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Mounting in female hamsters: Effects of different hormone regimens
Physiology & Behavior, Vol. 19, pp. 519--526. Pergamon Press and
Brain Research
Publ., 1977. Printed in the U.S.A.
Mounting in Female Hamsters: Effects of Different Hormone Regimens RALPH G. NOBLE
lCisconsin Regional Primate, Research Center, Madison, WI 53706 (Received 28 July •975) NOBLE, R. G. Mounting in female hamsters: effects of different hormone regimens. PHYSIOL. BEHAV. 19(4)519-526, 1977. - Adult female hamsters have substantial capacity to display male behavior without perinatal androgen administration and apparently without perinatal exposure to gonadal steroids. Seventy-two ovariectomized females were tested for male behavior during chronic treatment with either testosterone (T, N = 8); androstenedione (AD, N = 7); dihydrotestosterone (DHT, N = 8); estradiol (E2, N = 7); T + E2 (N = 9); AD + E2 (N = 6); DHT + E2 (N = 7); or no hormone (NoH, N = 19). Eighty-three percent of the females treated with T, T + E2, AD + E2, or DHT + E2 mounted during at least one test, and 70% displayed the intromission pattern; there were no significant differences among these treatment conditions. Forty-two percent of E2-treated females, 14% of AD-treated females, none of the DHT-treated females, and one of the NoH females mounted; these treatments were significantly less effective than T or any of the androgen-plus-estrogen combinations. Females treated with E~ or AD + E2 displayed lordosis responses when tested for female behavior, but females treated with T + E2, DHT + E~, T, DHT or NoH did not. In the hamster the capacity for the execution of male behavior develops independent of early androgen, and only the arousability of the system by hormonal and external stimulation is sex dimorphic. Testosterone Dihydrotestosterone Sex differences
Androstenedione
Mounting
Hamsters
Sex behavior
neural substrates underlying the execution of the components of the male copulatory pattern develop independent of genetic sex without requiring exposure to gonadal steroids during early development. Other studies, primarily with rats, have indicated that several variables which influence the male copulatory behavior of genetic males have essentially the same effect on the male copulatory behavior of genetic females (cf. [ 14] ). These findings would suggest that the consequences of early androgen action are restricted to altering the kinds and amounts of hormonal or proprioceptive stimulation required to elicit the execution of behavior patterns without altering the development of the capacity to execute the patterns. This interpretation depends on the assumption that the normal female develops without being exposed to gonadal steroids in significant amounts during early stages of development. This assumption is questionable at least for the rat. While attempts to determine if prenatal exposure to exogenous androgen alters the subsequent display of male behavior by female rats have not produced conclusive results (cf. [16]), there is evidence that the prenatal hormonal milieu can influence the capacity to display male behavior. Clemens and Coniglio [9] have demonstrated that the amount of male behavior displayed by female rats is highly correlated with the amount of exposure to testicular secretions from male siblings in utero. Some investigators have interpreted the available evidence as suggesting that, for both males and females, the capacity to display male behavior depends on exposure to gonadal steroids during early development with
WHILE FEMALES of many mammalian species have the capacity to display components of the male copulatory pattern [3], there are clear sex differences in the kinds and amounts of male sex behavior displayed when gonadectomized males and females are given standard regimens of exogenous androgen administration and tested for male behavior as adults [ 16]. In the species tested, this sex difference in the display of male copulatory behavior is related to the presence or absence of gonadal steroids during early stages of development [16]. Recent investigations strongly suggest that the facilitation of male copulatory behavior produced by exposure to androgen during early development results, at least in part, from effects of the androgen on the developing nervous system and cannot be explained on the basis of the effects of early androgen administration on the development of the genitalia (cf. [17]). Our thinking about the consequences of exposure to androgen during early development may be clarified by a careful analysis of the residual capacity of the normal adult female to display male behavior. Recent studies have demonstrated that the female rat has the capacity to display all the components of the male copulatory pattern. This capacity has been demonstrated both by testing the consequences for the display of male behavior of prolonged exposure to high doses of exogenous estrogen [ 14], and by providing peripheral electrical shock to females being tested for male behavior [21]. The residual capacity of the perinatally untreated female rat to display all the components of the male copulatory pattern suggests that the 519
520 both the testes of male siblings and the maternal adrenal being identified as possible sources of gonadal steroids [161. Further analysis of the effects of early exposure to gonadal steroids on psychosexual development requires an analysis of the capacity of animals which have not been exposed to gonadal steroids during early development to display male copulatory behavior. At present it is not clear that this analysis can be made in species in which the critical periods for psychosexual differentiation are partly prenatal [ 16]. The female hamster probably represents the closest available approximation to a rodent which develops in the absence of gonadal steroids during the critical period for psychosexual differentiation, although this has not been varified by direct measurements of hormone concentrations in the neonatal female. Prenatal androgen administration does not augment mounting by female hamsters [27]; castration of the male hamster on the day of birth virtually eliminates mounting following androgen treatment of the adult [7, 12, 24]; and postnatal administration of small quantities of androgen augments mounting of female hamsters [29,30]. Female hamsters rarely mount when exogenous androgen is administered, even in very high doses, for periods of time sufficient to restore the copulatory behavior of male hamsters castrated as adults [25]. Apparently the period of psychosexual differentiation is postnatal in the hamster. Therefore, it is important to carefully assess the capacity of female hamsters to display male behavior. A vigorous attempt to elicit male behavior from female hamsters may provide an estimate of the extent to which the capacity for male behavior develops in the absence of exposure to gonadal steroids early in development. It is also important to determine if in the female hamster extended periods of hormone treatment can facilitate male behavior, possibly producing changes similar to those which typically occur during exposure to gonadal steroids early in development. Evaluating the effects of various manipulations on the display of mounting by adult female hamsters may prove valuable both in explicating the effects of perinatal exposure to gonadal steroids and in evaluating hypotheses about the physiological mechanisms underlying sex differences in the display of male behavior. Recent studies suggest that the failure of female hamsters to m o u n t following adult treatment with testosterone for periods of time sufficient to restore the copulatory behavior of males castrated as adults results from the inability of female hamsters to respond to androgen treatment as rapidly as male hamsters and a failure to metabolize testosterone as effectively as male hamsters. Some indirect evidence suggests that the hypothesized deficiency of female hamsters to metabolize testosterone is a failure to convert testosterone to dihydrotestosterone rather than a failure to convert testosterone to estradiol. In both male and female hamsters chronic testosterone propionate administration reduces the dose of estradiol benzoate required to facilitate estrus when estradiol benzoate is administered in conjunction with progesterone. In male hamsters both chronic testosterone propionate administration and chronic dihydrotestosterone propionate administration impair the quality of estrogen-plus-progesteroneinduced estrus behavior, but in female hamsters only chronic dihydrotestosterone propionate administration impairs the quality of estrus behavior [26]. Daily injections of
~'i ~i~.l [ dihydrotestosterone propionate and either estradiol ben,'(>ate or estrone significantly increase mounting m re'male hamsters after 17 28 days of treatment. Estradiol benzoate and dihydrotestosterone propionate are not effective when administered alone, and testosterone propionate does not facilitate mounting. Estradiol benzoate combined with dihydrotestosterone propionate produce higher mount frequencies than do estrone plus dihydrotestosterone propionate [251. Since several studies have reported sex differences in steroid metabolism in the rat [10, 20, 231, the hypothesis was formed that the failure of female hamsters to m o u n t results from a reduced ability to convert testosterone to dihydrotestosterone. The present study compared the effects of testosterone, androstenedione, and dihydrotestosterone when administered alone or in combination with estradiol on mounting by female hamsters. Based on the hypothesis stated above, it was predicted that testosterone plus estradiol would be less effective in facilitating mounting by female hamsters than dihydrotestosterone plus estradiol. Androstenedione was included in the present study because it is at least as effective as testosterone in maintaining the copulatory behavior of castrate male hamsters [8]. Androgen implants made from polydimethylsiloxane tubing and estradiol implants made from stainless steel tubing sealed with polydimethylsiloxane elastomer were used in an attempt to provide reasonably low and constant hormone concentrations in the blood [5,15[. METHOD
Animals Female hamsters ( 9 1 - 1 0 0 g weight class) were purchased from Engle's Laboratory Animals, Inc. (Farmersburg, IN). Females were housed 4 - 5 per cage (38 x 33 x 17 cm) on a reversed light cycle (LD: 14: 10) with the lights turned off at 1200. After four weeks in the laboratory, females were ovariectomized using sodium pentobarbital anesthesia.
Design Females were assigned at random to one of the following groups: testosterone (T, N = 8); androstenedione (AD, N = 7); dihydrotestosterone (DHT, N = 8); estradiol (E 2, N = 7 ) ; T + E 2 ( N = 9 ) ; A D + E ~ (N = 6 ) ; D H T + E ~ (N = 7 ) and no hormone controls (NoH, N = 19). Two weeks after ovariectomy, hormone implants, described below, were p l a c e d subcutaneously between the scapulae while the animals were anesthetized. The implant was placed approximately 2 - 3 cm from the incision, which was closed with a wound clip. Females were tested for male behavior once a week for six weeks, beginning 17 days after the start of hormone treatment. The day after the last test for male behavior, they were tested for female behavior. Then the females were anesthetized, weighed, and the implant removed. At this time the length of the flank gland (intercostal sebaceous gland) on the left side was measured because it is androgen-dependent and the effectiveness of testosterone on this organ is dependent on its conversion to a 5~-metabolite, probably dihydrotestosterone [31 ].
Testing Procedure All tests were conducted between 1 3 0 0 - 1 8 0 0 during the first few hours of the dark phase of the cycle. The testing arenas were 5-% gallon all-glass aquaria.
MOUNTING IN FEMALE HAMSTERS The procedure for the testing and scoring of male behavior was similar to that described elsewhere [24]. When tested for male behavior, the experimental female was placed in the arena I0 min before the start of the tests, which were 10 min long. An ovariectomized female in hormone-induced estrus was placed in the chamber to start the test. The frequency and duration of anogenital examination, mounting and intromission were recorded using a keyboard connected to an array of counters and timers. In the present study, only mounts appropriately oriented to the caudal end of the stimulus female which included clasping the stimulus female with the forepaws and pelvic thrusting were recorded. Linguo-genital contact was used as the criterion for anogenital examination. When tested for female behavior, the female was placed in an arena containing two intact males. The tests were terminated either when the female assumed the lordosis posture or when she had received five appropriately oriented mounts. The number of mounts preceding the initial display of lordosis was recorded.
Implant Description Androgen implants were prepared from 3 cm sections of polydimethylsiloxane tubing (Medical Grade Silastic Tubing®, Dow Chemical Co.; inner diameter, 1.8 mm; outer diameter, 3.17 mm) following the procedure described by Gay [15]. Sections of tubing were rinsed in ethanol and allowed to dry. Then a 0.5 cm-long wood dowel was inserted flush with one end of the tubing. The lumen of the tube was filled with crystalline steroid and another 0.5 cm wood dowel inserted. Then the ends o f the implant were sealed with polydimethylsiloxane elastomer (382 Medical Grade Elastomer ®, Dow Chemical Co.). Estradiol implants were made from 1 cm sections of stainless steel in hypodermic tubing filled with crystalline hormone to within 1 mm of each end and sealed with elastomer. All implants were incubated for 4 8 - 7 2 hr in phosphate-buffered saline at 37 ° C. Several apparently good implants were rejected when moisture was observed within the lumen of the tubing. Each implant was rinsed With ethanol immediately before it was placed in the animal. The size of the androgen implants was selected to release less than 200 ~g of steroid per day as determined in a preliminary study using the procedure described by Berndston [5]. T, AD and DHT implants of this size are effective in maintaining the copulatory behavior of castrate male hamsters (Noble, unpublished observations). The estradiol implant selected is the smallest size implant which reliably facilitates estrous behavior in female hamsters when combined with a single subcutaneous injection of 0.5 mg progesterone, and implants this size do not significantly alter the copulatory performance of castrate male hamsters (Noble, unpublished observations). The radioimmunoassay [6] was used to determine the plasma concentrations of estradiol in four females which had each received three implants eight days before the blood was collected. F r o m these data, it was estimated that a single implant would produce an estradiol concentration of approximately 150 pg/ml of plasma, which is less than the plasma concentration of estradiol in proestrous female hamsters [ 1]. For this initial determination three implants were used in an attempt to guarantee measurable concentrations of hormone. Since the implants used in the experiment were still filled with crystalline hormone when
521 examined visually at autopsy, it is reasonable to assume that release rates were roughly constant during the experiment [5].
Statistical Procedure An unweighted-means Analysis of Variance was computed for b o d y weight and flank gland size. If the overall F was significant, the individual groups were compared using the Newman-Kuels procedure for comparison among hormone-treated groups, and using Dunnett's procedure for comparison between hormone-treated groups and the NoH controls [13]. Differences in behavior measures were analysed using appropriate nonparametric statistics as indicated in the text. RESULTS
Anogenital Examination Most hormone-treated females displayed anogenital licking during at least one test (72% of E 2 -treated females, 83% of AD + E~-treated females, and 100% of the females in all other treatment groups). Only 16% of the NoH females showed this response (E~ vs. NoH, p < 0 . 0 5 , Fisher's exact probability test) and each of these females did so during only one test. Mean durations of anogenital licking per 10 min period were computed from the last two tests for male behavior. This provided estimates of maximum performance under the different treatment regimens. All hormone-treated groups had mean anogenital licking durations longer than that of the NoH controls (p<0.01, Mann Whitney U). T-treated females had longer anogenital licking durations than any of the other hormone-treated females (p<0.01, in each case). Figure 1 shows the percentage of animals showing anogenital licking on each of the six tests. The percentage of females in the various androgen or androgen plus estradiol treatment conditions was fairly consistent across tests, but the performance of females treated with E2 was erratic, with 47% of the females engaging in this behavior on Tests 3 and 6 (on Days 31 and 52) and none of the females doing so on Days 24, 38 or 45. E~-treated females engaged in anogenital examination during significantly fewer tests than did females in androgen-treated groups (p<0.05, at least, Mann Whitney U). Examination of the mean duration of anogenital licking (Fig. 2) reflects a similar pattern and indicates that the significantly higher durations of the T-treated females were not observed during the first four tests.
Mounting Eighty-eight percent of the T + E2-treated females, 82% of the AD + E2-treated females, and 86% of the DHT + E2-treated females mounted. Seventy-five percent of Ttreated females mounted during at least one test, 14% of the AD-treated females, none of the DHT-treated females, and one of the NoH females mounted. Forty-one percent of the E 2-treated females mounted, each during one test. T, T + E~, AD + E 2 and DHT + E~-treated females mounted during a higher percentage of the tests than the NoH controls (p<0.01 in each case, Mann Whitney U), and the AD, DHT and E2-treated females did not differ from the controls. The percentage of females mounting increased gradually in all cases (see Fig. 3), and there were no
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FIG. 1. The effect of various hormone regimens on the percentage of ovariectomized females engaging in anogenital licking during testing for male behavior. significant differences among the different androgen plus estradiol-treated groups in the number of treatment days preceding the first mount. The median number of treatment days preceding the first mount was 35 for the androgen plus estradiol-treated females as a group. The onset of mounting among the T-treated females was similar with a median of 31 days preceding the first mount. Fifteen of the nineteen females which mounted during more than one test had higher mount frequencies on subsequent tests (p<0.01, sign test). During the last two tests for male behavior both the T and the T + E 2-treated females had higher m o u n t frequencies than did the females in the other treatment groups (see Fig. 4) and these differences were significant when assessed using a nonparametric statistic to compare the T-treated females and the T + E2-treated females as a group (p<0.02, Mann Whitney U).
In tromission Sixty-seven percent of T + E,-treated females, 82% of AD + E2-treated females, 57% of DHT + E2-treated females, 75% of T-treated females, 14% of AD-treated females, 14% of E2-treated females, none of the DHT-treated females and none of the NoH females displayed the intromission pattern (see Fig. 5). The intromission pattern was displayed in 87% of the tests during which females mounted, and the ratio of tests positive for intromission to tests positive for mounting did not vary significantly among treatment groups.
FIG. 2. The effect of various hormone regimens on the mean duration(s) of anogenital licking by ovariectomized female hamsters during tests for male behavior.
Female Behavior All of the AD-treated females and six of the seven E~-treated females displayed the lordosis response when tested for female behavior. In all cases, the display of lordosis was preceded by one or more mounts. None of the females in any of the other treatment conditions displayed the lordosis response.
Morphological Measurements Flank gland. The flank glands of females with either T or DHT implants were comparable in size to the flank glands of intact males (X -+ SE = 7.0 -+ 0.2 mm) or testosteronetreated castrate males (X -+ SE = 5.7 ± 0.2 mm) (Noble, in preparation), and larger than the flank glands of AD-treated females (p<0.002). The flank glands of the AD'treated females were larger than the flank glands of the E2-treated females or the NoH females (p< 0.002). Body weight. The females receiving hormone treatments which facilitated mounting (T, T + E 2 , AD + E~, and DHT + E 2 ) weighed less than females in other treatment groups (AD, DHT, E 2 , NoH; p< 0.01). AD-treated females weighed less than DHT-treated females (0.06>p>0.05), E~'treated females (p<0.001), or NoH females (p<0.001). Individual Differences Data from the T-treated females and the T + E 2-treated females were used to quantitatively assess the reliability of individual differences and to examine the covariation among mount frequency, the latency to the onset of
M O U N T I N G IN F E M A L E H A M S T E R S
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FIG. 3. The effect of various hormone regimens on the percentage of ovariectomized female hamsters mounting during tests for male behavior. anogenital licking, and b o d y weight. Correlations b e t w e e n flank gland size and o t h e r e n d p o i n t s were n o t assessed because individual differences in flank gland size were minimal. Individual differences in m o u n t f r e q u e n c y were highly consistent for b o t h T and T + E~ females (rho = .90; and rho = .80, respectively). The duration of t r e a t m e n t prior to anogenital licking was predictive of subsequent m o u n t i n g p e r f o r m a n c e of b o t h groups (T, rho = .90; T + E2, rho = .74). B o d y weight was also correlated with the duration of
FIG. 4. The effect of various hormone regimens on the mean frequency of mounting. t r e a t m e n t prior to the onset of anogenital licking (T, rho = .62; T + E 2 , rho = .84), but n o t with m o u n t f r e q u e n c y (T, rho = .44; T + E 2 , rho = .00). DISCUSSION In the hamster three different classes of sex differences in male behavior can be identified: (1) differences in the h o r m o n e regimens that facilitate male behavior in the adult; (2) differences in the duration of h o r m o n e administration adults require before displaying male behavior; and (3) differences in the m a x i m u m level o f male behavior display-
TABLE 1 EFFECTS OF DIFFERENT HORMONE TREATMENTS ON BODY WEIGHT AND FLANK GLAND LENGTH Measure
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*DHT, E2, NoH, AD (p<0.05); AD, AD + E2, DHT + E2, T + E2, T (p<0.05). Among hormone comparisons based on Newman-Kuels Procedure; all comparisons with control used Dunnett's Procedure. *DHT > DHT + E2, T + E2, T > AD + E2, AD > E2, NoH; (p<0.01, see above).
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FIG. 5. The effect of various hormone regimens on the percentage of ovariectomized female hamsters displaying the intromission pattern during tests for male behavior.
ed by the adult under favorable conditions of testing and hormone replacement. These three classes of differences are at least partially dissociable in the hamster. Following prolonged treatment with estradiol and any of the androgens used in the present study or with testosterone, which can be converted to both estradiol and other androgens (36), female hamsters m o u n t and display the intromission pattern. The differential effectiveness of the androgens in facilitating mounting when administered alone probably reflects differences either in the estrogenic properties of the androgens or in the extent to which these androgens are metabolized to estradiol. Seventy-five percent of testosterone-treated females mounted during at least one test compared to fourteen percent of androstenedione-treated females and none of the dihydrotestosterone-treated females. The differences among testosterone, a n d r o s t e n e d i o n e and dihydrotestosterone in facilitating mounting correspond to the relative convertability of these androgens to estradiol [18]. In rat brain tissue, testosterone is converted to estradiol but not estrone [34], while androstenedione is aromatized to estrone but not estradiol [ 28]. Apparently mounting in female hamsters is dependent on both androgenic and estrogenic stimulation. While all three androgen-plus-estrogen combinations were roughly equivalent in facilitating mounting, they probably provided different relative amounts of estrogenic and androgenic stimulation. Two observations suggest that combined treat-
ment with androstenedione and estradio] prowded icss androgenic stimulation than the other hormone regimen:~ which were effective in facilitating mounting. (t } Andnv stenedione-treated females had smaller flank glands. Dihydrotestosterone is the effective androgen in this target organ [31 ]. (2) Androstenedione-plus-estradiol-treated females displayed lordosis while testosterone-plus-estradioltreated females and dihydrotestosterone-plus-estradioltreated females did not. Chronic dihydrotestosterone treatment has been demonstrated to interfere with the lordosis behavior of female hamsters [ 26]. The equivalence of testosterone, testosterone plus estradiol, dihydrotestosterone plus estradiol, and androstenedione plus estradiol in facilitating mounting in female hamsters suggests that the requirement for mounting in female hamsters is that both androgenic stimulation and estrogenic stimulation be present in some minimal amount. In castrated male hamsters dally injections of either estradiol benzoate or dihydrotestosterone propionate are partly effective in reinstating components of the male copulatory pattern, though neither is as effective as testosterone propionate [26]. This suggests that the physiological mechanisms underlying mounting behavior in male and female hamsters are highly similar. Christensen e t al. [8] reported that daily injections of testosterone, androstenedione and dihydrotestosterone maintained mounting in castrate male hamsters; testosterone and androstenedione were equally effective and both were more effective than dihydrotestosterone. Tiefer and Johnson [33] also reported that daily injections of testosterone and androstenedione were equally effective in restoring the copulatory behavior of male hamsters. Subcutaneous implants of the same size used in the present study containing either testosterone, androstenedione or dihydrotestosterone maintain mounting in male hamsters. Dihydrotestosterone implants restore mounting behavior in castrate adrenalectomized male hamsters, suggesting that estrogen is not required to facilitate mounting by male hamsters. However, the relative effectiveness of the three hormones in maintaining ejaculatory frequency in males is similar to the relative effectiveness of the three hormones in facilitating mounting behavior in females, with testosterone being significantly more effective then dihydrotestosterone (Noble, in preparation). It is not clear why chronic subcutaneous implants of testosterone facilitated mounting by female hamsters when daily injections of testosterone propionate using amounts far in excess of what is required to fully restore mounting in males castrated at ten days of age or older [24], do not facilitate mounting under identical conditions [25]. Either the testosterone implants deliver a higher effective dose of androgen than the testosterone propionate injections, for example by producing a higher average concentration of testosterone in the blood, or the injection procedure itself has effects that interfere with the display of mounting by female hamsters, but do not interfere with the display of mounting by male hamsters. The available data suggest that the injection procedure itself has effects which interfere with the display of mounting by female hamsters. Silastic implants of testosterone propionate are as effective in facilitating mounting in female hamsters as silastic implants of testosterone (Noble, unpublished observations). Estimates of the total amounts of hormone involved in the two modes of hormone administration render a simple dose difference hypothesis
MOUNTING IN FEMALE HAMSTERS
525
implausible. In our previous study [25], 52 mg of testosterone propionate were injected over a 38-day period without facilitating mounting by female hamsters. Preliminary estimates of the release rates of implants of the size used in the present experiment, based on changes in the dry weight of the implants, indicate that over a 38-day period about 2.25 mg (standard deviation, 0.4 mg) of testosterone is released (Noble, in preparation). A direct test of the dose difference hypothesis would require determination of plasma concentrations of testosterone at several different times of day to permit an estimate of the average plasma concentration produced by the two different methods of hormone administration. The duration of hormone treatment preceding the onset of mounting by female hamsters far exceeds that required to produce maximal levels of mounting by male hamsters castrated either as adults [26] or prepubertally [24]. It also exceeds the duration of treatment required to induce mounting in immature male hamsters (Noble, unpublished observations). Also, the duration of hormone treatment required to induce mounting in the present study is comparable to the duration required in the prior experiment [25] in which high doses of estradiol benzoate and dihydrotestosterone propionate were injected daily. The extended period of hormone treatment preceding the display of mounting by females in the present study appears invariant across a variety of hormone regimens, each of which is effective in inducing mounting but which differ markedly in their effects on the other behavioral and anatomical endpoints examined. Prolonged hormone treatment of adult female hamsters may facilitate mounting by first producing changes in the responsiveness to exogenous hormones similar to the changes in responsiveness to hormones produced by perinatal hormone treatment [16] followed by hormone induced activation of mounting behavior. In a previous study [24], the duration of hormone treatment required prior to the display of mounting was inversely related to the age at castration during the first ten days of life. Males castrated five days postpartum did not differ from males castrated ten days postpartum in the maximum rate of mounting, but the Day 5 castrates did require more extended hormone treatment prior to the onset of mounting. The amount of time required to activate mounting in those Day 1 castrate males which did m o u n t is comparable to the amount of time required to activate mounting in
female hamsters. It appears possible that the female hamster may retain some limited ability to undergo organizational changes in response to extended hormone treatment as an adult. While the average m o u n t frequency of females is lower than that of intact males or that of testosterone-treated castrate males (Noble, in preparation), the m o u n t frequency of females is largely determined by highly stable individual differences in the predisposition to display male behavior. The determinants of these individual differences are not known. Female hamsters in the present study displayed the intromission pattern less frequently than comparably treated males and did not display the ejaculatory response. Since the occurrence of intromission and ejaculation are heavily dependent on appropriate sensory feedback in the male hamster (Noble, in preparation), it is not possible to interpret the failure of female hamsters to show the ejaculatory response. Taking into account the obvious anatomical differences between male and female hamsters, the similarity between males and females in the relative effectiveness of different hormone regimens in facilitating male behavior, and the capacity to display male behavior, is striking. In view of the evidence suggesting that female hamsters are not normally exposed to gonadal androgens during early development, these similarities are highly supportive of the hypothesis that the basic neural structures underlying the execution of male copulatory behavior develop independent of early androgen exposure, and only the arousability or sensitivity of the system to hormonal or external stimulation is affected. ACKNOWLEDGMENTS Publication No. 17-009 of the Wisconsin Regional Primate Research Center. This work was supported by Grant No. MH21312 from the National Institute of Mental Health and Grant No. RR00167 from the National Institutes of Health. The work was conducted while the author was supported by Public Health Service Training Grant No. 5-TO1-HD-00104-08 from the National Institute of Child Health and Human Development. Mr. Guenther Scheffler and Ms. Arlene Mitchell performed the radio-immunoassays, and their help is deeply appreciated. This manuscript was substantially improved as the result of the comments of an anonymous outside reviewer, whose assistance is greatly appreciated.
REFERENCES
1. Baranczuk, R. and G. S. Greenwald. Peripheral levels of estrogen in the cyclic hamster. Endocrinology 92: 805-812, 1973. 2. Baum, M. J. and J. T. M. Vreeburg. Copulation in castrated male rats following combined treatment with estradiol and dihydrotestosterone. Science 182: 283-284, 1973. 3. Beach, F. A. Factors involved in the control of mounting by female mammals. In: Perspectives in Reproduction and Sexual Behavior, edited by M. Diamond. Bloomington: Indiana University Press, 1968, pp. 83-131. 4. Beach, F. A., R. G. Noble and R. K. Orndoff. Effects of perinatal androgen treatment on responses of male rats to gonadal hormones in adulthood. J. comp. physiol. Psychol. 68: 490-497, 1969. 5. Berndston, W. E., C. Desjardins and L. L. Ewing. Inhibition and maintenance of spermatogenesis in rats with polydimethylsiloxane capsules containing various androgens. J. Endocr. 62: 125-136, 1974.
6. Bielert, C., J. A. Czaja, S. Eisele, G. Scheffler, J. A. Robinson and R. W. Goy. Mating in the rhesus monkey (Macaca mulatta) after conception and its relation to oestradiol and progesterone levels throughout pregnancy. J. Reprod. Fert. In Press. 7. Carter, C. S., L. G. Clemens and D. J. Hoekema. Neonatal androgen and adult sexual behavior in the golden hamster. Physiol. Behav. 9: 89-95, 1972. 8. Christensen, L. W., L. P. Coniglio, D. C. Paup and L. G. Clemens. Sexual behavior of male golden hamsters receiving diverse androgen treatments. Hormones Behav. 4: 208-211, 1973. 9. Clemens, L. G. and L. Coniglio. Influence of prenatal litter composition on mounting behavior of female rats. Am. Zool. 11: 617, 1971. 10. Denef, C. and P. DeMorr. Sexual differentiation of steroid metabolizing enzymes in the rat liver: Further studies on predetermination by testosterone at birth. Endocrinology 91: 374-384, 1972.
526 11. Drickamer, L. C. and J. G. Vandenbergh. Predictors of social dominance in the adult female golden hamster (Mesocricetus auratus). Anita. Behav. 21: 564-570, 1973. 12. Eaton, G. Effect of a single prepubertal injection of testosterone propionate on adult bisexual behavior of male hamsters castrated at birth. Endocrinology 87: 934-940, 1970. 13. Edwards, A. L. Experimental Design in Psychological Research. New York: Holt, Rinehart, and Winston, 1950. 14. Emery, D. E. and B. D. Sachs. Ejaculatory pattern in female rats without androgen treatment. Science 190: 484-485, 1975. 15. Gay, V. L. and R. L. Hauger. Inhibition of gonadotropin release in castrated rats by silastic implants containing testosterone, testosterone propionate, or estradiol: Acute response to removal of implants. In preparation. 16. Goy, R. W. and D. A. Goldfoot. Hormonal influences on sexually dimorphic behavior. In: Handbook of Physiology: Endocrinology VII, edited by R. O. Greep and E. B. Astwood. Washington, D.C.: American Physiological Society, Chapter 16. 17. Gray, G. D., H. N. Davis and D. A. Dewsbury. Masculine sexual behavior in male and female rats following perinatal manipulation of androgen: Effects of genital anaesthetization and sexual experience. Hormones Behav. 7: 317-330, 1976. 18. Gual, C., T. Morato, M. Hagan, M. Gut and R. I. Dorfman. Biosynthesis of estrogens. Endocrinology 71: 920-925, 1962. 19. Johnson, W. A. and L. Tiefer. Sexual preferences in neonatally castrated male golden hamsters. Physiol. Behav. 9: 213-217, 1972. 20. Kniewald, Z. and S. Milkovic. Testosterone, a regulator of 5c~ reductase activity in the pituitary of mate and female rats. Endocrinology 92: 1772-1775, 1973. 21. Krieger, M. S. and R. J. Barfield. Masculine sexual behavior: Pacing and ejaculatory patterns in female rats induced by electrical shock. Physiol. Behav. 16: 671-675, 1976. 22. Larsson, K., P. Sodersten and C. Beyer. Induction of male sexual behavior by oestradiol benzoate in combination with dihydrotestosterone. J. Endocr. 57: 563-564, 1973. 23. Naftolin, F., K. J. Ryan and Z. Petro. Aromatization of androstenedione by the anterior hypothalamus of adult male and female rats. Endocrinology 90" 295-298, 1972.
~t>t~! i 24. Noble, R. G. Fhe effects of castration at ditferent int,~r~als after birth on the copulatory behavior of male hamsters Hormones Behav. 4:45-- 52, 1973. 25. Noble, R. G. Estrogen plus androgen induced mountinke m adult female hamsters. Hormones Behav. 5 : 2 2 7 - 2 3 4 , 1974. 26. Noble, R. G. and P. B. Alsum. ttormone-dependent se.x dimorphisms in the golden hamster (Mesocricetus auratus) Physiol. Behav. 14: 567-574, 1975. 27. Nucci, L. P. and F. A. Beach. Effects of prenatal androgen treatment on mating behavior in female hamsters. Endocrinology 88: 1514-1515, 1971. 28. Reddi, V. V. R., F. Naftolin and K. J. Ryan. Conversion to estrone by neural tissues from fetal and neonatal rats. Endocrinology 94:117 121, 1974. 29. Swanson, H. H. Determination of the sex role in hamsters by the action of sex hormones in infancy. In: Influences on the Nervous System, Proc. Int. Soc. Psychoneuroendocrinology, Basel, Karger, 1971, pp. 424-440. 30. Swanson, H. H. and D. A. Crossley. Sexual behavior in golden hamster and its modification by neonatal administration of testosterone propionate. In: Hormones and Development, edited by M. Hamburgh and E. J. W. Barrington. New York: Appleton Century Crofts, 1971. 31. Takayasu, S. and K. Adachi. The in vivo and in vitro conversion of testosterone to 17c~-hydroxy-5c~-androsten-3-one (dthydrotestosterone) by the sebaceous gland of hamsters. Endocrinology 90: 73-80, 1972. 32. Tiefer, L. Gonadal hormones and mating behavior in the adult golden hamster. Hormones Behav. 1: 189-202, 1970. 33. Tiefer, L. and W. A. Johnson. Restorative effects of various androgens on copulatory behavior of male golden hamster. Behaviour 4: 359-364, 1973. 34. Weisz, J. and C. Gibbs. Metabolites of testosterone in the brain of the newborn female rat after injection of tritiated testosterone. Neuroendocrinology 14: 72-81, 1974. 35. Whalen, R. E. The ontogeny of sexuality. In: The Ontogeny of Vertebrate Behavior, edited by H. Moltz. New York: Academic Press, 229-261, 1971. 36. Wiltiams-Ashran, H. G. and A. H. Reddi. Androgenic regulation of tissue growth and function. In: Biochemical Actions of Hormones, edited by G. Litwach. New York: Academic Press, 257-294, 1972.
Brain Research
Publ., 1977. Printed in the U.S.A.
Mounting in Female Hamsters: Effects of Different Hormone Regimens RALPH G. NOBLE
lCisconsin Regional Primate, Research Center, Madison, WI 53706 (Received 28 July •975) NOBLE, R. G. Mounting in female hamsters: effects of different hormone regimens. PHYSIOL. BEHAV. 19(4)519-526, 1977. - Adult female hamsters have substantial capacity to display male behavior without perinatal androgen administration and apparently without perinatal exposure to gonadal steroids. Seventy-two ovariectomized females were tested for male behavior during chronic treatment with either testosterone (T, N = 8); androstenedione (AD, N = 7); dihydrotestosterone (DHT, N = 8); estradiol (E2, N = 7); T + E2 (N = 9); AD + E2 (N = 6); DHT + E2 (N = 7); or no hormone (NoH, N = 19). Eighty-three percent of the females treated with T, T + E2, AD + E2, or DHT + E2 mounted during at least one test, and 70% displayed the intromission pattern; there were no significant differences among these treatment conditions. Forty-two percent of E2-treated females, 14% of AD-treated females, none of the DHT-treated females, and one of the NoH females mounted; these treatments were significantly less effective than T or any of the androgen-plus-estrogen combinations. Females treated with E~ or AD + E2 displayed lordosis responses when tested for female behavior, but females treated with T + E2, DHT + E~, T, DHT or NoH did not. In the hamster the capacity for the execution of male behavior develops independent of early androgen, and only the arousability of the system by hormonal and external stimulation is sex dimorphic. Testosterone Dihydrotestosterone Sex differences
Androstenedione
Mounting
Hamsters
Sex behavior
neural substrates underlying the execution of the components of the male copulatory pattern develop independent of genetic sex without requiring exposure to gonadal steroids during early development. Other studies, primarily with rats, have indicated that several variables which influence the male copulatory behavior of genetic males have essentially the same effect on the male copulatory behavior of genetic females (cf. [ 14] ). These findings would suggest that the consequences of early androgen action are restricted to altering the kinds and amounts of hormonal or proprioceptive stimulation required to elicit the execution of behavior patterns without altering the development of the capacity to execute the patterns. This interpretation depends on the assumption that the normal female develops without being exposed to gonadal steroids in significant amounts during early stages of development. This assumption is questionable at least for the rat. While attempts to determine if prenatal exposure to exogenous androgen alters the subsequent display of male behavior by female rats have not produced conclusive results (cf. [16]), there is evidence that the prenatal hormonal milieu can influence the capacity to display male behavior. Clemens and Coniglio [9] have demonstrated that the amount of male behavior displayed by female rats is highly correlated with the amount of exposure to testicular secretions from male siblings in utero. Some investigators have interpreted the available evidence as suggesting that, for both males and females, the capacity to display male behavior depends on exposure to gonadal steroids during early development with
WHILE FEMALES of many mammalian species have the capacity to display components of the male copulatory pattern [3], there are clear sex differences in the kinds and amounts of male sex behavior displayed when gonadectomized males and females are given standard regimens of exogenous androgen administration and tested for male behavior as adults [ 16]. In the species tested, this sex difference in the display of male copulatory behavior is related to the presence or absence of gonadal steroids during early stages of development [16]. Recent investigations strongly suggest that the facilitation of male copulatory behavior produced by exposure to androgen during early development results, at least in part, from effects of the androgen on the developing nervous system and cannot be explained on the basis of the effects of early androgen administration on the development of the genitalia (cf. [17]). Our thinking about the consequences of exposure to androgen during early development may be clarified by a careful analysis of the residual capacity of the normal adult female to display male behavior. Recent studies have demonstrated that the female rat has the capacity to display all the components of the male copulatory pattern. This capacity has been demonstrated both by testing the consequences for the display of male behavior of prolonged exposure to high doses of exogenous estrogen [ 14], and by providing peripheral electrical shock to females being tested for male behavior [21]. The residual capacity of the perinatally untreated female rat to display all the components of the male copulatory pattern suggests that the 519
520 both the testes of male siblings and the maternal adrenal being identified as possible sources of gonadal steroids [161. Further analysis of the effects of early exposure to gonadal steroids on psychosexual development requires an analysis of the capacity of animals which have not been exposed to gonadal steroids during early development to display male copulatory behavior. At present it is not clear that this analysis can be made in species in which the critical periods for psychosexual differentiation are partly prenatal [ 16]. The female hamster probably represents the closest available approximation to a rodent which develops in the absence of gonadal steroids during the critical period for psychosexual differentiation, although this has not been varified by direct measurements of hormone concentrations in the neonatal female. Prenatal androgen administration does not augment mounting by female hamsters [27]; castration of the male hamster on the day of birth virtually eliminates mounting following androgen treatment of the adult [7, 12, 24]; and postnatal administration of small quantities of androgen augments mounting of female hamsters [29,30]. Female hamsters rarely mount when exogenous androgen is administered, even in very high doses, for periods of time sufficient to restore the copulatory behavior of male hamsters castrated as adults [25]. Apparently the period of psychosexual differentiation is postnatal in the hamster. Therefore, it is important to carefully assess the capacity of female hamsters to display male behavior. A vigorous attempt to elicit male behavior from female hamsters may provide an estimate of the extent to which the capacity for male behavior develops in the absence of exposure to gonadal steroids early in development. It is also important to determine if in the female hamster extended periods of hormone treatment can facilitate male behavior, possibly producing changes similar to those which typically occur during exposure to gonadal steroids early in development. Evaluating the effects of various manipulations on the display of mounting by adult female hamsters may prove valuable both in explicating the effects of perinatal exposure to gonadal steroids and in evaluating hypotheses about the physiological mechanisms underlying sex differences in the display of male behavior. Recent studies suggest that the failure of female hamsters to m o u n t following adult treatment with testosterone for periods of time sufficient to restore the copulatory behavior of males castrated as adults results from the inability of female hamsters to respond to androgen treatment as rapidly as male hamsters and a failure to metabolize testosterone as effectively as male hamsters. Some indirect evidence suggests that the hypothesized deficiency of female hamsters to metabolize testosterone is a failure to convert testosterone to dihydrotestosterone rather than a failure to convert testosterone to estradiol. In both male and female hamsters chronic testosterone propionate administration reduces the dose of estradiol benzoate required to facilitate estrus when estradiol benzoate is administered in conjunction with progesterone. In male hamsters both chronic testosterone propionate administration and chronic dihydrotestosterone propionate administration impair the quality of estrogen-plus-progesteroneinduced estrus behavior, but in female hamsters only chronic dihydrotestosterone propionate administration impairs the quality of estrus behavior [26]. Daily injections of
~'i ~i~.l [ dihydrotestosterone propionate and either estradiol ben,'(>ate or estrone significantly increase mounting m re'male hamsters after 17 28 days of treatment. Estradiol benzoate and dihydrotestosterone propionate are not effective when administered alone, and testosterone propionate does not facilitate mounting. Estradiol benzoate combined with dihydrotestosterone propionate produce higher mount frequencies than do estrone plus dihydrotestosterone propionate [251. Since several studies have reported sex differences in steroid metabolism in the rat [10, 20, 231, the hypothesis was formed that the failure of female hamsters to m o u n t results from a reduced ability to convert testosterone to dihydrotestosterone. The present study compared the effects of testosterone, androstenedione, and dihydrotestosterone when administered alone or in combination with estradiol on mounting by female hamsters. Based on the hypothesis stated above, it was predicted that testosterone plus estradiol would be less effective in facilitating mounting by female hamsters than dihydrotestosterone plus estradiol. Androstenedione was included in the present study because it is at least as effective as testosterone in maintaining the copulatory behavior of castrate male hamsters [8]. Androgen implants made from polydimethylsiloxane tubing and estradiol implants made from stainless steel tubing sealed with polydimethylsiloxane elastomer were used in an attempt to provide reasonably low and constant hormone concentrations in the blood [5,15[. METHOD
Animals Female hamsters ( 9 1 - 1 0 0 g weight class) were purchased from Engle's Laboratory Animals, Inc. (Farmersburg, IN). Females were housed 4 - 5 per cage (38 x 33 x 17 cm) on a reversed light cycle (LD: 14: 10) with the lights turned off at 1200. After four weeks in the laboratory, females were ovariectomized using sodium pentobarbital anesthesia.
Design Females were assigned at random to one of the following groups: testosterone (T, N = 8); androstenedione (AD, N = 7); dihydrotestosterone (DHT, N = 8); estradiol (E 2, N = 7 ) ; T + E 2 ( N = 9 ) ; A D + E ~ (N = 6 ) ; D H T + E ~ (N = 7 ) and no hormone controls (NoH, N = 19). Two weeks after ovariectomy, hormone implants, described below, were p l a c e d subcutaneously between the scapulae while the animals were anesthetized. The implant was placed approximately 2 - 3 cm from the incision, which was closed with a wound clip. Females were tested for male behavior once a week for six weeks, beginning 17 days after the start of hormone treatment. The day after the last test for male behavior, they were tested for female behavior. Then the females were anesthetized, weighed, and the implant removed. At this time the length of the flank gland (intercostal sebaceous gland) on the left side was measured because it is androgen-dependent and the effectiveness of testosterone on this organ is dependent on its conversion to a 5~-metabolite, probably dihydrotestosterone [31 ].
Testing Procedure All tests were conducted between 1 3 0 0 - 1 8 0 0 during the first few hours of the dark phase of the cycle. The testing arenas were 5-% gallon all-glass aquaria.
MOUNTING IN FEMALE HAMSTERS The procedure for the testing and scoring of male behavior was similar to that described elsewhere [24]. When tested for male behavior, the experimental female was placed in the arena I0 min before the start of the tests, which were 10 min long. An ovariectomized female in hormone-induced estrus was placed in the chamber to start the test. The frequency and duration of anogenital examination, mounting and intromission were recorded using a keyboard connected to an array of counters and timers. In the present study, only mounts appropriately oriented to the caudal end of the stimulus female which included clasping the stimulus female with the forepaws and pelvic thrusting were recorded. Linguo-genital contact was used as the criterion for anogenital examination. When tested for female behavior, the female was placed in an arena containing two intact males. The tests were terminated either when the female assumed the lordosis posture or when she had received five appropriately oriented mounts. The number of mounts preceding the initial display of lordosis was recorded.
Implant Description Androgen implants were prepared from 3 cm sections of polydimethylsiloxane tubing (Medical Grade Silastic Tubing®, Dow Chemical Co.; inner diameter, 1.8 mm; outer diameter, 3.17 mm) following the procedure described by Gay [15]. Sections of tubing were rinsed in ethanol and allowed to dry. Then a 0.5 cm-long wood dowel was inserted flush with one end of the tubing. The lumen of the tube was filled with crystalline steroid and another 0.5 cm wood dowel inserted. Then the ends o f the implant were sealed with polydimethylsiloxane elastomer (382 Medical Grade Elastomer ®, Dow Chemical Co.). Estradiol implants were made from 1 cm sections of stainless steel in hypodermic tubing filled with crystalline hormone to within 1 mm of each end and sealed with elastomer. All implants were incubated for 4 8 - 7 2 hr in phosphate-buffered saline at 37 ° C. Several apparently good implants were rejected when moisture was observed within the lumen of the tubing. Each implant was rinsed With ethanol immediately before it was placed in the animal. The size of the androgen implants was selected to release less than 200 ~g of steroid per day as determined in a preliminary study using the procedure described by Berndston [5]. T, AD and DHT implants of this size are effective in maintaining the copulatory behavior of castrate male hamsters (Noble, unpublished observations). The estradiol implant selected is the smallest size implant which reliably facilitates estrous behavior in female hamsters when combined with a single subcutaneous injection of 0.5 mg progesterone, and implants this size do not significantly alter the copulatory performance of castrate male hamsters (Noble, unpublished observations). The radioimmunoassay [6] was used to determine the plasma concentrations of estradiol in four females which had each received three implants eight days before the blood was collected. F r o m these data, it was estimated that a single implant would produce an estradiol concentration of approximately 150 pg/ml of plasma, which is less than the plasma concentration of estradiol in proestrous female hamsters [ 1]. For this initial determination three implants were used in an attempt to guarantee measurable concentrations of hormone. Since the implants used in the experiment were still filled with crystalline hormone when
521 examined visually at autopsy, it is reasonable to assume that release rates were roughly constant during the experiment [5].
Statistical Procedure An unweighted-means Analysis of Variance was computed for b o d y weight and flank gland size. If the overall F was significant, the individual groups were compared using the Newman-Kuels procedure for comparison among hormone-treated groups, and using Dunnett's procedure for comparison between hormone-treated groups and the NoH controls [13]. Differences in behavior measures were analysed using appropriate nonparametric statistics as indicated in the text. RESULTS
Anogenital Examination Most hormone-treated females displayed anogenital licking during at least one test (72% of E 2 -treated females, 83% of AD + E~-treated females, and 100% of the females in all other treatment groups). Only 16% of the NoH females showed this response (E~ vs. NoH, p < 0 . 0 5 , Fisher's exact probability test) and each of these females did so during only one test. Mean durations of anogenital licking per 10 min period were computed from the last two tests for male behavior. This provided estimates of maximum performance under the different treatment regimens. All hormone-treated groups had mean anogenital licking durations longer than that of the NoH controls (p<0.01, Mann Whitney U). T-treated females had longer anogenital licking durations than any of the other hormone-treated females (p<0.01, in each case). Figure 1 shows the percentage of animals showing anogenital licking on each of the six tests. The percentage of females in the various androgen or androgen plus estradiol treatment conditions was fairly consistent across tests, but the performance of females treated with E2 was erratic, with 47% of the females engaging in this behavior on Tests 3 and 6 (on Days 31 and 52) and none of the females doing so on Days 24, 38 or 45. E~-treated females engaged in anogenital examination during significantly fewer tests than did females in androgen-treated groups (p<0.05, at least, Mann Whitney U). Examination of the mean duration of anogenital licking (Fig. 2) reflects a similar pattern and indicates that the significantly higher durations of the T-treated females were not observed during the first four tests.
Mounting Eighty-eight percent of the T + E2-treated females, 82% of the AD + E2-treated females, and 86% of the DHT + E2-treated females mounted. Seventy-five percent of Ttreated females mounted during at least one test, 14% of the AD-treated females, none of the DHT-treated females, and one of the NoH females mounted. Forty-one percent of the E 2-treated females mounted, each during one test. T, T + E~, AD + E 2 and DHT + E~-treated females mounted during a higher percentage of the tests than the NoH controls (p<0.01 in each case, Mann Whitney U), and the AD, DHT and E2-treated females did not differ from the controls. The percentage of females mounting increased gradually in all cases (see Fig. 3), and there were no
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FIG. 1. The effect of various hormone regimens on the percentage of ovariectomized females engaging in anogenital licking during testing for male behavior. significant differences among the different androgen plus estradiol-treated groups in the number of treatment days preceding the first mount. The median number of treatment days preceding the first mount was 35 for the androgen plus estradiol-treated females as a group. The onset of mounting among the T-treated females was similar with a median of 31 days preceding the first mount. Fifteen of the nineteen females which mounted during more than one test had higher mount frequencies on subsequent tests (p<0.01, sign test). During the last two tests for male behavior both the T and the T + E 2-treated females had higher m o u n t frequencies than did the females in the other treatment groups (see Fig. 4) and these differences were significant when assessed using a nonparametric statistic to compare the T-treated females and the T + E2-treated females as a group (p<0.02, Mann Whitney U).
In tromission Sixty-seven percent of T + E,-treated females, 82% of AD + E2-treated females, 57% of DHT + E2-treated females, 75% of T-treated females, 14% of AD-treated females, 14% of E2-treated females, none of the DHT-treated females and none of the NoH females displayed the intromission pattern (see Fig. 5). The intromission pattern was displayed in 87% of the tests during which females mounted, and the ratio of tests positive for intromission to tests positive for mounting did not vary significantly among treatment groups.
FIG. 2. The effect of various hormone regimens on the mean duration(s) of anogenital licking by ovariectomized female hamsters during tests for male behavior.
Female Behavior All of the AD-treated females and six of the seven E~-treated females displayed the lordosis response when tested for female behavior. In all cases, the display of lordosis was preceded by one or more mounts. None of the females in any of the other treatment conditions displayed the lordosis response.
Morphological Measurements Flank gland. The flank glands of females with either T or DHT implants were comparable in size to the flank glands of intact males (X -+ SE = 7.0 -+ 0.2 mm) or testosteronetreated castrate males (X -+ SE = 5.7 ± 0.2 mm) (Noble, in preparation), and larger than the flank glands of AD-treated females (p<0.002). The flank glands of the AD'treated females were larger than the flank glands of the E2-treated females or the NoH females (p< 0.002). Body weight. The females receiving hormone treatments which facilitated mounting (T, T + E 2 , AD + E~, and DHT + E 2 ) weighed less than females in other treatment groups (AD, DHT, E 2 , NoH; p< 0.01). AD-treated females weighed less than DHT-treated females (0.06>p>0.05), E~'treated females (p<0.001), or NoH females (p<0.001). Individual Differences Data from the T-treated females and the T + E 2-treated females were used to quantitatively assess the reliability of individual differences and to examine the covariation among mount frequency, the latency to the onset of
M O U N T I N G IN F E M A L E H A M S T E R S
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FIG. 3. The effect of various hormone regimens on the percentage of ovariectomized female hamsters mounting during tests for male behavior. anogenital licking, and b o d y weight. Correlations b e t w e e n flank gland size and o t h e r e n d p o i n t s were n o t assessed because individual differences in flank gland size were minimal. Individual differences in m o u n t f r e q u e n c y were highly consistent for b o t h T and T + E~ females (rho = .90; and rho = .80, respectively). The duration of t r e a t m e n t prior to anogenital licking was predictive of subsequent m o u n t i n g p e r f o r m a n c e of b o t h groups (T, rho = .90; T + E2, rho = .74). B o d y weight was also correlated with the duration of
FIG. 4. The effect of various hormone regimens on the mean frequency of mounting. t r e a t m e n t prior to the onset of anogenital licking (T, rho = .62; T + E 2 , rho = .84), but n o t with m o u n t f r e q u e n c y (T, rho = .44; T + E 2 , rho = .00). DISCUSSION In the hamster three different classes of sex differences in male behavior can be identified: (1) differences in the h o r m o n e regimens that facilitate male behavior in the adult; (2) differences in the duration of h o r m o n e administration adults require before displaying male behavior; and (3) differences in the m a x i m u m level o f male behavior display-
TABLE 1 EFFECTS OF DIFFERENT HORMONE TREATMENTS ON BODY WEIGHT AND FLANK GLAND LENGTH Measure
T + E~
AD + E2
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DHT
E2
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5.9 0.2
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1.0 0.2
*DHT, E2, NoH, AD (p<0.05); AD, AD + E2, DHT + E2, T + E2, T (p<0.05). Among hormone comparisons based on Newman-Kuels Procedure; all comparisons with control used Dunnett's Procedure. *DHT > DHT + E2, T + E2, T > AD + E2, AD > E2, NoH; (p<0.01, see above).
524
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FIG. 5. The effect of various hormone regimens on the percentage of ovariectomized female hamsters displaying the intromission pattern during tests for male behavior.
ed by the adult under favorable conditions of testing and hormone replacement. These three classes of differences are at least partially dissociable in the hamster. Following prolonged treatment with estradiol and any of the androgens used in the present study or with testosterone, which can be converted to both estradiol and other androgens (36), female hamsters m o u n t and display the intromission pattern. The differential effectiveness of the androgens in facilitating mounting when administered alone probably reflects differences either in the estrogenic properties of the androgens or in the extent to which these androgens are metabolized to estradiol. Seventy-five percent of testosterone-treated females mounted during at least one test compared to fourteen percent of androstenedione-treated females and none of the dihydrotestosterone-treated females. The differences among testosterone, a n d r o s t e n e d i o n e and dihydrotestosterone in facilitating mounting correspond to the relative convertability of these androgens to estradiol [18]. In rat brain tissue, testosterone is converted to estradiol but not estrone [34], while androstenedione is aromatized to estrone but not estradiol [ 28]. Apparently mounting in female hamsters is dependent on both androgenic and estrogenic stimulation. While all three androgen-plus-estrogen combinations were roughly equivalent in facilitating mounting, they probably provided different relative amounts of estrogenic and androgenic stimulation. Two observations suggest that combined treat-
ment with androstenedione and estradio] prowded icss androgenic stimulation than the other hormone regimen:~ which were effective in facilitating mounting. (t } Andnv stenedione-treated females had smaller flank glands. Dihydrotestosterone is the effective androgen in this target organ [31 ]. (2) Androstenedione-plus-estradiol-treated females displayed lordosis while testosterone-plus-estradioltreated females and dihydrotestosterone-plus-estradioltreated females did not. Chronic dihydrotestosterone treatment has been demonstrated to interfere with the lordosis behavior of female hamsters [ 26]. The equivalence of testosterone, testosterone plus estradiol, dihydrotestosterone plus estradiol, and androstenedione plus estradiol in facilitating mounting in female hamsters suggests that the requirement for mounting in female hamsters is that both androgenic stimulation and estrogenic stimulation be present in some minimal amount. In castrated male hamsters dally injections of either estradiol benzoate or dihydrotestosterone propionate are partly effective in reinstating components of the male copulatory pattern, though neither is as effective as testosterone propionate [26]. This suggests that the physiological mechanisms underlying mounting behavior in male and female hamsters are highly similar. Christensen e t al. [8] reported that daily injections of testosterone, androstenedione and dihydrotestosterone maintained mounting in castrate male hamsters; testosterone and androstenedione were equally effective and both were more effective than dihydrotestosterone. Tiefer and Johnson [33] also reported that daily injections of testosterone and androstenedione were equally effective in restoring the copulatory behavior of male hamsters. Subcutaneous implants of the same size used in the present study containing either testosterone, androstenedione or dihydrotestosterone maintain mounting in male hamsters. Dihydrotestosterone implants restore mounting behavior in castrate adrenalectomized male hamsters, suggesting that estrogen is not required to facilitate mounting by male hamsters. However, the relative effectiveness of the three hormones in maintaining ejaculatory frequency in males is similar to the relative effectiveness of the three hormones in facilitating mounting behavior in females, with testosterone being significantly more effective then dihydrotestosterone (Noble, in preparation). It is not clear why chronic subcutaneous implants of testosterone facilitated mounting by female hamsters when daily injections of testosterone propionate using amounts far in excess of what is required to fully restore mounting in males castrated at ten days of age or older [24], do not facilitate mounting under identical conditions [25]. Either the testosterone implants deliver a higher effective dose of androgen than the testosterone propionate injections, for example by producing a higher average concentration of testosterone in the blood, or the injection procedure itself has effects that interfere with the display of mounting by female hamsters, but do not interfere with the display of mounting by male hamsters. The available data suggest that the injection procedure itself has effects which interfere with the display of mounting by female hamsters. Silastic implants of testosterone propionate are as effective in facilitating mounting in female hamsters as silastic implants of testosterone (Noble, unpublished observations). Estimates of the total amounts of hormone involved in the two modes of hormone administration render a simple dose difference hypothesis
MOUNTING IN FEMALE HAMSTERS
525
implausible. In our previous study [25], 52 mg of testosterone propionate were injected over a 38-day period without facilitating mounting by female hamsters. Preliminary estimates of the release rates of implants of the size used in the present experiment, based on changes in the dry weight of the implants, indicate that over a 38-day period about 2.25 mg (standard deviation, 0.4 mg) of testosterone is released (Noble, in preparation). A direct test of the dose difference hypothesis would require determination of plasma concentrations of testosterone at several different times of day to permit an estimate of the average plasma concentration produced by the two different methods of hormone administration. The duration of hormone treatment preceding the onset of mounting by female hamsters far exceeds that required to produce maximal levels of mounting by male hamsters castrated either as adults [26] or prepubertally [24]. It also exceeds the duration of treatment required to induce mounting in immature male hamsters (Noble, unpublished observations). Also, the duration of hormone treatment required to induce mounting in the present study is comparable to the duration required in the prior experiment [25] in which high doses of estradiol benzoate and dihydrotestosterone propionate were injected daily. The extended period of hormone treatment preceding the display of mounting by females in the present study appears invariant across a variety of hormone regimens, each of which is effective in inducing mounting but which differ markedly in their effects on the other behavioral and anatomical endpoints examined. Prolonged hormone treatment of adult female hamsters may facilitate mounting by first producing changes in the responsiveness to exogenous hormones similar to the changes in responsiveness to hormones produced by perinatal hormone treatment [16] followed by hormone induced activation of mounting behavior. In a previous study [24], the duration of hormone treatment required prior to the display of mounting was inversely related to the age at castration during the first ten days of life. Males castrated five days postpartum did not differ from males castrated ten days postpartum in the maximum rate of mounting, but the Day 5 castrates did require more extended hormone treatment prior to the onset of mounting. The amount of time required to activate mounting in those Day 1 castrate males which did m o u n t is comparable to the amount of time required to activate mounting in
female hamsters. It appears possible that the female hamster may retain some limited ability to undergo organizational changes in response to extended hormone treatment as an adult. While the average m o u n t frequency of females is lower than that of intact males or that of testosterone-treated castrate males (Noble, in preparation), the m o u n t frequency of females is largely determined by highly stable individual differences in the predisposition to display male behavior. The determinants of these individual differences are not known. Female hamsters in the present study displayed the intromission pattern less frequently than comparably treated males and did not display the ejaculatory response. Since the occurrence of intromission and ejaculation are heavily dependent on appropriate sensory feedback in the male hamster (Noble, in preparation), it is not possible to interpret the failure of female hamsters to show the ejaculatory response. Taking into account the obvious anatomical differences between male and female hamsters, the similarity between males and females in the relative effectiveness of different hormone regimens in facilitating male behavior, and the capacity to display male behavior, is striking. In view of the evidence suggesting that female hamsters are not normally exposed to gonadal androgens during early development, these similarities are highly supportive of the hypothesis that the basic neural structures underlying the execution of male copulatory behavior develop independent of early androgen exposure, and only the arousability or sensitivity of the system to hormonal or external stimulation is affected. ACKNOWLEDGMENTS Publication No. 17-009 of the Wisconsin Regional Primate Research Center. This work was supported by Grant No. MH21312 from the National Institute of Mental Health and Grant No. RR00167 from the National Institutes of Health. The work was conducted while the author was supported by Public Health Service Training Grant No. 5-TO1-HD-00104-08 from the National Institute of Child Health and Human Development. Mr. Guenther Scheffler and Ms. Arlene Mitchell performed the radio-immunoassays, and their help is deeply appreciated. This manuscript was substantially improved as the result of the comments of an anonymous outside reviewer, whose assistance is greatly appreciated.
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