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Androgens and reproductive behavior in ovariectomized ring doves
Physiology & Behavior, Vol. 32, pp. 697--699. Copyright © Pergamon Press Ltd., 1984. Printed in the U.S.A.
0031-9384/84 $3.00 + .00
BRIEF COMMUNICATION
Androgens and Reproductive Behavior in Ovariectomized Ring Doves E M I L I E F. R I S S M A N A N D E L I Z A B E T H A D K I N S - R E G A N
Department of Psychology, Uris Hall, Cornell University, Ithaca, N Y 14853 Received 22 June 1983 RISSMAN, E. F. AND E. ADKINS-REGAN. Androgens and reproductive behavior in ovariectomized ring doves. PHYSIOL BEHAV 32(4) 697-699, 1984.--Eighteen ovariectomized ring doves (Streptopelia risoria) received subcutaneous silastic implants of either testosterone propionate (TP), 5a-dihydrotestosterone propionate (DHTP) or cholesterol. Birds were paired daily for l-hr with intact males. Eleven days after implantation the pairs were observed. Both TP and DHTP activated wingflippingbehavior in the females. None of the females showed receptive crouching. Aromatization of testosterone to estrogen does not appear to be involved in wingflipping in female doves. The results suggest that wingtlipping in females is not as hormone specific as it appears to be in male doves. Reproductive behavior
Androgens
Aromatization
HORMONAL specificity of behavior is best examined in animals which demonstrate a repertoire of different steroid dependent reproductive behavior patterns. During their reproductive cycle, ring doves (Streptopelia risoria) exhibit several such patterns which can be analyzed separately with respect to hormonal control. Two of these, copulatory behavior and wingflipping, a nest solicitation posture which can be accompanied by nest-cooing, can be examined in terms of specificity in both sexes. Copulatory patterns are dimorphic; females show receptive crouching while males demonstrate mounting and cloacal contact. Thus, the hormonal control of copulatory behavior might be expected to differ between the sexes. Wingflipping is a monomorphic behavior in ring dotes, in that both members of a pair perform this behavior, primarily during the courtship phase of the cycle. In contrast to copulatory behavior, wingllipping might be expected to have the same hormonal correlates in male and female doves. Thus far, most studies have investigated the ability of estrogens and androgens to reinstate reproductive behavior in castrated male doves. These studies indicate that estradiol can activate wingflipping [1, 4, 11] in castrates. Testosterone (T) will also effectively restore wingflipping and copulatory behavior when given via either injection or hypothalamic implantation [1, 2, 6, 11]. Dihydrotestosterone (DHT) and dihydrotestosterone propionate (DHTP) are not as effective as T in activating these behaviors in castrates [1, 6, 15]. Thus in males T may activate wingflipping via aromatization to E (estradiol). The steroid specificity of reproductive behavior in the female dove is less clear. Cheng and Lehrman [4] found reinstatement of wingflipping when they gave injections of
Ring doves
Wingflipping
Nest solicitation
testosterone propionate (TP) or estradiol benzoate (EB) to ovariectomized females. Only estrogen treated females showed sexual crouching, indicative of receptivity. One interpretation of these data is that TP acts through aromatization to E in females, as it may in males, to activate wingflipping. Aromatase enzyme activity has been measured in male doves [8, 9, 16], and is present in female doves as well [9]. We tested this aromatization hypothesis in females by treating ovariectomized doves with either an aromatizable (TP) or a non-aromatizable (DHTP) androgen. We expected that TP but not DHTP treated females would wingflip, and that neither group would show receptive crouching. METHOD
Animals The subjects were eighteen female ring doves. Six males served as stimulus animals. All the birds were experienced breeders (having reared at least 2 squabs to fledging). The birds were housed in the same room throughout the study. Females were housed singly in metal cages with wire mesh fronts and backs (45x50x50 cm). Males were housed in cages identical to females', with 2 males per cage. All birds were visually isolated from birds in other cages. Birds received food, water and grit ad lib. Lights were on for 14 hours per day (0600-2000).
Surgery Ovariectomies were performed according to Cheng [3] except that ovaries were made to regress prior to surgery by placing the birds on a light cycle of 10 hours light: 14 hours
697
698
RISSMAN AND ADKINS-REGAN TABLE I WlNGFLIPPINGBEHAVIOROF IMPLANTEDOVARIECTOM1ZEDRING DOVES Implant Cholesterol Number Showing Behavior Mean Duration of Behavior in Minutes Standard Deviation of the Mean
DHTP
-fP
0
4*
5+
0
3.67*
5.75"t
0
2.07
2.55
N =6 in each group. *Significantly different from cholesterol, p<0.04 level at least. tSignificantly different from cholesterol, p <0.008 level.
dark for 3 weeks. Regression of the avian ovary facilitates the complete removal of the organ without excessive bleeding, an otherwise difficult procedure. At the completion of the experiment the females were laparotomized to check for possible ovarian regeneration. Two to four weeks after the ovariectomies were completed each female was implanted (under Fluothane anesthesia) with a 1.3 cm length of Silastic tubing (o.d. 2.413 mm, i.d. 1.575 mm). Implants were placed subcutaneously in the back of the neck. Implants contained 1.0 cm of either TP (Sigma Chemical Co.), 5a-DHTP (Steraloids, Inc.) or cholesterol. Each treatment group contained six females. Procedure
Each female was paired with an intact male for one hour daily beginning the morning after implantation. Pairing was accomplished by placing a nest bowl with straw and a male into the female's cage. The same male was placed into the female's cage each time. All pairing occurred between 0800-1200 hr. The order in which each stimulus male was paired with his 3 different females was randomized. On the eleventh day after implantation of the Silastic capsule the pair was observed for their first fifteen minutes together. Female behavior recorded included: number of receptive crouches, slaps and pecks, latency to begin wingflipping, and length of time spent wingflipping (see [12,13] for a description of wingflipping). Male behavior was also noted. Data were analyzed with Fisher Exact and Mann-Whitney U-tests. One-tailed tests were used except where indicated. RESULTS As Table 1 illustrates, more females wingflipped when implanted with TP (p<0.008, Fisher Exact Test) or DHTP (p <0.040) than when implanted with cholesterol. Duration of wingflipping was longer in hormone treated birds (TP, p <0.008 and DHTP, p <0.032, Mann-Whitney U-test) than in control birds. The duration of wingflipping and latency to begin wingflipping did not differ between the two hormonetreated groups (p=0.322 for duration and p=0.278 for latency, Mann-Whitney U-test, two-tailed). None of the females showed any receptive crouching, in spite of the fact that some of their male partners bow-cooed during the tests.
One female (a control bird) slapped the stimulus male during the observation period. Several females pecked their males. The number of females that pecked did not vary with hormone treatment. DISCUSSION The results clearly show that both aromatizable (TP) and non-aromatizable (DHTP) androgens can activate wingflipping in ovariectomized ring doves, and this differs from resuits seen in male doves. In castrated males TP and EB reinstate wingttipping, while DHT and DHTP are less effective [1, 7, 15]. Thus in the male wingflipping may be dependent upon the aromatization of T to E, because detectable levels of E are not found in the plasma of male doves during any phase of the breeding cycle [10]. Our data supplement those of Cheng and Lehrman [4] and suggest that several hormonal pathways can initiate wingtlipping in females. The data also support the view of receptive crouching as an exclusively estrogen-dependent behavior. There is currently no evidence that DHT is aromatizable in vivo in either birds or mammals [14]. Thus it is very likely that the hormonal specificity of wingflipping is broader in female than in male doves. The significance of DHTP (or TP) stimulation of wingflipping for intact females is less clear. II is possible that androgens (T and DHT) found in low concentrations in females throughout the breeding cycle [5] may help maintain low levels of wingflipping in females after high estrogen levels associated with follicular development have declined. For example, we have seen wingflipping during incubation, a period when estradiol is undetectable in female dove plasma [10]. ACKNOWLEDGEMENTS We thank Mary Ascenzi, Jayne Tishman, and Len Rodriguez for their technical assistance. E.F.R. w i s h e s to thank Drs, John Hutchison and Ari van Tienhoven for helpful discussions of these data, Dr. Rae Silver for technical advice, and Julie McCollister for typing the manuscript. We are especially grateful to Dr. Carl J. Erickson for his helpful comments on an early draft of this paper. This work was funded by NSF grant BNS 76-24308to Dr. E. Adkins-Regan. EiF.R. was supported by an NSF predoctoral fellowship while involved with this work.
A N D R O G E N S A N D R E P R O D U C T I V E B E H A V I O R IN D O V E S
699
REFERENCES 1. Adkins-Regan, E. Effect of sex steroids on the reproductive behavior of castrated male ring doves (Streptopelia sp.). Physiol Behav 26: 561-565, 1981. 2. Barfield, R. J. Activation of sexual and aggressive behavior by androgen implanted into the male ring dove brain. Endocrinology 89: 1470-1476, 1971. 3. Cheng, M. F. Effect ofovariectomy on the reproductive behavior of female ring doves (Streptopelia risoria). J Comp Physiol Psychol 83: 221-233, 1973. 4. Cheng, M. F. and D. Lehrman. Gonadal hormone specificity in the sexual behavior of ring doves. Psychoneuroendocrinology l: 95-102, 1975. 5. Feder, H. H., A. Storey, D. Goodwin, C. Reboulleau and R. Silver. Testosterone and "Sa-dihydrotestosterone" levels in peripheral plasma of male and female ring doves (Streptopelia risoria) during the reproductive cycle. Biol Reprod 16: 666-667, 1977. 6. Hutchison, J. B. Hypothalamic mechanisms of sexual behavior, with special reference to birds. In: Advances in the Study of Behavior, vol 6, edited by J. S. Rosenblatt, R. A. Hinde, E. Shaw and C. Beer. New York: Academic Press, 1976, pp. 159200. 7. Hutchison, J. B. Hypothalamic regulation of male sexual responsiveness to androgen. In: Biological Determinants of Sexual Behavior, edited by J. B. Hutchison. Chichester: Wiley, 1978, pp. 277-318.
8. Hutchison, J. B. and T. Steimer. Brain 5t~-reductase: A correlate of behavioral sensitivity to androgen. Science 213: 244--246, 1981. 9. Hutchison, J. B. and T. Steimer. Hormone-mediated behavioral transitions: a role for brain aromatase in the dove. Paper presented at the Symposium on Hormones and Behavior in Higher Vertebrates, September 8--11, 1982, Bielefeld, West Germany. 10. Korenbrot, C. C., D. W. Schomber and C. J. Erickson. Radioimmunoassay of plasma estradiol during the breeding cycle of ring doves (Streptopelia risoria). Endocrinology 94: 1126-1132, 1974. l 1. Martinez-Vargas, M. C. The induction of nest building in the ring dove (Streptopelia risoria): Hormonal and social factors. Behaviour 50: 123-151, 1974. 12. Miller, W. J. and L. S. Miller. Synopsis of behaviour traits of the ring-neck dove. Anita Behav 18: 3-8, 1958. 13. Rissman, E. F. Detection of cuckoldry in ring doves. Anita Behav 31: 449-456, 1983. 14. Ryan, K. J. Biogenesis of estrogens. Proc Int Congr Biochem 5th 7: 381-394, 1963. 15. Silver, R., M. O'Connell and R. Saad. The effect of androgens on the behavior of birds. In: Endocrine Control of Sexual Behavior, edited by C. Beyer. New York: Raven Press, 1979, pp. 223-278. 16. Steimer, T. and J. B. Hutchison. Androgen increases formation of behaviorally effective oestrogen in dove brain. Nature 292: 345-347, 1981.
0031-9384/84 $3.00 + .00
BRIEF COMMUNICATION
Androgens and Reproductive Behavior in Ovariectomized Ring Doves E M I L I E F. R I S S M A N A N D E L I Z A B E T H A D K I N S - R E G A N
Department of Psychology, Uris Hall, Cornell University, Ithaca, N Y 14853 Received 22 June 1983 RISSMAN, E. F. AND E. ADKINS-REGAN. Androgens and reproductive behavior in ovariectomized ring doves. PHYSIOL BEHAV 32(4) 697-699, 1984.--Eighteen ovariectomized ring doves (Streptopelia risoria) received subcutaneous silastic implants of either testosterone propionate (TP), 5a-dihydrotestosterone propionate (DHTP) or cholesterol. Birds were paired daily for l-hr with intact males. Eleven days after implantation the pairs were observed. Both TP and DHTP activated wingflippingbehavior in the females. None of the females showed receptive crouching. Aromatization of testosterone to estrogen does not appear to be involved in wingflipping in female doves. The results suggest that wingtlipping in females is not as hormone specific as it appears to be in male doves. Reproductive behavior
Androgens
Aromatization
HORMONAL specificity of behavior is best examined in animals which demonstrate a repertoire of different steroid dependent reproductive behavior patterns. During their reproductive cycle, ring doves (Streptopelia risoria) exhibit several such patterns which can be analyzed separately with respect to hormonal control. Two of these, copulatory behavior and wingflipping, a nest solicitation posture which can be accompanied by nest-cooing, can be examined in terms of specificity in both sexes. Copulatory patterns are dimorphic; females show receptive crouching while males demonstrate mounting and cloacal contact. Thus, the hormonal control of copulatory behavior might be expected to differ between the sexes. Wingflipping is a monomorphic behavior in ring dotes, in that both members of a pair perform this behavior, primarily during the courtship phase of the cycle. In contrast to copulatory behavior, wingllipping might be expected to have the same hormonal correlates in male and female doves. Thus far, most studies have investigated the ability of estrogens and androgens to reinstate reproductive behavior in castrated male doves. These studies indicate that estradiol can activate wingflipping [1, 4, 11] in castrates. Testosterone (T) will also effectively restore wingflipping and copulatory behavior when given via either injection or hypothalamic implantation [1, 2, 6, 11]. Dihydrotestosterone (DHT) and dihydrotestosterone propionate (DHTP) are not as effective as T in activating these behaviors in castrates [1, 6, 15]. Thus in males T may activate wingflipping via aromatization to E (estradiol). The steroid specificity of reproductive behavior in the female dove is less clear. Cheng and Lehrman [4] found reinstatement of wingflipping when they gave injections of
Ring doves
Wingflipping
Nest solicitation
testosterone propionate (TP) or estradiol benzoate (EB) to ovariectomized females. Only estrogen treated females showed sexual crouching, indicative of receptivity. One interpretation of these data is that TP acts through aromatization to E in females, as it may in males, to activate wingflipping. Aromatase enzyme activity has been measured in male doves [8, 9, 16], and is present in female doves as well [9]. We tested this aromatization hypothesis in females by treating ovariectomized doves with either an aromatizable (TP) or a non-aromatizable (DHTP) androgen. We expected that TP but not DHTP treated females would wingflip, and that neither group would show receptive crouching. METHOD
Animals The subjects were eighteen female ring doves. Six males served as stimulus animals. All the birds were experienced breeders (having reared at least 2 squabs to fledging). The birds were housed in the same room throughout the study. Females were housed singly in metal cages with wire mesh fronts and backs (45x50x50 cm). Males were housed in cages identical to females', with 2 males per cage. All birds were visually isolated from birds in other cages. Birds received food, water and grit ad lib. Lights were on for 14 hours per day (0600-2000).
Surgery Ovariectomies were performed according to Cheng [3] except that ovaries were made to regress prior to surgery by placing the birds on a light cycle of 10 hours light: 14 hours
697
698
RISSMAN AND ADKINS-REGAN TABLE I WlNGFLIPPINGBEHAVIOROF IMPLANTEDOVARIECTOM1ZEDRING DOVES Implant Cholesterol Number Showing Behavior Mean Duration of Behavior in Minutes Standard Deviation of the Mean
DHTP
-fP
0
4*
5+
0
3.67*
5.75"t
0
2.07
2.55
N =6 in each group. *Significantly different from cholesterol, p<0.04 level at least. tSignificantly different from cholesterol, p <0.008 level.
dark for 3 weeks. Regression of the avian ovary facilitates the complete removal of the organ without excessive bleeding, an otherwise difficult procedure. At the completion of the experiment the females were laparotomized to check for possible ovarian regeneration. Two to four weeks after the ovariectomies were completed each female was implanted (under Fluothane anesthesia) with a 1.3 cm length of Silastic tubing (o.d. 2.413 mm, i.d. 1.575 mm). Implants were placed subcutaneously in the back of the neck. Implants contained 1.0 cm of either TP (Sigma Chemical Co.), 5a-DHTP (Steraloids, Inc.) or cholesterol. Each treatment group contained six females. Procedure
Each female was paired with an intact male for one hour daily beginning the morning after implantation. Pairing was accomplished by placing a nest bowl with straw and a male into the female's cage. The same male was placed into the female's cage each time. All pairing occurred between 0800-1200 hr. The order in which each stimulus male was paired with his 3 different females was randomized. On the eleventh day after implantation of the Silastic capsule the pair was observed for their first fifteen minutes together. Female behavior recorded included: number of receptive crouches, slaps and pecks, latency to begin wingflipping, and length of time spent wingflipping (see [12,13] for a description of wingflipping). Male behavior was also noted. Data were analyzed with Fisher Exact and Mann-Whitney U-tests. One-tailed tests were used except where indicated. RESULTS As Table 1 illustrates, more females wingflipped when implanted with TP (p<0.008, Fisher Exact Test) or DHTP (p <0.040) than when implanted with cholesterol. Duration of wingflipping was longer in hormone treated birds (TP, p <0.008 and DHTP, p <0.032, Mann-Whitney U-test) than in control birds. The duration of wingflipping and latency to begin wingflipping did not differ between the two hormonetreated groups (p=0.322 for duration and p=0.278 for latency, Mann-Whitney U-test, two-tailed). None of the females showed any receptive crouching, in spite of the fact that some of their male partners bow-cooed during the tests.
One female (a control bird) slapped the stimulus male during the observation period. Several females pecked their males. The number of females that pecked did not vary with hormone treatment. DISCUSSION The results clearly show that both aromatizable (TP) and non-aromatizable (DHTP) androgens can activate wingflipping in ovariectomized ring doves, and this differs from resuits seen in male doves. In castrated males TP and EB reinstate wingttipping, while DHT and DHTP are less effective [1, 7, 15]. Thus in the male wingflipping may be dependent upon the aromatization of T to E, because detectable levels of E are not found in the plasma of male doves during any phase of the breeding cycle [10]. Our data supplement those of Cheng and Lehrman [4] and suggest that several hormonal pathways can initiate wingtlipping in females. The data also support the view of receptive crouching as an exclusively estrogen-dependent behavior. There is currently no evidence that DHT is aromatizable in vivo in either birds or mammals [14]. Thus it is very likely that the hormonal specificity of wingflipping is broader in female than in male doves. The significance of DHTP (or TP) stimulation of wingflipping for intact females is less clear. II is possible that androgens (T and DHT) found in low concentrations in females throughout the breeding cycle [5] may help maintain low levels of wingflipping in females after high estrogen levels associated with follicular development have declined. For example, we have seen wingflipping during incubation, a period when estradiol is undetectable in female dove plasma [10]. ACKNOWLEDGEMENTS We thank Mary Ascenzi, Jayne Tishman, and Len Rodriguez for their technical assistance. E.F.R. w i s h e s to thank Drs, John Hutchison and Ari van Tienhoven for helpful discussions of these data, Dr. Rae Silver for technical advice, and Julie McCollister for typing the manuscript. We are especially grateful to Dr. Carl J. Erickson for his helpful comments on an early draft of this paper. This work was funded by NSF grant BNS 76-24308to Dr. E. Adkins-Regan. EiF.R. was supported by an NSF predoctoral fellowship while involved with this work.
A N D R O G E N S A N D R E P R O D U C T I V E B E H A V I O R IN D O V E S
699
REFERENCES 1. Adkins-Regan, E. Effect of sex steroids on the reproductive behavior of castrated male ring doves (Streptopelia sp.). Physiol Behav 26: 561-565, 1981. 2. Barfield, R. J. Activation of sexual and aggressive behavior by androgen implanted into the male ring dove brain. Endocrinology 89: 1470-1476, 1971. 3. Cheng, M. F. Effect ofovariectomy on the reproductive behavior of female ring doves (Streptopelia risoria). J Comp Physiol Psychol 83: 221-233, 1973. 4. Cheng, M. F. and D. Lehrman. Gonadal hormone specificity in the sexual behavior of ring doves. Psychoneuroendocrinology l: 95-102, 1975. 5. Feder, H. H., A. Storey, D. Goodwin, C. Reboulleau and R. Silver. Testosterone and "Sa-dihydrotestosterone" levels in peripheral plasma of male and female ring doves (Streptopelia risoria) during the reproductive cycle. Biol Reprod 16: 666-667, 1977. 6. Hutchison, J. B. Hypothalamic mechanisms of sexual behavior, with special reference to birds. In: Advances in the Study of Behavior, vol 6, edited by J. S. Rosenblatt, R. A. Hinde, E. Shaw and C. Beer. New York: Academic Press, 1976, pp. 159200. 7. Hutchison, J. B. Hypothalamic regulation of male sexual responsiveness to androgen. In: Biological Determinants of Sexual Behavior, edited by J. B. Hutchison. Chichester: Wiley, 1978, pp. 277-318.
8. Hutchison, J. B. and T. Steimer. Brain 5t~-reductase: A correlate of behavioral sensitivity to androgen. Science 213: 244--246, 1981. 9. Hutchison, J. B. and T. Steimer. Hormone-mediated behavioral transitions: a role for brain aromatase in the dove. Paper presented at the Symposium on Hormones and Behavior in Higher Vertebrates, September 8--11, 1982, Bielefeld, West Germany. 10. Korenbrot, C. C., D. W. Schomber and C. J. Erickson. Radioimmunoassay of plasma estradiol during the breeding cycle of ring doves (Streptopelia risoria). Endocrinology 94: 1126-1132, 1974. l 1. Martinez-Vargas, M. C. The induction of nest building in the ring dove (Streptopelia risoria): Hormonal and social factors. Behaviour 50: 123-151, 1974. 12. Miller, W. J. and L. S. Miller. Synopsis of behaviour traits of the ring-neck dove. Anita Behav 18: 3-8, 1958. 13. Rissman, E. F. Detection of cuckoldry in ring doves. Anita Behav 31: 449-456, 1983. 14. Ryan, K. J. Biogenesis of estrogens. Proc Int Congr Biochem 5th 7: 381-394, 1963. 15. Silver, R., M. O'Connell and R. Saad. The effect of androgens on the behavior of birds. In: Endocrine Control of Sexual Behavior, edited by C. Beyer. New York: Raven Press, 1979, pp. 223-278. 16. Steimer, T. and J. B. Hutchison. Androgen increases formation of behaviorally effective oestrogen in dove brain. Nature 292: 345-347, 1981.