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Vasotocin, prostaglandin, and female reproductive behavior in the frog, Rana pipiens
HORMONES
AND
BEHAVIOR
Vasotocin,
15, 86-93 (1981)
Prostaglandin, and Female Reproductive Behavior in the Frog, Rana pipiens CAROL DIAKOW
Biology
Department.
Adelphi
AND AUDREY University.
NEMIROFF
Garden City. Newa York 11530
Receptive behavior in the female frog, Rana pipiens, as indicated by inhibition of the release call, was potentiated by administration of 5 &gbw (ip) prostaglandin EL or prostaglandin F2-. Arginine-8 vasotocin (1 &g body wt) also inhibited the release call, but its effect was delayed by a single injection of 10@g/gbody wt of the prostaglandin synthetase inhibitor, indomethacin. These results suggest that both vasotocin and prostaglandin may be involved in the manifestation of female mating behavior in R. pipiens and that vasotocin may act, at least in part, through a mechanism that involves prostaglandin synthesis.
An unreceptive female Rana pipiens emits a release call when clasped by a sexually active male. Contractions of the trunk muscles produce this call and stimulate the male to release the female. In contrast, a receptive female is silent, so the male can maintain his clasp, and oviposition and fertilization can occur (Noble and Aronson, 1942). The release call, which has been studied extensively by Schmidt (196.5, 1966, 1972, 1973, 1974), has been inhibited by administration of arginine-8 vasotocin (AVT) and it has been suggested that this hormone may play a role in potentiating reproductive behavior in this species (Diakow, 1978). The neurohypophysial polypeptide, arginine-8 vasotocin is the antidiuretie hormone in R. pipicrzs (Munsick, 1966; Acher, Chauvet, and Chauvet, 1969), and one mechanism whereby AVT might inhibit the call could involve water accumulation and/or consequent pressure within the body (Diakow, 1978; Diakow and Raimondi, 1980). The release call has been inhibited without AVT administration by fluid retention caused by cloaca1 ligature (Diakow, 1977). The relationship between AVT function and gonadal activity is unknown. Although inhibition of the release call normally occurs around the time of ovulation (personal observation), AVT can inhibit the call in the absence of the ovaries (Diakow, Wilcox, and Woltmann, 1978) and even in male frogs (Raimondi and Diakow, 1981). The gonadal steroids, estrogen and progesterone, do not inhibit the call (Diakow, et al., 1978). Indeed, these hormones seem to retard AVT’s action in release call inhibition (Diakow and Raimondi, 1980). 86 00 18-506X/8l/010086-08$01.00/0 Copyright All rights
Q 1981 by Academic Press. Inc of reproduction in any form reserved.
NONSTEROIDS
AND FEMALE
SEX BEHAVIOR
87
Besides its possible role in potentiating reproductive behavior in the female frog, AVT seems to play a role in reproductive behaviors of other nonmammalian vertebrates. These behaviors include spawning of teleosts (Pickford and Strecker, 1977, review by Crews and Silver, 1981), oviposition in the domestic hen (Rzasa, 1978) and clasping by male newts (Moore and Zoeller, 1979). < The fatty acids characterized as prostaglandins (PG) are another group of nonsteroid hormones that seem to be important for reproductive behavior. PGE,, PGI$, and PGF,, induce oviposition in the domestic hen (Hertelendy, Yeh, and Biellier, 1974), and though several prostaglandins also induce oviposition in quail, PGE, is the most effective (Hertelendy, 1974). The observation that indomethacin, a prostaglandin synthetase inhibitor, delays oviposition in the hen (Day and Nalbandov, 1977) supports the hypothesis that PG is involved in oviposition in birds. Similarly, there is evidence that PGF,, and PGE, induce oviposition behavior in goldfish (Stacey, 1976, 1980; Stacey and Peter, 1979). In mammals, PGF,, potentiates the mating postures of female rats (Hall, Luttge, and Berry, 1975; Dudley and Moss, 1976; Rodriguez-Sierra and Komisaruk, 1977)and hamsters (Buntin and Lisk, 1979). In the frog, PGF,, induces a leg adduction posture in females that enables X~nopus faevis males to maintain their clasps on them (Kelley, personal communication). The experiments reported in this paper investigate the relationship between AVT function and prostaglandin action in reproductive behavior of the leopard frog. Experiment 1 tests if the prostaglandins Q and F,, inhibit the release call, and Experiment 2 tests whether AVT might act through a mechanism utilizing prostaglandin in inhibiting the release call. EXPERIMENT
1
Design
Three groups of 10 ovariectomized R. pipiens each were tested for the release call by manual stimulation, injected, and retested at 30 min, then hourly, for the first 5 hr after injection. One group was injected with prostaglandin & (PGIQ; the second, with prostaglandin Fzu (PGF,,); and the third, with deionized water (DI). Methods Subjects and housing. All subjects were female, northern leopard frogs, R. pipiens. Median and range snout-vent lengths were 86 mm (82-94
mm). The frogs were collected in Vermont in the autumn and stored in hibernation until they were shipped to the laboratory in March. In the laboratory, they were housed in groups in large tanks containing a dilute solution of Agristrep (streptomycin sulfate) and floating Styrofoam pads upon which they could sit to emerge from the water. During the experi-
88
DIAKOW
AND NEMIROFF
ment, they were housed individually in plastic containers (30 x 17 x 21 cm). The Styrofoam pad was removed during the testing period. Laboratory temperature was 20-24°C and the animals were housed under constant fluorescent overhead lighting. Ovariectomies. The subjects were ovariectomized in order to prevent any ovarian response to PG from confounding the results. Surgery was performed under ether anesthesia through ventral, parasaggital incisions 3-7 days after arrival in the laboratory. Median and range ovarian volumes were 12 ml (8-15 ml). Tests and injections. Tests began 21-25 days after ovariectomy. For testing, the frogs were held between the thumb and forefinger of the experimenter on either side of the vertebral column posterior to the pectoral girdle. The number of release calls elicited in the first 30 set of clasping was recorded, and then the frog was weighed to the nearest gram on a Mettler P1200 balance. On the first day of testing, the females were tested twice; on the second day, they were tested twice, injected, then tested 30 mitt, and 1,2,3,4, and 5 hr after injection. Prostaglandin (5 pg/g body wt) (Upjohn) was administered ip. Injections were prepared from solutions of 10 mg prostaglandin in 20 cc deionized water. These solutions were prepared on the day before injection and stored at 5°C. Results
There was a significant decline in the number of release calls after injection of both prostaglandins & and F,,, but not after injection with deionized water (Table 1). The median number of release calls was 0 by 30 min after injection of prostaglandin; recovery of the release call had begun 4 hr after injection. There was no significant change in the weight of any group after injection. TABLE 1 Effect of F’rostaglandin on the Release Call Group”
Before injectioti
After injection
Release callsc
PGE, FGF,” DI
32 (14-43)d 31 (12-42) 31 (10-45)
0 (0-16fe 0 (O-4y 3.5 (22-47)
Weight (g)
p-k PGF*a DI
41 (35-48) 40 (37-44) 39 (35-50)
39 (36-49) 40 (37-48) 39 (33-54)
a N = 10 for each group. b Four preinjection tests; six postinjection tests. c Number of release calls in 30 set of manual clasping around trunk. d Median and range of the medians for each female. e Significant difference between the pre- and postinjection tests using the Wilcoxon matched-pairs signed-ranks test; p 5 0.05, two tail.
NONSTEROIDS
AND FEMALE
EXPERIMENT
SEX BEHAVIOR
89
2
Design
Three groups of females were tested for the release call by manual stimulation, injected, and retested hourly l-9 and 22-26 hr after injection. One group was injected with AVT (AVT-only, N = 8); the second, with AVT and indomethacin, a prostaglandin synthesis inhibitor (AVT-Indo, N = 9); and a third, with indomethacin alone Undo-only, N = 8). Methods
Twenty-one subjects were Northern leopard frogs, R. @piens, Callected in Vermont in the autumn and stored in hibernation until they were shipped to the laboratory in March. They were introduced into the experiment 3 1 days after arrival in the laboratory. Four subjects had been collected and shipped to the laboratory in the autumn and stored under standard laboratory conditions (Diakow, 1977). All subjects were large, mature females with well-developed ovaries. Subjects were tested twice, on the next day tested twice again, then injected and retested hourly l-9 and 22-26 hr after injection. All subjects croaked more than 10 times during three of the four preinjection tests. All injections were intraperitoneal. The AVT (Calbiochem) dose was 1.0 pg/g body wt from a solution containing 1 mg AVT in 9.5 ml deionized water. The indomethacin (Sigma) dose was 10.0 pg/g body wt. Indomethacin injections were drawn from a solution containing 0.05 g indomethacin, 0.1 g sodium carbonate, and enough deionized water to make up 50 cc. The solution was agitated intermittently for at least an hour before injection. At the time of injection, the median and range body weights were 58 g (43-72 g) for the AVT only group, 56 g (45-59 g) for the AVT-Indo group, and 46g (41-65 g) for the Indo-only group. Results
Figure 1 shows the median number of release calls for each test after injection for the three groups. Inspection suggests a decline in the number of release calls after AVT injection in both the AVT-only and AVT-Indo groups. Statistical comparison of the preinjection and postinjection medians for each subject confirms that the release call was lower after injection in both groups that received AVT but not in the group receiving indomethacin only (Wilcoxin test). Table 2 presents the median and range of the medians for each female. The decline in the release calls occurred faster in the AVT-only group than in the AVT-Indo group. The best fit lines for the decline in release calls over the first 3 hr after injection for the two groups are shown in Fig. 2. The slopes (m) are significantly different (t = 19.06, p < .OOl; two tail). Figure 3 shows that water accumulation occurred in both groups receiv-
90
DIAKOW AND NEMIROFF l -*
AVT
o----o
only
”
8
AVT+ lndo n 9
t.....*
lndo ”
only 0
?
HOURS
AFTER
INJECTION
FIG. 1. Median number of release calls elicited in 30 set of manual stimulation of the trunk after injection of 1&g body wt vasotocin (AVT-only), 1pg/g body wt vasotocin and 10 &g body wt indomethacin (AVT + Indo), and IO pg/g body wt indomethacin alone (Indo-only).
ing AVT, but water did not accumulate in the Indo-only group. There was no difference in the pattern of water accumulation between the AVT-only and AVT-Indo groups (Fig. 3). DISCUSSION
The results of Experiment 1 suggest that prostaglandins may be involved in inhibition of the release call in female leopard frogs. Because indomethacin retarded the ability of AVT to inhibit the call, AVT’s effect on female receptivity may be mediated, in part, through prostaglandins. TABLE 2 Effect of AVT on the Release Call Group AVT only (N = 8) AVT + Indo (N = 9) Indo Only (N = 8)
Before injectiona
After injection
28b.c (7-40) 24 (15-43) 17 (6-33)
lad (l-36) 216 (l-34) 18 (14-34)
a 4 Preinjection tests; 13 postinjection tests. b Number of release calls in 30 set of manual clasping around the trunk. c Median and range of the medians for each female. d Significant difference between the pre- and postinjection tests using the Wilcoxon matched-pairs signed-ranks test; p 5 0.05; two tail.
91
NONSTEROIDS AND FEMALE SEX BEHAVIOR
-*AVT
only
2
I HOURS
3
AFTER
INJECTION
FIG. 2. The rate of decline of t1le release call after 1pg/g body wt vasotocin injection is slower when 10pg/g body wt indomethacin is also administered. See Fig. 1for description of groups.
Rzgsa (1978) has reported a similar interaction between AVT and prostaglandin in oviposition and oviduct contractility of the domestic hen. Both hormones enhanced oviposition; and indomethacin prevented the stimulation of oviduct contractions by AVT. Rzgsa proposed that AVT exerted its effect on oviposition by acting through PG to cause oviduct contractions. It has been suggested that the silence of the receptive female is due to an increase in intraabdominal pressure or distension of the body wall
o---o l
-•
t..-..* ‘t
. .l
l
l
l ‘,,.
* ,,,_ l
AVT + lndo AVT only lndo
only
_,__
.f..
l -” I
2
3
4 HOURS
5
6
7
AFTER
8
9
“h
22 23 21 25 26
INJECTION
FIG. 3. Median weight after injection expressed as a percentage of body weight at the time of injection. See Fig. I for description of groups.
92
DIAKOW
AND NEMIROFF
resulting from ovulation of the egg mass into the uterus (Noble and Aronson, 1942) or from water accumulation due to AVT action (Diakow, 1978; Diakow and Raimondi, 1980). There is, indeed, much evidence that an increase in girth which causes intraabdominal pressure and distension of the body wall will also inhibit the release call; effective pressure and distension has been provided artificially by water accumulation after cloaca1 ligature (Diakow, 1977, 1978; Diakow and Raimondi, 1980) and inflation of an intraabdominal balloon (Diakow, 1978). The present studies add a new dimension for understanding the silence of the receptive female because it is the first to disassociate inhibition of the call from an increase in girth. In Experiment 1, the decline in the release call after prostaglandin administration occurred without swelling of the body due to water accumulation; and in Experiment 2, indomethatin retarded the inhibition of the release call, but not swelling accompanying water accumulation, after AVT administration. We are presented, then, with the necessity of evaluating many possible physiological mechanisms for inhibition of the release call in R. pipiens. In order to test Noble and Aronson’s hypothesis, it is necessary (1) to find an increase in intraabdominal pressure associated with ovulation; (2) to quantify this pressure increase if it exists; and (3) to show that this amount of intraabdominal pressure will, indeed, inhibit the release call. If their hypothesis is correct, it can only account for initiation of the inhibition of the release call, for Diakow & Raimondi have already presented evidence that if the egg mass is removed from the uterus of receptive females, they remain silent. In order to provide evidence for Diakow’s suggestion that AVT might inhibit the release call it is necessary to show elevated AVT levels associated with receptivity, increased responsivity to AVT at the time of breeding, or at least, increased water accumulation associated with receptivity. The present experiments have expanded the possibilities to be evaluated by making it necessary to measure prostaglandin levels associated with receptivity. ACKNOWLEDGMENTS We gratefully acknowledge encouragement for initiating Experiment 1 from David Crews, Darcy Kelley, and Norman Stacey, extensive consultation with J. Wayne Lazar and Louise Leotta, and the gift of prostaglandin from John E. Pike of the Upjohn Company.
REFERENCES Acher, R., Chauvet, J., and Chauvet, M. T. (1969). Evolution of neurohypophysial hormone with reference to amphibian. Nature (London.) 221, 759-760. Buntin, J., and Lisk, R. D. (1981). Prostaglandin &-induced lordosis in estrogen-primed female hamsters: Relationship to progesterone action. Physiol. Behav. 23, 569-575. Crews, D., and Silver, R. (1981). Reproductive physiology and behavior interactions in nonmammahan vertebrates. In R. W. Goy and D. W. Pfaff (Eds.), Handbook of Behavioral Neurobiology. Plenum, New York, in press.
NONSTEROIDS AND FEMALE SEX BEHAVIOR
93
Day, S. L., and Nalbandov, A. V. (1977). Presence of prostaglandin F (PGF) in hen follicles and its physiological role in ovulation and oviposition. Biol. Reprod. 16, 486-494. Diakow, C. (1977). Initiation and inhibition of the release croak of Rana pipiens. Physiol. Behav.
19, 607-610.
Diakow, C. (1978). A hormonal basis for breeding behavior in female frogs: Vasotocin inhibits the release call of Rana pipiens. Science 199, 145661457. Diakow, C. and Raimondi, D. (1981). Physiology of Rana pipiens reproductive behavior: A proposed mechanism for inhibition of the release call. Amer. Zoo/., 21(l) in press. Diakow, C., Wilcox, J. N., and Woltmann, R. (1978). Female frog reproductive behavior elicited in the absence of the ovaries. Horm. Behut,. 11, 183-189. Dudley, C. A., and Moss, R. L. (1976). Prostaglandin I$: facilitatory action on the lordotic female rats. Prostaglandins 10, 877-888. Hall, N. R., Luttge, W. G., and Berry, R. B. (1975). Intracerebral prostaglandin I$: Effect upon sexual behavior, open field activity, and body temperature in ovariectomized 10, 877-888. female rats. Prostaglandins Hertelendy, F. (1974). Effects of prostaglandins, cyclic AMP, seminal plasma, indomethacin and other factors on oviposition in the Japanese quail, Cotrtrnix corrrrnix Japonicu. J. Reprod.
Fert. 40, 87-93.
Hertelendy, F., Yeh, M., and Biellier, H. V. (1974). Induction of oviposition in the domestic hen by prostaglandins. Gen. Camp. Endocrinol. 22, 529-531. Moore, F. L., and Zoeller, R. T. (1979). Endocrine control of amphibian sexual behavior: evidence for a neurohormone-androgen interaction. Harm. Behav. 13, 207-213. Munsick, R. A. (1966). Chromatographic and pharmacological characterization of the neurohypophysial hormones of an amphibian and a reptile. Endocrinology 78,591-599. Noble, G. K., and Aronson, L. R. (1942). The sexual behavior of Anura I. The normal mating pattern of Rana pipiens. Bull. Amer. Mus. Natur. Hisr. 80, 127-142. Pickford, G. E., and Strecker, E. L. (1977). The spawning reflex response of the killifish, Fundulus hereroclifus: Isotocin is relatively inactive in comparison with arginine vasotocin. Gen. Comp. Endocrinol. 32, 132-137. Raimondi, D., and Diakow, C. (1981). Sex dimorphism in responsiveness to hormonal induction of female behavior in frogs. In press. Rodriguez-Sierra, J. F., and Komisaruk, B. R. (1977). Effects of prostaglandin F$ and indomethacin on sexual behavior in the female rat. Horm. Behav. 9, 281-289. Rz?sa, J. (1978). Effects of arginine vasotocin and prostaglandin E, on the hen uterus, Prostaglandins
16, 357-372.
Schmidt, R. S. (1965). Larynx control and call production in frogs. Copeia 1965, 143-147. Schmidt, R. S. (1966). Central mechanisms of frog calling. Behaviour 26, 251-285. Schmidt, R. S. (1972). Action of intrinsic laryngeal muscles during release calling in leopard frog. .I. Exp. Zool. 181, 233-243. Schmidt, R. S. (1973). Central mechanisms of frog calling. Amer. Zool. 13, 1169-l 177. Schmidt, R. S. (1974). Neural correlates of frog calling. Trigeminal tegmentum. J. Camp. Physiol.
92, 229-254.
Stacey, N. E. (1976). Effects of indomethacin and prostaglandins on the spawning behavior of female goldfish. Prosraglandins 12, 113-120. Stacey, N. E. (1981). Hormonal regulation of female reproductive behavior in fish. Amer. Zoo/., 21(l) in press. Stacey, N. E., and Peter, R. E. (1979). Central action of prostaglandins in spawning behavior of female goldfish. Physiol. Behav. 22, 1191-I 196.
AND
BEHAVIOR
Vasotocin,
15, 86-93 (1981)
Prostaglandin, and Female Reproductive Behavior in the Frog, Rana pipiens CAROL DIAKOW
Biology
Department.
Adelphi
AND AUDREY University.
NEMIROFF
Garden City. Newa York 11530
Receptive behavior in the female frog, Rana pipiens, as indicated by inhibition of the release call, was potentiated by administration of 5 &gbw (ip) prostaglandin EL or prostaglandin F2-. Arginine-8 vasotocin (1 &g body wt) also inhibited the release call, but its effect was delayed by a single injection of 10@g/gbody wt of the prostaglandin synthetase inhibitor, indomethacin. These results suggest that both vasotocin and prostaglandin may be involved in the manifestation of female mating behavior in R. pipiens and that vasotocin may act, at least in part, through a mechanism that involves prostaglandin synthesis.
An unreceptive female Rana pipiens emits a release call when clasped by a sexually active male. Contractions of the trunk muscles produce this call and stimulate the male to release the female. In contrast, a receptive female is silent, so the male can maintain his clasp, and oviposition and fertilization can occur (Noble and Aronson, 1942). The release call, which has been studied extensively by Schmidt (196.5, 1966, 1972, 1973, 1974), has been inhibited by administration of arginine-8 vasotocin (AVT) and it has been suggested that this hormone may play a role in potentiating reproductive behavior in this species (Diakow, 1978). The neurohypophysial polypeptide, arginine-8 vasotocin is the antidiuretie hormone in R. pipicrzs (Munsick, 1966; Acher, Chauvet, and Chauvet, 1969), and one mechanism whereby AVT might inhibit the call could involve water accumulation and/or consequent pressure within the body (Diakow, 1978; Diakow and Raimondi, 1980). The release call has been inhibited without AVT administration by fluid retention caused by cloaca1 ligature (Diakow, 1977). The relationship between AVT function and gonadal activity is unknown. Although inhibition of the release call normally occurs around the time of ovulation (personal observation), AVT can inhibit the call in the absence of the ovaries (Diakow, Wilcox, and Woltmann, 1978) and even in male frogs (Raimondi and Diakow, 1981). The gonadal steroids, estrogen and progesterone, do not inhibit the call (Diakow, et al., 1978). Indeed, these hormones seem to retard AVT’s action in release call inhibition (Diakow and Raimondi, 1980). 86 00 18-506X/8l/010086-08$01.00/0 Copyright All rights
Q 1981 by Academic Press. Inc of reproduction in any form reserved.
NONSTEROIDS
AND FEMALE
SEX BEHAVIOR
87
Besides its possible role in potentiating reproductive behavior in the female frog, AVT seems to play a role in reproductive behaviors of other nonmammalian vertebrates. These behaviors include spawning of teleosts (Pickford and Strecker, 1977, review by Crews and Silver, 1981), oviposition in the domestic hen (Rzasa, 1978) and clasping by male newts (Moore and Zoeller, 1979). < The fatty acids characterized as prostaglandins (PG) are another group of nonsteroid hormones that seem to be important for reproductive behavior. PGE,, PGI$, and PGF,, induce oviposition in the domestic hen (Hertelendy, Yeh, and Biellier, 1974), and though several prostaglandins also induce oviposition in quail, PGE, is the most effective (Hertelendy, 1974). The observation that indomethacin, a prostaglandin synthetase inhibitor, delays oviposition in the hen (Day and Nalbandov, 1977) supports the hypothesis that PG is involved in oviposition in birds. Similarly, there is evidence that PGF,, and PGE, induce oviposition behavior in goldfish (Stacey, 1976, 1980; Stacey and Peter, 1979). In mammals, PGF,, potentiates the mating postures of female rats (Hall, Luttge, and Berry, 1975; Dudley and Moss, 1976; Rodriguez-Sierra and Komisaruk, 1977)and hamsters (Buntin and Lisk, 1979). In the frog, PGF,, induces a leg adduction posture in females that enables X~nopus faevis males to maintain their clasps on them (Kelley, personal communication). The experiments reported in this paper investigate the relationship between AVT function and prostaglandin action in reproductive behavior of the leopard frog. Experiment 1 tests if the prostaglandins Q and F,, inhibit the release call, and Experiment 2 tests whether AVT might act through a mechanism utilizing prostaglandin in inhibiting the release call. EXPERIMENT
1
Design
Three groups of 10 ovariectomized R. pipiens each were tested for the release call by manual stimulation, injected, and retested at 30 min, then hourly, for the first 5 hr after injection. One group was injected with prostaglandin & (PGIQ; the second, with prostaglandin Fzu (PGF,,); and the third, with deionized water (DI). Methods Subjects and housing. All subjects were female, northern leopard frogs, R. pipiens. Median and range snout-vent lengths were 86 mm (82-94
mm). The frogs were collected in Vermont in the autumn and stored in hibernation until they were shipped to the laboratory in March. In the laboratory, they were housed in groups in large tanks containing a dilute solution of Agristrep (streptomycin sulfate) and floating Styrofoam pads upon which they could sit to emerge from the water. During the experi-
88
DIAKOW
AND NEMIROFF
ment, they were housed individually in plastic containers (30 x 17 x 21 cm). The Styrofoam pad was removed during the testing period. Laboratory temperature was 20-24°C and the animals were housed under constant fluorescent overhead lighting. Ovariectomies. The subjects were ovariectomized in order to prevent any ovarian response to PG from confounding the results. Surgery was performed under ether anesthesia through ventral, parasaggital incisions 3-7 days after arrival in the laboratory. Median and range ovarian volumes were 12 ml (8-15 ml). Tests and injections. Tests began 21-25 days after ovariectomy. For testing, the frogs were held between the thumb and forefinger of the experimenter on either side of the vertebral column posterior to the pectoral girdle. The number of release calls elicited in the first 30 set of clasping was recorded, and then the frog was weighed to the nearest gram on a Mettler P1200 balance. On the first day of testing, the females were tested twice; on the second day, they were tested twice, injected, then tested 30 mitt, and 1,2,3,4, and 5 hr after injection. Prostaglandin (5 pg/g body wt) (Upjohn) was administered ip. Injections were prepared from solutions of 10 mg prostaglandin in 20 cc deionized water. These solutions were prepared on the day before injection and stored at 5°C. Results
There was a significant decline in the number of release calls after injection of both prostaglandins & and F,,, but not after injection with deionized water (Table 1). The median number of release calls was 0 by 30 min after injection of prostaglandin; recovery of the release call had begun 4 hr after injection. There was no significant change in the weight of any group after injection. TABLE 1 Effect of F’rostaglandin on the Release Call Group”
Before injectioti
After injection
Release callsc
PGE, FGF,” DI
32 (14-43)d 31 (12-42) 31 (10-45)
0 (0-16fe 0 (O-4y 3.5 (22-47)
Weight (g)
p-k PGF*a DI
41 (35-48) 40 (37-44) 39 (35-50)
39 (36-49) 40 (37-48) 39 (33-54)
a N = 10 for each group. b Four preinjection tests; six postinjection tests. c Number of release calls in 30 set of manual clasping around trunk. d Median and range of the medians for each female. e Significant difference between the pre- and postinjection tests using the Wilcoxon matched-pairs signed-ranks test; p 5 0.05, two tail.
NONSTEROIDS
AND FEMALE
EXPERIMENT
SEX BEHAVIOR
89
2
Design
Three groups of females were tested for the release call by manual stimulation, injected, and retested hourly l-9 and 22-26 hr after injection. One group was injected with AVT (AVT-only, N = 8); the second, with AVT and indomethacin, a prostaglandin synthesis inhibitor (AVT-Indo, N = 9); and a third, with indomethacin alone Undo-only, N = 8). Methods
Twenty-one subjects were Northern leopard frogs, R. @piens, Callected in Vermont in the autumn and stored in hibernation until they were shipped to the laboratory in March. They were introduced into the experiment 3 1 days after arrival in the laboratory. Four subjects had been collected and shipped to the laboratory in the autumn and stored under standard laboratory conditions (Diakow, 1977). All subjects were large, mature females with well-developed ovaries. Subjects were tested twice, on the next day tested twice again, then injected and retested hourly l-9 and 22-26 hr after injection. All subjects croaked more than 10 times during three of the four preinjection tests. All injections were intraperitoneal. The AVT (Calbiochem) dose was 1.0 pg/g body wt from a solution containing 1 mg AVT in 9.5 ml deionized water. The indomethacin (Sigma) dose was 10.0 pg/g body wt. Indomethacin injections were drawn from a solution containing 0.05 g indomethacin, 0.1 g sodium carbonate, and enough deionized water to make up 50 cc. The solution was agitated intermittently for at least an hour before injection. At the time of injection, the median and range body weights were 58 g (43-72 g) for the AVT only group, 56 g (45-59 g) for the AVT-Indo group, and 46g (41-65 g) for the Indo-only group. Results
Figure 1 shows the median number of release calls for each test after injection for the three groups. Inspection suggests a decline in the number of release calls after AVT injection in both the AVT-only and AVT-Indo groups. Statistical comparison of the preinjection and postinjection medians for each subject confirms that the release call was lower after injection in both groups that received AVT but not in the group receiving indomethacin only (Wilcoxin test). Table 2 presents the median and range of the medians for each female. The decline in the release calls occurred faster in the AVT-only group than in the AVT-Indo group. The best fit lines for the decline in release calls over the first 3 hr after injection for the two groups are shown in Fig. 2. The slopes (m) are significantly different (t = 19.06, p < .OOl; two tail). Figure 3 shows that water accumulation occurred in both groups receiv-
90
DIAKOW AND NEMIROFF l -*
AVT
o----o
only
”
8
AVT+ lndo n 9
t.....*
lndo ”
only 0
?
HOURS
AFTER
INJECTION
FIG. 1. Median number of release calls elicited in 30 set of manual stimulation of the trunk after injection of 1&g body wt vasotocin (AVT-only), 1pg/g body wt vasotocin and 10 &g body wt indomethacin (AVT + Indo), and IO pg/g body wt indomethacin alone (Indo-only).
ing AVT, but water did not accumulate in the Indo-only group. There was no difference in the pattern of water accumulation between the AVT-only and AVT-Indo groups (Fig. 3). DISCUSSION
The results of Experiment 1 suggest that prostaglandins may be involved in inhibition of the release call in female leopard frogs. Because indomethacin retarded the ability of AVT to inhibit the call, AVT’s effect on female receptivity may be mediated, in part, through prostaglandins. TABLE 2 Effect of AVT on the Release Call Group AVT only (N = 8) AVT + Indo (N = 9) Indo Only (N = 8)
Before injectiona
After injection
28b.c (7-40) 24 (15-43) 17 (6-33)
lad (l-36) 216 (l-34) 18 (14-34)
a 4 Preinjection tests; 13 postinjection tests. b Number of release calls in 30 set of manual clasping around the trunk. c Median and range of the medians for each female. d Significant difference between the pre- and postinjection tests using the Wilcoxon matched-pairs signed-ranks test; p 5 0.05; two tail.
91
NONSTEROIDS AND FEMALE SEX BEHAVIOR
-*AVT
only
2
I HOURS
3
AFTER
INJECTION
FIG. 2. The rate of decline of t1le release call after 1pg/g body wt vasotocin injection is slower when 10pg/g body wt indomethacin is also administered. See Fig. 1for description of groups.
Rzgsa (1978) has reported a similar interaction between AVT and prostaglandin in oviposition and oviduct contractility of the domestic hen. Both hormones enhanced oviposition; and indomethacin prevented the stimulation of oviduct contractions by AVT. Rzgsa proposed that AVT exerted its effect on oviposition by acting through PG to cause oviduct contractions. It has been suggested that the silence of the receptive female is due to an increase in intraabdominal pressure or distension of the body wall
o---o l
-•
t..-..* ‘t
. .l
l
l
l ‘,,.
* ,,,_ l
AVT + lndo AVT only lndo
only
_,__
.f..
l -” I
2
3
4 HOURS
5
6
7
AFTER
8
9
“h
22 23 21 25 26
INJECTION
FIG. 3. Median weight after injection expressed as a percentage of body weight at the time of injection. See Fig. I for description of groups.
92
DIAKOW
AND NEMIROFF
resulting from ovulation of the egg mass into the uterus (Noble and Aronson, 1942) or from water accumulation due to AVT action (Diakow, 1978; Diakow and Raimondi, 1980). There is, indeed, much evidence that an increase in girth which causes intraabdominal pressure and distension of the body wall will also inhibit the release call; effective pressure and distension has been provided artificially by water accumulation after cloaca1 ligature (Diakow, 1977, 1978; Diakow and Raimondi, 1980) and inflation of an intraabdominal balloon (Diakow, 1978). The present studies add a new dimension for understanding the silence of the receptive female because it is the first to disassociate inhibition of the call from an increase in girth. In Experiment 1, the decline in the release call after prostaglandin administration occurred without swelling of the body due to water accumulation; and in Experiment 2, indomethatin retarded the inhibition of the release call, but not swelling accompanying water accumulation, after AVT administration. We are presented, then, with the necessity of evaluating many possible physiological mechanisms for inhibition of the release call in R. pipiens. In order to test Noble and Aronson’s hypothesis, it is necessary (1) to find an increase in intraabdominal pressure associated with ovulation; (2) to quantify this pressure increase if it exists; and (3) to show that this amount of intraabdominal pressure will, indeed, inhibit the release call. If their hypothesis is correct, it can only account for initiation of the inhibition of the release call, for Diakow & Raimondi have already presented evidence that if the egg mass is removed from the uterus of receptive females, they remain silent. In order to provide evidence for Diakow’s suggestion that AVT might inhibit the release call it is necessary to show elevated AVT levels associated with receptivity, increased responsivity to AVT at the time of breeding, or at least, increased water accumulation associated with receptivity. The present experiments have expanded the possibilities to be evaluated by making it necessary to measure prostaglandin levels associated with receptivity. ACKNOWLEDGMENTS We gratefully acknowledge encouragement for initiating Experiment 1 from David Crews, Darcy Kelley, and Norman Stacey, extensive consultation with J. Wayne Lazar and Louise Leotta, and the gift of prostaglandin from John E. Pike of the Upjohn Company.
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Day, S. L., and Nalbandov, A. V. (1977). Presence of prostaglandin F (PGF) in hen follicles and its physiological role in ovulation and oviposition. Biol. Reprod. 16, 486-494. Diakow, C. (1977). Initiation and inhibition of the release croak of Rana pipiens. Physiol. Behav.
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