/120)=3.09, p<0.05, and test 5, F(3/120)=17.27, p<0.01. Further analysis indicated that during the 4th test only the "fifty-fifty" females differed significantly from all other females. During the 5th test "predominantly male" females and "fifty-fifty" females were significantly different, higher and lower, respectively, from all other females.
DISCUSSION The present experiment clearly demonstrates that the prenatal presence of male fetuses is not a prerequisite for TP-induced mounting behavior in adult female rats. These data corroborate recent findings by van de Poll et al. [22]. The present data are not in line with the results reported by Clemens and co-workers [7-9]. There is no easy explanation for this discrepancy. However, it might be of importance that in the experiments of Clemens the procedure was quite different from ours, and that their animals might have been exposed to considerable prenatal and postnatal stress. First of all "between Day 10 and 13 of gestation one horn of the uterus was r e m o v e d . . . " , and secondly "cesarian sections were performed 21 days after m a t i n g . . . " ([9], p. 41). Furthermore, the possible problems with cesarian sections became apparent through the following statement: "In the present experiment (Experiment 3; [9]), animals were not
1009 delivered by cesarian section and this alleviated their susceptibility to respiratory infection" ([9], p. 45) might indicate a substantial morbidity among the experimental females. Recently, some support for the hypothesis that fetal female rats are masculinized by male littermates was published by Meisel and Ward [16]. These authors found that TP-induced mounting behavior in adult female rats was related only to the presence of males on the caudal side of the females in the same uterine horn. However, if the experimental females (delivered by cesarian section) were grouped according to fetal male contiguity, as Clemens and coworkers did [7,9], no significant effects for mounting were found. The Meisel and Ward [16] experiment differed from the present investigation in that the percentage of females displaying mounting behavior (about 55%) was rather low. This difference might be explained by the method of TP administration and subsequent behavioral testing: 0.25 mg TP/day for 14 days. In the present investigation the equivalent of 0.1 mg TP/day was injected for 8 weeks, which resulted in virtually all of the females displaying mounting behavior. The results of the present investigation are in line with the idea that female rat fetuses are normally exposed to endogenous androgens (see also [19,30]). The origin of the relatively high levels of testosterone in female fetal plasma could very well be the placenta rather than male fetal siblings (e.g., [12,21]). In vitro studies have shown that the placenta is capable of converting pregnenolone and progesterone to androstenedione and testosterone [6,17]. Recent findings of high levels of androsterone (a metabolite of testosterone and androstenedione) in placentae of female rat fetuses (Day 20 of gestation) further support this idea (J. T. M. Vreeburg, personal communication). The hypothesis that female rats produce androgens at the end of their prenatal life, which couM affect CNS development, is consistent with the present findings. However, the consistency as such is no proof that androgens are required for development of CNS systems involved in the display of mounting behavior in adulthood. Such statement would require the analysis of behavior in animals in which prenatal androgen production and/or exposure is diminished or inhibfled. Present work addresses the latter question. The general picture which emerges from this study is, that female rats show mounting behavior in adulthood independent of litter composition at birth. There was only one group out of 4 ("predominantly male") that showed increased numbers of mounts in only one out of 3 tests. Thus in one test situation out of 12 would the results appear to support Clemens' hypothesis. As can be expected when several groups of rats are tested on a number of occasions, every once in a while an individual testresult may differ from the general picture. The consistent finding of large numbers of mounts in most of the tests with females from "all female" litters provides in fact the best evidence that the presence of males cannot be a large contribution to the development of CNS systems involved in adult mounting behavior. Finally, it seems inappropriate to talk about prenatal "masculinization" since organization of the nervous system of female rat fetuses through endogenous androgens seems to be a naturally occurring phenomenon. The finding that normal female mammals of many species will mount female and male partners (including pelvic thrusting and sometimes intromission and "orgasm" behavior) [3, 4, 13] indicate that this so-called "masculine" behavior is an intrinsic female behavioral characteristic.
1010
SLOB AND VAN DER SCHOOT ACKNOWLEDGEMENT
Special thanks are due to G. H. Ketting, Department of Anatomy, Faculty of Medicine, Erasmus University Rotterdam, for careful selection and taking care of the new born litters. We thank P. E.
Schenck for statistical advice and Professor J. J. van der Werff ten Bosch, and A. Bot for critical reading of the manuscript. This experiment was carded out in part with the able assistance of medical students during a course in endocrinology.
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