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The relationship between levels of exogenous hormones and the display of lordosis by the female rat
Hormones and Behavior 2, 287-297 (1971)
The Relationship Between Levels of Exogenous Hormones and the Display of Lordosis by the Female Rat DONNA FITZROY HARDY and JOSEPH F. DeBOLD Department of Psychology, San Fernando Valley State College, Northridge, California 91324
In three experiments, the duration, intensity and probability of display of lordosis by ovariectomized female rats were measured in response to administration of progesterone and various levels of eStndiO1 benzoate (ED). Behaviors of females in natural heat were compared with those receiving exogenous hormones. Lordosis duration was shown to be directly proportional to the amount of EB administered, although mean duration of hormone-treated females did not reach that shown by females in natural heat. Both Lordosis Quotient and intensity of lordosis rcachcd asymptote with relatively low doses of EB when animals were treated with progesterone. Ovariectomy reduced the duration and intensity of lordosis even when amounts of EB and progesterone capable of restoring Lordosis Quotients were administered. Finally, the well-known synergistic relationship between estrogen and progesterone in bringing about lordosis in ovariectomized females was found to influence duration and intensity of lordosis as well as the probability of its display. Auxiliary heat behaviors (earwiggling, hopping, and display of “spontaneous” lordosis) were found to be related to level of replacement-EB as well as to the presence of progesterone. These results suggest that hormonally induced heat can be differentiated from natural heat on the basis of duration of lordosis.
Sexual receptivity in the female rat has usually been defined in terms of the display of a characteristic spinal reflex, the lordosis response. Although some early studies (Boling and Blandau, 1939) have equated a single lordosis as indicating sexual receptivity, most recent studies (Beach, 1943; Kuehn and Beach, 1963; Edwards, Whalen and Nadler, 1967) base level of receptivity upon the proportion of mounts by the male rat which elicits lordosis by the female. Various authors express this proportion as Copulatory Quotient, Sensitivity Index, Receptivity Quotient, Lordosis-to-Mount Ratio, L/M or Lordosis Quotient. It is generally assumed that the higher the proportion of mounts which result in lordosis, the higher the level of receptivity. Relatively few systematic attempts have been made to characterize sexual behavior with any index of lordosis other than its display in response to mounting by the male. Kuehn and Beach (1963) and Moss (1971) measured duration of 287
288
HARDYANDDeBOLD
lordosis. Other authors (Hemmingsen, 1933; Hart, 1969; Gerald and Kenney, 1970; Whalen, Luttge and Gorzalka, 1971; Hardy and DeBold, 1971) noted differences in intensity of lordosis, although intensity has not been a common measure of level of receptivity. The purpose of the present study was to investigate quality of lordosis as well as its display to mounting by the male. In addition to behavior of the intact rat during natural heat, behavior was studied in ovariectomized females displaying lordosis while receiving various levels of replacement estrogen, ranging from a “physiological dose” (0.5 ,ug) to a “pharmacological dose” (500 ng). The behavior of individual females during natural heat was compared to their behavior during hormone-induced heat following ovariectomy. An attempt was made to classify the intensity of each lordosis response, and the duration of each lordosis was measured. The study differs from previous studies in that differences in behavior of the female which are due to intromission and ejaculation by the male (Kuehn and Beach, 1963; Dewsbury, 1967; Moss, 1971; Hardy and DeBold, in press) were prevented by a technique which eliminates intromission (Hardy, 1969).
MATEFUALS AND METHODS Female Sprague-Dawley rats obtained from Simonsen Laboratories (Gilroy, CA) were housed in pairs with food and water present at all times. A reversed light-dark schedule was employed with fluorescent ceiling lights on from 6:00 PM to 6:00 AM. During behavioral tests, all females received 10 mounts by sexually experienced male rats in glass observation jars (12 in. across and 18 in. high). Vaginal masks made of pliable masking tape (1 in. wide and 2-3 in. long) were affixed to the fur of the ventral surface of all females. When secured to the base of the tail, masks covered the perineal region and occluded the vaginal orifice. It had been previously demonstrated that these masks prevent intromission by the male without interfering with the normal display of estrus behavior by the female (Hardy, 1969). Although there is little reason to believe that the masks interfered with the actual mounting by the males (cf. Kaufman, 1953), males were frequently “primed” with unmasked stimulus females to prevent the waning of sexual behavior observed by Whalen (1961) in males that were prevented from achieving intromission. During tests, presence or absence of three behaviors characteristic of estrus in this species were noted: ear-wiggling, hopping, and lordosis to mounting by the male. In addition, the occurrence of “spontaneous lordosis” was noted in Experiment III; this behavior was defined as lordosis in response to sniffing, grasping with the forepaws or any tactile stimulus from the male rat other than mounts with thrusting.
HORMONESAND DISPLAY OF LORDOSIS
289
The index of sexual responsiveness was the Lordosis Quotient: total number of lordosis responses/test __-L.Q. = --total number of mounts with thrusting by the male Sexual responsiveness was further characterized by two measures of lordosis quality-duration and intensity. Total duration of all lordosis responses for each test was timed by independent observers using cumulative stopwatches. Mean duration (in seconds) of lordosis was then recorded for each test: total duration of lordosis responses/test _--.-_---__ Mean Lordosis Duration = -total number of lordosis responses/test The intensity scale:
of each lordosis was rated by these observers using a 3-point
1 point
= marginal lordosis (slight flex of spine, slightly raised head and hips with tailbase elevated from the floor). 2 points = normal lordosis (spinal flex, head at an approximate angle of 30” with the horizontal, front paws placed slightly forward and hind legs straightened up stiffly). 3 points = exaggerated lordosis (pronounced spinal flex, head at an angle of 45” or more with the horizontal). These postures are illustrated in another paper (Hardy and DeBold, 1971). Total points were accumulated for each test and the mean calculated: total number of lordosis points/test _-.-.-___ Mean Lordosis Intensity = ----total number of Iordosis responses/test EXPERIMENT I Relationship Between Level of Estradiol Benzoate and the Display of Lordosis Procedure. In this experiment, 60 female rats were used. Twelve females were intact and displaying regular 4-day estrous cycles (as confirmed by vaginal smearing), while the remaining forty-eight had been ovariectomized 3 months prior to the experiment. One hour before testing of intact females, heat was established by their behavioral response to mounting by the males: females were masked and allowed to receive several mounts by the males. It had been previously found that normally cycling female rats display lordosis to most mounts during the onset of heat (Hardy, in press). Intact females that displayed lordosis to each mount by the male were considered to be in heat and were tested later in the morning. The ovariectomized female rats were divided into four equal groups which received 2 subcutaneous doses of estradiol benzoate (EB) in oil 24 hr apart; each dose was 0.5, 5, 50, or
290
HARDY AND DeBOLD
500 pg. EB was administered in two series; the first series was one week prior to testing. The second two injections of EB were given 44 and 20 hr before testing. Five hours before testing, 500 pg progesterone in oil was administered subcutaneously. This injection regimen roughly approximates that used in other laboratories, and the 4 doses of EB encompass the range of exogenous estrogen administered by other workers; Tests were conducted between 8:OO AM and noon. Results. Data from Experiment I are summarized in Table 1. There was an overall difference between Lordosis Quotients of the five groups (F(4,55) = 7.79, p < 0.001) with the females receiving 0.5 yg EB showing the lowest mean L.Q. and the groups receiving 5, 50, and 5OOpg EB the highest mean L.Q.‘s. Lordosis Quotients of intact females were significantly higher than the 0.5 pg EB group (t (12.2)1 = 2.37, p < 0.05) but lower than that of the 50 pg EB group (t (11) 1 = 2.80, p < 0.02). In addition, there was a clear relationship between level of replacement hormone and Mean Lordosis Duration (F(4,55) = 20.28, p < 0.001) (Fig. 1). The Mean Lordosis Duration of females in natural heat was significantly longer than the duration of lordosis of females receiving the highest level of replacement hormone, 500 pg EB (t (22) = 2.90, p < 0.01). A test for linearity showed a significant linear component for the duration of lordosis (F(1,55) = 80.75, p < 0.001). There were overall differences between Mean Lordosis Intensity of intact females and ovariectomized females receiving replacement hormones (Kruskal-Wallis, H = 14.74, p < O.Ol), and Mean Lordosis Intensity of intact females was significantly higher than that of ovariectomized females receiving 0.5 pg EB (Mann-Whitney U test, p < 0.01). Finally, there were general increases in percentage of females displaying ear-wiggling and hopping behaviors with increases in level of EB replacement. More females receiving the higher doses of EB (50 and 5OOpg) displayed hopping than did intact females in natural heat.
EXPERIMENT II Quality of Lordosis Before and After Ovariectomy Procedure. This experiment compared the behavior of females displaying natural heat with their behavior following ovariectomy during hormonally induced heat. Twenty intact females were tested during the morning of their natural heat (between 8:00 AM and noon with a reversed light cycle). Lordosis Quotient, Mean Lordosis Duration, Mean Lordosis Intensity and display of ear-wiggling and hopping were recorded for each female. Females were ovariectomized immediately following the test of the twentieth female, IDegrees of freedom compensated for unequal variance.
.90-1.00 .70-I .oo
1.00
70-1.00
O-1-00
+.03 -+o k.01 +.03
.61+ .13
.98 1.00 .99 .92
range
mean k SE
Lordosis Quotient
2~.08 + .02 2 .06 +-.04
1.11 * .06
.45 59 .75 .90
mean -t SE O-.72 .50-.78 54-1.25 .67-1.15 .75-1.55
range
-
Mean
1.44 c .lO
1.25 k.09 1.56 f .09 1.30 + .08
.79 k.14
mean k SE
1.00-2.11
1.10-2.00 1.00-1.90
1.00-1.90
O-1.20
range
Lordosis Intensity (scale = l-3)
100
92 83 92
42
Percent showing eare&w
1FemaIesreceived estradiol benzoate (EB) 44 and 20 hr before testing and 500 fig progesterone 5 hr before testing.
50 kg EB 500 pg EB Intact
0.5 j.ig EB 5%EB
Treatment group
Mean Lordosis Duration (in seconds)
Behavior of Ovariectomized Female Rats Displaying Hormonal-induced HeatI and Intact Females Displaying Natural Heat (SE = standard error, )?-- 12 per group)
TABLE 1
58
50 83 83
17
Percent showing hopping
5; 7a
%
5
; 3 tY 3
292
HARDY AND DeBOLD
.-u) m g.40;; -I c.20L 5 -
0.5
5
50
500
intact
Fig. 1. The relationship between duration of lordosis and level of estradiol benzoate (EB) in pg. The four groups of females receiving EB had been ovariectomized and were administered 500 ~g progesterone before testing. The fifth group of females were intact and displaying natural heat. The vertical lines indicate standard error (n = 12 in each group).
then the vaginas of all females were smeared with a fine wire loop for 10 days. These smears confirmed the diestrus vaginal condition and the absence of ovarian tissue. On the 10th day following ovariectomy, hormone administration was begun: females were administered I gg EB 44 and 20 hr before the start of testing and 5OOpg progesterone 5 hr before testing. Previous experiments in our laboratory had established that two injections of 1 grg EB followed by 500 @g progesterone result in Lordosis Quotients which closely approximate those of females in natura1 heat. Results. The behavior of females during natural and hormone-induced heat is summarized in Table 2. There were no significant differences between Lordosis Quotients of females before and after ovariectomy, and hormone treatment (paired t (19) = 0.66, NS), but Lordosis Duration was significantly longer when females were displaying natural heat (paired t (19) = 9.60, p < 0.001). Furthermore, Mean Lordosis Intensity was greater during natural heat than during hormonal-induced heat (sign test, p < 0.001). There were no differences between the two conditions with respect to percent of females showing ear-wiggling or hopping.
.86 It.06
.95 + .02
mean + SE
.55 k .02
1.03 f .04
.70-1.00
.10-l .oo
mean + SE
range
Lordosis Quotient
.30-.75
.70-1.55
range
Mean
1.20 t .06
1.43 k.07
mean k SE
80
1.00-l .80
50
70
100 1.00-2.11
range
Percent showing hopping Percent showing ear-wiggling
Lordosis Intensity (scale = 1-3)
IFemales received 1 pg EB 44 and 20 hr before testing and 500 pg progesterone 5 hr before testing.
induced beat
Intact, natural heat Ovariectomized, EB + progesterone
Condition of females
Mean Lordosis Duration (in seconds)
Behavior of Female Rats During Natural Heat and Following Ovariectomy During Hormonal-induced Heat,1 (n = 20)
TABLE 2
CJ Ez 5
r
ss
z
T 3 a !$
294
HARDY
AND DeBOLD
EXPERIMENT III Relationships between Progesterone and Behavior of Estrogen-treated Fernale Rats Procedure. Sixteen female rats that had been ovariectomized for about 2 months were administered 2 subcutaneous doses of 5 pg EB 24 hr apart one week prior to testing, then 5 pg EB 42 and 18 hr before the start of testing. Half of them received 500 pg progesterone 3 hr before testing. This dose of EB had been previously found to be the lowest level to bring about lordosis in all females after two injections in the absence of progesterone. Each female was then tested 3 times between 6:00 AM and noon. Lordosis Quotient, Mean Lordosis Duration and Mean Lordosis Intensity were averaged for the series of three tests of each female. Females then received the same injections of hormones and the same series of behavioral tests one week and two weeks later for a total of three series of tests at weekly intervals. Results. The behavior of the EB only and the EB + progesterone groups is summarized in Table 3 which gives overall means for the three series of tests of each group. As was expected, the well-known synergism between estrogen and progesterone in bringing about lordosis was demonstrated: the group receiving EB + progesterone had a significantly higher L.Q. than the group receiving EB alone (t (14) = 4.38, p < 0.001). Progesterone resulted in an increase in Mean Lordosis Duration (r (14) = 2.59, p < 0.05) as well as in Mean Lordosis Intensity (Mann-Whitney U test, p < 0.05). Progesterone also influenced other behaviors that are characteristic of heat: more females receiving EB + progesterone showed ear-wiggling, hopping, and spontaneous lordosis on at least one test during the three series of tests than did females receiving EB alone.
DISCUSSION As has been noted by other authors (Edwards, Whalen and Nadler, 1967), sexual receptivity is a graded rather than an all-or-none phenomenon, and the assessment of level of receptivity requires the use of a continuous measure. The *most common behavioral index, the proportion of mounts by the male which elicits lordosis by the female (Lordosis Quotient in this study) is one such measure. It is thus a better index of sexual receptivity than is the simple presence or absence of lordosis or the presence of copulatory plugs in the vagina. If each mount elicits lordosis, then females are usually considered to be maximally receptive. Two recent studies have demonstrated the close relationship between amounts of replacement estrogen and levels of display of lordosis when estrogen is used alone (Davidson, Smith, Rodgers, and Bloch, 1968) or in combination with progesterone (Whalen and Hardy, 1970). The
.40 27.lO .87 ?r .04
-
.45 + .02 s3 If: .02
Mean Lordosis Duration (in seconds) e SE 1.01 + .Ol 1.06 + .02
Mean Lordosis Intensity (scale = l-3) *SE 25 88
Percent showing ear-wiggling
25 100
Percent showing hopping
63 88
Percent showing spontaneous lordosis
IFemales received injections of 5 pg EB 42 and 18 hr before the start of testing. 2Females received injections of 5 pg EB 42 and 18 hr before the start of testing and 500 yg progesterone 3 hr before the start of testing.
EB only1 EB + prog.2
Treatment group
Lordosis Quotient *SE
Behavior of Ovariectomized Females Displaying Hormonal-induced Heat. Lordosis Quotients, Mean Lordosis Durations and Mean Lordosis Intensities are Averages of the Mean Scores Obtained During Each of Three Weekly Series of Tests. Other Data are Based on Percent of Females Displaying Ear-wiggling, Hopping or Spontaneous Lordosis at Least Once During the Total Testing Period (n = 8 per group)
TABLE 3
r? r z i? E
_(
z 2 $6-8
296
HARDY AND DeBOLD
present study indicates that female rats also differ in other measures of lordosis in addition to the probability of its display-namely, they differ in the duration and intensity of the lordosis response. Perhaps the most interesting finding is that when sexual receptivity is judged to be maximal by the Lordosis Quotient, it can be submaximal according to Lordosis Duration. This may suggest a ceiling effect with Lordosis Quotient as an index of sexual receptivity. It also suggests that sexual behavior cannot be characterized by single measures of behavior. Judging from the Lordosis Quotient, lordosis intensity, and hopping, the 50 pg EB females are more receptive than are intact females. Judging from duration of lordosis and ear-wiggling, the intact females are more receptive than any other group. It seems that different measures of sexual responsiveness are associated with different dose-response curves. It is interesting, however, that the lordosis-duration measure indicates that intact female rats may be receiving a higher “dose” of endogenous estrogen than are ovariectomized females given “pharmacological” levels of estradiol benzoate. It seems possible that the relative insensitivity of Lordosis Quotient as an index of behavior has contributed to the common misconception that steroid therapy to ovariectomized female rats restores behavior which is indistinguishable from that of natural heat (cf. Powers, 1970). Indeed, hormone therapy restores other behaviors characteristic of natural heat, such as ear-wiggling, hopping and display of spontaneous lordosis (Experiments I and III). The present study confirms the finding of Gerall and Kenney (1970) that these behaviors, as well as display of lordosis, are related to the level of replacement estrogen. In addition, this study reveals that the duration of lordosis displayed during natural heat is greater than duration of lordosis during hormone-induced heat. This same difference has been found by other workers: Kuehn and Beach (1963) reported that the duration of lordosis to mounting by the male rat was 1.3 set in intact females in natural heat; Moss (1971) found that lordosis of hormone-primed ovariectomized females in response to mounting by males ranged from 0.5-1.0 set in length. It seems reasonable to assume that the reason for differences in intensity and duration of lordosis during natural and hormonal-induced heat is that injection of synthetic steroids does not duplicate the pattern of hormonal output by the ovary. In view of the fact that there is little correspondence between level, time course of administration, or type of hormone during “replacement” therapy, and the endocrine events of the estrous cycle, it does not seem surprising that there are behavioral differences between females in hormonalinduced heat and females’ in natural heat. It seems more plausible to relate the dissimilarities in the duration and intensity of lordosis described in this paper to the use of exogenous hormones than to assume that there is something unique about natural, spontaneous sexual receptivity in the female rat. In any event, we suggest that workers using replacement therapy be more cautious about generalizing to the natural state of heat.
HORMONES AND DISPLAY OF LORDOSIS
297
ACKNOWLEDGMENTS We express our thanks to John A. Reinschmidt for his technical assistance and to Lauren K. Gerbrandt and Richard E. Whalen for suggestions with regard to the manuscript. Hormones were generously provided by Dr. Preston Pcrlman, Schering Corporafion, Bloomfield, NJ. REFERENCES Beach, F. A. (1943). Effects of injury to the cerebral cortex upon the display of masculine and feminine mating behavior by female rats. J. Copnp. Psychol. 36, 169-188. Boling, J. L., and Blandau, R. J. (1939). The estrogen-progesterone induction of mating responses in the spayed female rat. Endocrinology 25, 359-364. Davidson, J. M., Smith, E. R., Rodgers, C. H., and Bloch, G. J. (1968). Relative thresholds of behavioral and somatic responses to estrogen. Physiol. Behav. 3, 221-229. Dcwsbury, D. A. (1967). A quantitative description of the behavior of rats during copulation. Behaviour 29, 154-178. Edwards, D. A., Whalen, R. E., and Nadler, R. D. (1967). Induction of estrus: estrogen-progesterone interactions. Physiol. Behav. 3, 29-33. Cerall. A. A., and Kenney, A. McM. (1970). Nconatally androgcnized females’ rcsponsiveness to estrogen and progesterone. Endocrinology 81, 560-566. Hardy, D. F. Sexual behavior in continuously cycling rats. Behaviow, in press. Hardy, D. F. (1969). The estrous cycle of the female rat (Rartcrs norueqicus). Ph.D. Dissertation, University of California, Irvine. Hardy D. F., and DeBold, J. 1~.(1971). Effects of mounts without intromission upon the behavior of female rats during the onset of estrogen-induced heat. Physiol. Behau., in press. Hardy, D. F., and DeBold, J. F. Effects of coital stimulation upon the behavior of the fem,ale rat. J. Comp. Physiol. Psychol., in press. Hart, B. L. (1969). Gonadal hormones and sexual reflexes in the female rat. Horns. Behav. 1, 65-71. Hemmingsen, A. M. (1933). Studies on the ocstrous-producing hormone (oestrin). Skan. Arch, Physiol.
65, 91-250.
Kaufman, R. S. (1953). Effects of preventing intromission upon sexual behavior of rats. J. Comp. Physiol. Psychol. 46, 209-211. Kuehn, R. E., and Beach, 1’. A. (1963). Quantitative measurement of sexual receptivity in female rats. Behaviour 21, 282-299. Moss, R. L. (1971). Modification of copulatory behavior in the female rat following olfactory bulb removal. J. Comp. Physiol. Psychol. 74, 374-382. Powers, J. B. (1970). Hormonal control of sexual receptivity during the estrous cycle of the rat. Physiol. Behav. 5, 831-835. Whalen, R. 13.(1961). Effects of mounting without intromission and intromission without ejaculation upon sexual behavior and maze learning. J. Camp. Physiol. Psycho!. 54. 409415. Whalen, R. E., and Hardy, D. F. (1970). induction of receptivity in female rats and cats with estrogen and testosterone. Physiol. Behav. 5, 529-533. Whalen, R. E., Luttge, W. C., and Gorzalka, B. B. (197 1). Neonatal androgenization and the development of estrogen responsivity in male and female rats. fform. Bchav. 2 83-90.
The Relationship Between Levels of Exogenous Hormones and the Display of Lordosis by the Female Rat DONNA FITZROY HARDY and JOSEPH F. DeBOLD Department of Psychology, San Fernando Valley State College, Northridge, California 91324
In three experiments, the duration, intensity and probability of display of lordosis by ovariectomized female rats were measured in response to administration of progesterone and various levels of eStndiO1 benzoate (ED). Behaviors of females in natural heat were compared with those receiving exogenous hormones. Lordosis duration was shown to be directly proportional to the amount of EB administered, although mean duration of hormone-treated females did not reach that shown by females in natural heat. Both Lordosis Quotient and intensity of lordosis rcachcd asymptote with relatively low doses of EB when animals were treated with progesterone. Ovariectomy reduced the duration and intensity of lordosis even when amounts of EB and progesterone capable of restoring Lordosis Quotients were administered. Finally, the well-known synergistic relationship between estrogen and progesterone in bringing about lordosis in ovariectomized females was found to influence duration and intensity of lordosis as well as the probability of its display. Auxiliary heat behaviors (earwiggling, hopping, and display of “spontaneous” lordosis) were found to be related to level of replacement-EB as well as to the presence of progesterone. These results suggest that hormonally induced heat can be differentiated from natural heat on the basis of duration of lordosis.
Sexual receptivity in the female rat has usually been defined in terms of the display of a characteristic spinal reflex, the lordosis response. Although some early studies (Boling and Blandau, 1939) have equated a single lordosis as indicating sexual receptivity, most recent studies (Beach, 1943; Kuehn and Beach, 1963; Edwards, Whalen and Nadler, 1967) base level of receptivity upon the proportion of mounts by the male rat which elicits lordosis by the female. Various authors express this proportion as Copulatory Quotient, Sensitivity Index, Receptivity Quotient, Lordosis-to-Mount Ratio, L/M or Lordosis Quotient. It is generally assumed that the higher the proportion of mounts which result in lordosis, the higher the level of receptivity. Relatively few systematic attempts have been made to characterize sexual behavior with any index of lordosis other than its display in response to mounting by the male. Kuehn and Beach (1963) and Moss (1971) measured duration of 287
288
HARDYANDDeBOLD
lordosis. Other authors (Hemmingsen, 1933; Hart, 1969; Gerald and Kenney, 1970; Whalen, Luttge and Gorzalka, 1971; Hardy and DeBold, 1971) noted differences in intensity of lordosis, although intensity has not been a common measure of level of receptivity. The purpose of the present study was to investigate quality of lordosis as well as its display to mounting by the male. In addition to behavior of the intact rat during natural heat, behavior was studied in ovariectomized females displaying lordosis while receiving various levels of replacement estrogen, ranging from a “physiological dose” (0.5 ,ug) to a “pharmacological dose” (500 ng). The behavior of individual females during natural heat was compared to their behavior during hormone-induced heat following ovariectomy. An attempt was made to classify the intensity of each lordosis response, and the duration of each lordosis was measured. The study differs from previous studies in that differences in behavior of the female which are due to intromission and ejaculation by the male (Kuehn and Beach, 1963; Dewsbury, 1967; Moss, 1971; Hardy and DeBold, in press) were prevented by a technique which eliminates intromission (Hardy, 1969).
MATEFUALS AND METHODS Female Sprague-Dawley rats obtained from Simonsen Laboratories (Gilroy, CA) were housed in pairs with food and water present at all times. A reversed light-dark schedule was employed with fluorescent ceiling lights on from 6:00 PM to 6:00 AM. During behavioral tests, all females received 10 mounts by sexually experienced male rats in glass observation jars (12 in. across and 18 in. high). Vaginal masks made of pliable masking tape (1 in. wide and 2-3 in. long) were affixed to the fur of the ventral surface of all females. When secured to the base of the tail, masks covered the perineal region and occluded the vaginal orifice. It had been previously demonstrated that these masks prevent intromission by the male without interfering with the normal display of estrus behavior by the female (Hardy, 1969). Although there is little reason to believe that the masks interfered with the actual mounting by the males (cf. Kaufman, 1953), males were frequently “primed” with unmasked stimulus females to prevent the waning of sexual behavior observed by Whalen (1961) in males that were prevented from achieving intromission. During tests, presence or absence of three behaviors characteristic of estrus in this species were noted: ear-wiggling, hopping, and lordosis to mounting by the male. In addition, the occurrence of “spontaneous lordosis” was noted in Experiment III; this behavior was defined as lordosis in response to sniffing, grasping with the forepaws or any tactile stimulus from the male rat other than mounts with thrusting.
HORMONESAND DISPLAY OF LORDOSIS
289
The index of sexual responsiveness was the Lordosis Quotient: total number of lordosis responses/test __-L.Q. = --total number of mounts with thrusting by the male Sexual responsiveness was further characterized by two measures of lordosis quality-duration and intensity. Total duration of all lordosis responses for each test was timed by independent observers using cumulative stopwatches. Mean duration (in seconds) of lordosis was then recorded for each test: total duration of lordosis responses/test _--.-_---__ Mean Lordosis Duration = -total number of lordosis responses/test The intensity scale:
of each lordosis was rated by these observers using a 3-point
1 point
= marginal lordosis (slight flex of spine, slightly raised head and hips with tailbase elevated from the floor). 2 points = normal lordosis (spinal flex, head at an approximate angle of 30” with the horizontal, front paws placed slightly forward and hind legs straightened up stiffly). 3 points = exaggerated lordosis (pronounced spinal flex, head at an angle of 45” or more with the horizontal). These postures are illustrated in another paper (Hardy and DeBold, 1971). Total points were accumulated for each test and the mean calculated: total number of lordosis points/test _-.-.-___ Mean Lordosis Intensity = ----total number of Iordosis responses/test EXPERIMENT I Relationship Between Level of Estradiol Benzoate and the Display of Lordosis Procedure. In this experiment, 60 female rats were used. Twelve females were intact and displaying regular 4-day estrous cycles (as confirmed by vaginal smearing), while the remaining forty-eight had been ovariectomized 3 months prior to the experiment. One hour before testing of intact females, heat was established by their behavioral response to mounting by the males: females were masked and allowed to receive several mounts by the males. It had been previously found that normally cycling female rats display lordosis to most mounts during the onset of heat (Hardy, in press). Intact females that displayed lordosis to each mount by the male were considered to be in heat and were tested later in the morning. The ovariectomized female rats were divided into four equal groups which received 2 subcutaneous doses of estradiol benzoate (EB) in oil 24 hr apart; each dose was 0.5, 5, 50, or
290
HARDY AND DeBOLD
500 pg. EB was administered in two series; the first series was one week prior to testing. The second two injections of EB were given 44 and 20 hr before testing. Five hours before testing, 500 pg progesterone in oil was administered subcutaneously. This injection regimen roughly approximates that used in other laboratories, and the 4 doses of EB encompass the range of exogenous estrogen administered by other workers; Tests were conducted between 8:OO AM and noon. Results. Data from Experiment I are summarized in Table 1. There was an overall difference between Lordosis Quotients of the five groups (F(4,55) = 7.79, p < 0.001) with the females receiving 0.5 yg EB showing the lowest mean L.Q. and the groups receiving 5, 50, and 5OOpg EB the highest mean L.Q.‘s. Lordosis Quotients of intact females were significantly higher than the 0.5 pg EB group (t (12.2)1 = 2.37, p < 0.05) but lower than that of the 50 pg EB group (t (11) 1 = 2.80, p < 0.02). In addition, there was a clear relationship between level of replacement hormone and Mean Lordosis Duration (F(4,55) = 20.28, p < 0.001) (Fig. 1). The Mean Lordosis Duration of females in natural heat was significantly longer than the duration of lordosis of females receiving the highest level of replacement hormone, 500 pg EB (t (22) = 2.90, p < 0.01). A test for linearity showed a significant linear component for the duration of lordosis (F(1,55) = 80.75, p < 0.001). There were overall differences between Mean Lordosis Intensity of intact females and ovariectomized females receiving replacement hormones (Kruskal-Wallis, H = 14.74, p < O.Ol), and Mean Lordosis Intensity of intact females was significantly higher than that of ovariectomized females receiving 0.5 pg EB (Mann-Whitney U test, p < 0.01). Finally, there were general increases in percentage of females displaying ear-wiggling and hopping behaviors with increases in level of EB replacement. More females receiving the higher doses of EB (50 and 5OOpg) displayed hopping than did intact females in natural heat.
EXPERIMENT II Quality of Lordosis Before and After Ovariectomy Procedure. This experiment compared the behavior of females displaying natural heat with their behavior following ovariectomy during hormonally induced heat. Twenty intact females were tested during the morning of their natural heat (between 8:00 AM and noon with a reversed light cycle). Lordosis Quotient, Mean Lordosis Duration, Mean Lordosis Intensity and display of ear-wiggling and hopping were recorded for each female. Females were ovariectomized immediately following the test of the twentieth female, IDegrees of freedom compensated for unequal variance.
.90-1.00 .70-I .oo
1.00
70-1.00
O-1-00
+.03 -+o k.01 +.03
.61+ .13
.98 1.00 .99 .92
range
mean k SE
Lordosis Quotient
2~.08 + .02 2 .06 +-.04
1.11 * .06
.45 59 .75 .90
mean -t SE O-.72 .50-.78 54-1.25 .67-1.15 .75-1.55
range
-
Mean
1.44 c .lO
1.25 k.09 1.56 f .09 1.30 + .08
.79 k.14
mean k SE
1.00-2.11
1.10-2.00 1.00-1.90
1.00-1.90
O-1.20
range
Lordosis Intensity (scale = l-3)
100
92 83 92
42
Percent showing eare&w
1FemaIesreceived estradiol benzoate (EB) 44 and 20 hr before testing and 500 fig progesterone 5 hr before testing.
50 kg EB 500 pg EB Intact
0.5 j.ig EB 5%EB
Treatment group
Mean Lordosis Duration (in seconds)
Behavior of Ovariectomized Female Rats Displaying Hormonal-induced HeatI and Intact Females Displaying Natural Heat (SE = standard error, )?-- 12 per group)
TABLE 1
58
50 83 83
17
Percent showing hopping
5; 7a
%
5
; 3 tY 3
292
HARDY AND DeBOLD
.-u) m g.40;; -I c.20L 5 -
0.5
5
50
500
intact
Fig. 1. The relationship between duration of lordosis and level of estradiol benzoate (EB) in pg. The four groups of females receiving EB had been ovariectomized and were administered 500 ~g progesterone before testing. The fifth group of females were intact and displaying natural heat. The vertical lines indicate standard error (n = 12 in each group).
then the vaginas of all females were smeared with a fine wire loop for 10 days. These smears confirmed the diestrus vaginal condition and the absence of ovarian tissue. On the 10th day following ovariectomy, hormone administration was begun: females were administered I gg EB 44 and 20 hr before the start of testing and 5OOpg progesterone 5 hr before testing. Previous experiments in our laboratory had established that two injections of 1 grg EB followed by 500 @g progesterone result in Lordosis Quotients which closely approximate those of females in natura1 heat. Results. The behavior of females during natural and hormone-induced heat is summarized in Table 2. There were no significant differences between Lordosis Quotients of females before and after ovariectomy, and hormone treatment (paired t (19) = 0.66, NS), but Lordosis Duration was significantly longer when females were displaying natural heat (paired t (19) = 9.60, p < 0.001). Furthermore, Mean Lordosis Intensity was greater during natural heat than during hormonal-induced heat (sign test, p < 0.001). There were no differences between the two conditions with respect to percent of females showing ear-wiggling or hopping.
.86 It.06
.95 + .02
mean + SE
.55 k .02
1.03 f .04
.70-1.00
.10-l .oo
mean + SE
range
Lordosis Quotient
.30-.75
.70-1.55
range
Mean
1.20 t .06
1.43 k.07
mean k SE
80
1.00-l .80
50
70
100 1.00-2.11
range
Percent showing hopping Percent showing ear-wiggling
Lordosis Intensity (scale = 1-3)
IFemales received 1 pg EB 44 and 20 hr before testing and 500 pg progesterone 5 hr before testing.
induced beat
Intact, natural heat Ovariectomized, EB + progesterone
Condition of females
Mean Lordosis Duration (in seconds)
Behavior of Female Rats During Natural Heat and Following Ovariectomy During Hormonal-induced Heat,1 (n = 20)
TABLE 2
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294
HARDY
AND DeBOLD
EXPERIMENT III Relationships between Progesterone and Behavior of Estrogen-treated Fernale Rats Procedure. Sixteen female rats that had been ovariectomized for about 2 months were administered 2 subcutaneous doses of 5 pg EB 24 hr apart one week prior to testing, then 5 pg EB 42 and 18 hr before the start of testing. Half of them received 500 pg progesterone 3 hr before testing. This dose of EB had been previously found to be the lowest level to bring about lordosis in all females after two injections in the absence of progesterone. Each female was then tested 3 times between 6:00 AM and noon. Lordosis Quotient, Mean Lordosis Duration and Mean Lordosis Intensity were averaged for the series of three tests of each female. Females then received the same injections of hormones and the same series of behavioral tests one week and two weeks later for a total of three series of tests at weekly intervals. Results. The behavior of the EB only and the EB + progesterone groups is summarized in Table 3 which gives overall means for the three series of tests of each group. As was expected, the well-known synergism between estrogen and progesterone in bringing about lordosis was demonstrated: the group receiving EB + progesterone had a significantly higher L.Q. than the group receiving EB alone (t (14) = 4.38, p < 0.001). Progesterone resulted in an increase in Mean Lordosis Duration (r (14) = 2.59, p < 0.05) as well as in Mean Lordosis Intensity (Mann-Whitney U test, p < 0.05). Progesterone also influenced other behaviors that are characteristic of heat: more females receiving EB + progesterone showed ear-wiggling, hopping, and spontaneous lordosis on at least one test during the three series of tests than did females receiving EB alone.
DISCUSSION As has been noted by other authors (Edwards, Whalen and Nadler, 1967), sexual receptivity is a graded rather than an all-or-none phenomenon, and the assessment of level of receptivity requires the use of a continuous measure. The *most common behavioral index, the proportion of mounts by the male which elicits lordosis by the female (Lordosis Quotient in this study) is one such measure. It is thus a better index of sexual receptivity than is the simple presence or absence of lordosis or the presence of copulatory plugs in the vagina. If each mount elicits lordosis, then females are usually considered to be maximally receptive. Two recent studies have demonstrated the close relationship between amounts of replacement estrogen and levels of display of lordosis when estrogen is used alone (Davidson, Smith, Rodgers, and Bloch, 1968) or in combination with progesterone (Whalen and Hardy, 1970). The
.40 27.lO .87 ?r .04
-
.45 + .02 s3 If: .02
Mean Lordosis Duration (in seconds) e SE 1.01 + .Ol 1.06 + .02
Mean Lordosis Intensity (scale = l-3) *SE 25 88
Percent showing ear-wiggling
25 100
Percent showing hopping
63 88
Percent showing spontaneous lordosis
IFemales received injections of 5 pg EB 42 and 18 hr before the start of testing. 2Females received injections of 5 pg EB 42 and 18 hr before the start of testing and 500 yg progesterone 3 hr before the start of testing.
EB only1 EB + prog.2
Treatment group
Lordosis Quotient *SE
Behavior of Ovariectomized Females Displaying Hormonal-induced Heat. Lordosis Quotients, Mean Lordosis Durations and Mean Lordosis Intensities are Averages of the Mean Scores Obtained During Each of Three Weekly Series of Tests. Other Data are Based on Percent of Females Displaying Ear-wiggling, Hopping or Spontaneous Lordosis at Least Once During the Total Testing Period (n = 8 per group)
TABLE 3
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_(
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296
HARDY AND DeBOLD
present study indicates that female rats also differ in other measures of lordosis in addition to the probability of its display-namely, they differ in the duration and intensity of the lordosis response. Perhaps the most interesting finding is that when sexual receptivity is judged to be maximal by the Lordosis Quotient, it can be submaximal according to Lordosis Duration. This may suggest a ceiling effect with Lordosis Quotient as an index of sexual receptivity. It also suggests that sexual behavior cannot be characterized by single measures of behavior. Judging from the Lordosis Quotient, lordosis intensity, and hopping, the 50 pg EB females are more receptive than are intact females. Judging from duration of lordosis and ear-wiggling, the intact females are more receptive than any other group. It seems that different measures of sexual responsiveness are associated with different dose-response curves. It is interesting, however, that the lordosis-duration measure indicates that intact female rats may be receiving a higher “dose” of endogenous estrogen than are ovariectomized females given “pharmacological” levels of estradiol benzoate. It seems possible that the relative insensitivity of Lordosis Quotient as an index of behavior has contributed to the common misconception that steroid therapy to ovariectomized female rats restores behavior which is indistinguishable from that of natural heat (cf. Powers, 1970). Indeed, hormone therapy restores other behaviors characteristic of natural heat, such as ear-wiggling, hopping and display of spontaneous lordosis (Experiments I and III). The present study confirms the finding of Gerall and Kenney (1970) that these behaviors, as well as display of lordosis, are related to the level of replacement estrogen. In addition, this study reveals that the duration of lordosis displayed during natural heat is greater than duration of lordosis during hormone-induced heat. This same difference has been found by other workers: Kuehn and Beach (1963) reported that the duration of lordosis to mounting by the male rat was 1.3 set in intact females in natural heat; Moss (1971) found that lordosis of hormone-primed ovariectomized females in response to mounting by males ranged from 0.5-1.0 set in length. It seems reasonable to assume that the reason for differences in intensity and duration of lordosis during natural and hormonal-induced heat is that injection of synthetic steroids does not duplicate the pattern of hormonal output by the ovary. In view of the fact that there is little correspondence between level, time course of administration, or type of hormone during “replacement” therapy, and the endocrine events of the estrous cycle, it does not seem surprising that there are behavioral differences between females in hormonalinduced heat and females’ in natural heat. It seems more plausible to relate the dissimilarities in the duration and intensity of lordosis described in this paper to the use of exogenous hormones than to assume that there is something unique about natural, spontaneous sexual receptivity in the female rat. In any event, we suggest that workers using replacement therapy be more cautious about generalizing to the natural state of heat.
HORMONES AND DISPLAY OF LORDOSIS
297
ACKNOWLEDGMENTS We express our thanks to John A. Reinschmidt for his technical assistance and to Lauren K. Gerbrandt and Richard E. Whalen for suggestions with regard to the manuscript. Hormones were generously provided by Dr. Preston Pcrlman, Schering Corporafion, Bloomfield, NJ. REFERENCES Beach, F. A. (1943). Effects of injury to the cerebral cortex upon the display of masculine and feminine mating behavior by female rats. J. Copnp. Psychol. 36, 169-188. Boling, J. L., and Blandau, R. J. (1939). The estrogen-progesterone induction of mating responses in the spayed female rat. Endocrinology 25, 359-364. Davidson, J. M., Smith, E. R., Rodgers, C. H., and Bloch, G. J. (1968). Relative thresholds of behavioral and somatic responses to estrogen. Physiol. Behav. 3, 221-229. Dcwsbury, D. A. (1967). A quantitative description of the behavior of rats during copulation. Behaviour 29, 154-178. Edwards, D. A., Whalen, R. E., and Nadler, R. D. (1967). Induction of estrus: estrogen-progesterone interactions. Physiol. Behav. 3, 29-33. Cerall. A. A., and Kenney, A. McM. (1970). Nconatally androgcnized females’ rcsponsiveness to estrogen and progesterone. Endocrinology 81, 560-566. Hardy, D. F. Sexual behavior in continuously cycling rats. Behaviow, in press. Hardy, D. F. (1969). The estrous cycle of the female rat (Rartcrs norueqicus). Ph.D. Dissertation, University of California, Irvine. Hardy D. F., and DeBold, J. 1~.(1971). Effects of mounts without intromission upon the behavior of female rats during the onset of estrogen-induced heat. Physiol. Behau., in press. Hardy, D. F., and DeBold, J. F. Effects of coital stimulation upon the behavior of the fem,ale rat. J. Comp. Physiol. Psychol., in press. Hart, B. L. (1969). Gonadal hormones and sexual reflexes in the female rat. Horns. Behav. 1, 65-71. Hemmingsen, A. M. (1933). Studies on the ocstrous-producing hormone (oestrin). Skan. Arch, Physiol.
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Kaufman, R. S. (1953). Effects of preventing intromission upon sexual behavior of rats. J. Comp. Physiol. Psychol. 46, 209-211. Kuehn, R. E., and Beach, 1’. A. (1963). Quantitative measurement of sexual receptivity in female rats. Behaviour 21, 282-299. Moss, R. L. (1971). Modification of copulatory behavior in the female rat following olfactory bulb removal. J. Comp. Physiol. Psychol. 74, 374-382. Powers, J. B. (1970). Hormonal control of sexual receptivity during the estrous cycle of the rat. Physiol. Behav. 5, 831-835. Whalen, R. 13.(1961). Effects of mounting without intromission and intromission without ejaculation upon sexual behavior and maze learning. J. Camp. Physiol. Psycho!. 54. 409415. Whalen, R. E., and Hardy, D. F. (1970). induction of receptivity in female rats and cats with estrogen and testosterone. Physiol. Behav. 5, 529-533. Whalen, R. E., Luttge, W. C., and Gorzalka, B. B. (197 1). Neonatal androgenization and the development of estrogen responsivity in male and female rats. fform. Bchav. 2 83-90.