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Effects of Vasoactive Intestinal Peptide on Plasma Prolactin in Passerines
General and Comparative Endocrinology 113, 323–330 (1999) Article ID gcen.1998.7220, available online at http://www.idealibrary.com on
Effects of Vasoactive Intestinal Peptide on Plasma Prolactin in Passerines Donna L. Maney,1 Stephan J. Schoech,* Peter J. Sharp,† and John C. Wingfield Department of Zoology, Box 351800, University of Washington, Seattle, Washington 98105; *Department of Biology and Center for the Integrative Study of Animal Behavior, Indiana University, Bloomington, Indiana 47405; and †Roslin Institute, Roslin, Midlothian EH25 9PS, United Kingdom Accepted November 2, 1998
Vasoactive intestinal peptide (VIP) is a potent releaser of prolactin (PRL) in domestic fowl, turkey, and ring doves. However, few comparative studies have investigated this in wild species. We tested the effects of intravenously administered chicken VIP on plasma PRL concentrations in four passerine species: the white-crowned sparrow (Zonotrichia leucophrys gambelii), the dark-eyed junco (Junco hyemalis), the Florida scrub-jay (Aphelocoma coerulescens), and the western scrub-jay (A. californica). In the white-crowned sparrow, junco, and Florida scrub-jay, which were tested during the breeding season, VIP induced a rapid increase in plasma PRL. Serial plasma samples taken after VIP injection in the white-crowned sparrow show a 10-fold increase in PRL within 2 min of treatment, followed by a gradual decline. Effects of VIP, as compared to saline, remained significant for at least 20 min after treatment. Western scrub-jays did not respond to intravenous VIP with a significant rise in PRL secretion, possibly because they were tested after termination of the breeding season. This study indicates that VIP control of PRL release may be widespread among avian species, and that seasonal changes in plasma PRL may be mediated in part at the level of the pituitary. In addition, analysis of the control data revealed no increase in plasma PRL as a result of injection or restraint, suggesting that unlike in mammals, PRL is not released during acute stress in passerines. r 1999 Academic Press
Key Words: prolactin; vasoactive intestinal peptide; avian; releasing factor. In birds, the pituitary hormone prolactin (PRL) is involved in many aspects of reproductive physiology and behavior. It is widely thought to play a role in parental behavior by mediating increases in incubation, crop milk secretion, feeding of young, and nest defense (Silver, 1984; Janik and Buntin, 1985; Lea et al., 1986; Buntin et al., 1991). After eggs are laid, PRL may inhibit GnRH and thus facilitate gonadal regression (Rozenboim et al., 1993). PRL is also thought to play a role in postnuptial molt at the termination of breeding in passerines (Dawson and Sharp, 1998). Secretion of PRL follows a highly seasonal pattern in most avian species studied (e.g., Hiatt et al., 1987). Increasing day length in spring triggers a slow rise in plasma PRL, which then increases sharply as parental behavior commences. In passerines a surge of PRL accompanies the onset of photorefractoriness, which is characterized by the termination of breeding and hypothalamic insensitivity to long day length (see Nicholls et al., 1988, for review). In domestic fowl (Gallus domesticus), turkey (Meleagris gallopavo), and ring doves (Streptopelia risoria), PRL release is controlled by vasoactive intestinal peptide (VIP), a hypothalamic releasing factor (Macnamee et al., 1986; Lea and Vowles, 1986; Opel and Proudman, 1988; El Halawani et al., 1990). Until recently, the effects of VIP on PRL secretion
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Current address: Department of Psychology, Johns Hopkins University, Baltimore, MD 21218. 0016-6480/99 $30.00 Copyright r 1999 by Academic Press All rights of reproduction in any form reserved.
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had not been tested in a passerine or a wild avian species. Dawson and Sharp (1998) found an inhibition of PRL secretion after immunization against VIP in European starlings (Sturnus vulgaris). Here we report that VIP acts as a PRL releasing factor in three other wild passerines: white-crowned sparrows (Zonotrichia leucophrys gambelii), Florida scrub-jays (Aphelocoma coerulescens), and dark-eyed juncos (Junco hyemalis). Our results are consistent with those of Vleck and Patrick (1999), who report that intravenous VIP elevates plasma PRL significantly within minutes of treatment in blue jays (Cyanocita cristata), zebra finches (Poephilla guttata), and Mexican jays (A. ultramarina). Because the protocol to test the effects of VIP on PRL release requires that animals be restrained, we were also able to test the effects of capture and handling stress on PRL secretion.
MATERIALS AND METHODS White-Crowned Sparrows (Zonotrichia leucophrys) Male Z. l. gambelii were captured during their autumnal migration in Central Washington State in 1994 and 1995. They were maintained in outdoor aviaries for 2 months before being transferred to individual cages in an environmental chamber with controlled light and temperature (20°C). Sex was determined by laparotomy prior to transfer. Day length in the chamber approximated a natural winter photoperiod (8 L:16 D) while the birds acclimated to captive conditions. After 1 week, the light cycle was changed to long days (20 L:4 D) to simulate conditions during breeding in the Arctic. Injections began 30 days after transfer to long days, at which time gonadal development reaches its peak (see Wingfield and Farner, 1993). Immediately prior to control or VIP injection, a blood sample was taken by alar venipuncture to determine baseline PRL. Birds then received an intravenous (jugular) injection of 0, 50, 100, or 500 ng chicken VIP (chVIP; Peninsula Laboratories, Belmont, CA) in 20 µl saline. Additional blood samples were taken at 2, 5, 10, and 20 min after injection. These doses correspond approximately to 2, 4, and 20 µg/kg. There were eight birds in each dosage group, and each bird was injected only once. Birds
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Maney et al.
were held in a cloth bag during the intervals between samples. Once the first bird was removed from the chamber for sampling, the chamber was entered approximately every 6 to 15 min until sampling was completed. The time of sampling with respect to when the chamber door was first opened was carefully recorded. Regression analysis revealed no relationship between initial preinjection PRL levels and the time since the chamber door was first opened (r2 ⫽ 0.006, P ⫽ 0.72). Samples were kept cool on ice until they were centrifuged and the plasma harvested. Plasma samples were stored at ⫺20°C until PRL assay in the laboratory of J.C.W. (see below).
Dark-Eyed Juncos (Junco hyemalis) Juncos were captured during April and May of 1997 in the immediate vicinity of Mountain Lake Biological Station in western Virginia (for a more complete description of the study site see Ketterson et al., 1990; Schoech et al., 1999). All birds were free-living at the time of treatment; they were captured in the field and held temporarily before being released at the capture site. Treatment occurred 1–3 weeks prior to nest initiation. All birds were captured singly in mist nets and removed as soon as possible. Nine juncos were injected intravenously with 1000 ng chVIP (approximately 50 µg/kg) in 25 µl saline, and six were injected with saline only. Blood samples were collected immediately prior to injection and at 15 min after. In all cases the initial preinjection sample was collected between 1 and 2 min of the time the bird first entered the net. Plasma samples were shipped to the United Kingdom for PRL assay in the laboratory of P.J.S. (see below).
Florida Scrub-Jays (Aphelocoma coerulescens) During March and April of 1997, Florida scrub-jays were trapped at Archbold Biological Station in southcentral Florida (see Schoech, 1996, for details). At this time, the jays were at the incubation stage of their reproductive cycle. Individual jays were captured in continuously monitored Potter traps. Eight jays were injected with 1000 ng chVIP (approximately 13 µg/kg) in 50 µl saline, and six jays received saline only. Blood samples were collected immediately prior to and at 5 and 15 min following injection. In all cases, birds were removed from the traps and the initial sample col-
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VIP and PRL in Passerines
lected within 1 min of time of capture. All birds were free-living at the time of treatment and were released shortly after the last blood sample was collected. Plasma samples were shipped on dry ice to the United Kingdom for PRL assay in the laboratory of P.J.S. (see below).
Western Scrub-Jays (Aphelocoma californica) Western scrub-jays were captured in Sunland, California, in July of 1994 and transported to Seattle, Washington. They were housed in an outdoor aviary under ambient conditions and given ad libitum food (peanuts and dog food) and water until treatment in August of 1994. Twelve jays received an intravenous injection of 500 ng chVIP (approximately 6 µg/kg) in 20 µl saline, and 12 jays received saline only. Blood samples were collected immediately prior to and at 5, 10, and 20 min after injection. When tested, each individual was removed from the aviary and an initial sample collected within 2 min of entering the aviary. Upon completion of treatment (i.e., injection with saline or VIP and collection of serial samples), the jay was returned to the aviary and another individual was captured. The nearly 30 min that passed between visits to the aviary to release one jay and capture the subsequent one was most likely sufficient for any hormonal effects of our previous visit to pass. Samples were kept cool on ice until they were centrifuged and the plasma harvested. Plasma samples were stored at ⫺20°C until PRL assay in the lab of J.C.W. (see below).
PRL Assay Z. leucophrys and A. californica plasma PRL levels were determined by a postprecipitation, double antibody radioimmunoassay (RIA) using purified chicken PRL as a standard and an anti-serum against PRL raised in rabbits. Both standard and antiserum were generously donated by Dr. A. F. Parlow (Pituitary Hormones and Antisera Center, UCLA-Harbor Medical Center, Torrance, CA). We have shown previously that this assay can be used to determine plasma PRL levels in Aphelocoma jays (Schoech et al., 1996). Because we had not measured PRL levels in sparrows, we validated the assay for Z. leucophrys by comparing inhibition curves for chicken standard and pooled white-crowned sparrow plasma (see below, Fig. 1).
A. coerulescens and J. hyemalis PRL levels were assayed in the laboratory of P.J.S. using antisera against recombinant-derived starling PRL (Bentley et al., 1997). Inhibition curves for plasma of both species are parallel to the standard curve in this assay, which has been used previously to measure PRL levels in J. hyemalis (Schoech et al., 1998). Although the absolute levels of PRL determined by this assay usually exceed those determined by the Parlow PRL assay, relative differences in PRL levels are well conserved (see Maney et al., 1999). The two assays have similar coefficients of variation; interassay variation is approximately 15 and 16% for the Parlow and starling assays, respectively, and intraassay variation is approximately 10 and 8%, respectively.
Statistical Analysis PRL levels were normalized by dividing each posttreatment level by the pretreatment level. Data were then log-transformed to reduce heteroscedasticity. Most analyses consisted of a two-way repeated measures ANOVA with time as the repeated measure and VIP dose as the independent variable. If this test showed significant effects (P ⬍ 0.05), post hoc tests (F tests followed by Scheffe tests or t tests) were used to examine within- and between-treatment differences. A difference was considered significant if the P value remained below 0.05 after correction for the number of comparisons. Since PRL was measured at only one posttreatment time in the junco study, these data were analyzed by unpaired t test only. To test the effects of restraint stress on PRL secretion, we also looked for an effect of time on PRL levels following saline injection. Analyses were performed using a one-way repeated measured ANOVA followed by Scheffe tests where appropriate, with the exception of the junco data, which were analyzed using paired t tests.
RESULTS Radioimmunoassay Validation White-crowned sparrow plasma inhibited the binding of radiolabeled chicken PRL to the chicken PRL
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antibody (Fig. 1). The near-parallel dilution curves (F1,11 ⫽ 1.064, P ⫽ 0.324, test for homogeneity of slopes) of chicken standard and white-crowned sparrow plasma indicate that this assay provides accurate measurement of relative PRL levels in Z. leucophrys. In addition, we have used this assay to quantify photoinduced PRL secretion in three subspecies of Z. leucophrys (Maney et al., 1999). PRL levels during photostimulation follow a typical avian pattern when measured by this assay. In this study, PRL for both whitecrowned sparrows and western scrub-jays fell within the physiological range reported for white-crowned sparrows and Aphelocoma jays when using this assay (Maney et al., 1999; Schoech, 1996; Schoech et al., 1996). The junco and Florida scrub-jay samples were measured with the recombinant starling PRL assay, which measures PRL levels in these species as approximately one order of magnitude higher than levels in the species measured with the Parlow PRL assay. Both assays reliably detect similar relationships among samples measured by both methods (Maney et al., 1999). The levels reported here are similar to those previously obtained using the recombinant starling
FIG. 1. Standard curve for chicken PRL (chPRL) and the inhibition curve from pooled white-crowned sparrow plasma. The standard curve shows displacement of 125I-labeled chPRL by the reference standard. The near-parallel dilution curve from white-crowned sparrow plasma indicates that this RIA can be used to assess relative levels of plasma PRL in this species (F1,11 ⫽ 1.064, P ⫽ 0.324, test for homogeneity of slopes).
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Maney et al.
PRL assay for European starlings (Bentley et al., 1997), juncos (Schoech et al., 1998), and white-crowned sparrows (Maney et al., 1999).
Intravenous VIP Injections In photostimulated white-crowned sparrows, intravenous VIP injection significantly affected plasma PRL (Fig. 2A; effect of dose, F ⫽ 9.505, P ⫽ 0.0002; effect of time, F ⫽ 6.164, P ⫽ 0.0008; interaction, F ⫽ 1.953, P ⫽ 0.0558). The higher two doses of VIP significantly elevated plasma PRL, compared to saline treatment, within 2 min of injection (for 100 ng, F ⫽ 5.068, P ⫽ 0.026; for 500 ng, F ⫽ 5.68, P ⫽ 0.0152). This effect was still significant at 5 min following treatment (for 100 ng, F ⫽ 4.776, P ⫽ 0.034; for 500 ng, F ⫽ 5.403, P ⫽ 0.0192). At 10 min after injection, 100 ng VIP continued to elevate plasma PRL compared with saline (F ⫽ 5.748, P ⫽ 0.0144), and with 50 ng VIP (F ⫽ 4.882, P ⫽ 0.0308), whereas the effect of 500 ng VIP was no longer significant. One hundred nanograms of VIP continued to elevate plasma PRL compared with saline at 20 min after treatment (F ⫽ 5.203, P ⫽ 0.0232). In dark-eyed juncos (J. hyemalis), plasma PRL was significantly higher 15 min after injection with 1000 ng VIP than after saline injection (Fig. 2B; unpaired t test, t ⫽ ⫺2.997, P ⫽ 0.0103). In Florida scrub-jays (A. coerulescens), injection of 1000 ng VIP significantly increased plasma PRL (Fig. 2C; effect of treatment, F ⫽ 13.545, P ⫽ 0.0031; effect of time, F ⫽ 15.931, P ⫽ 0.0018; interaction, F ⫽ 0.274, P ⫽ 0.6104). Plasma PRL increased significantly within 5 min of VIP injection compared with saline injection (t ⫽ ⫺3.663, P ⫽ 0.0066). This effect remained significant at 15 min following treatment (t ⫽ ⫺3.45, P ⫽ .0096). In Western scrub-jays (A. californica) there was no effect of VIP injection on plasma PRL (Fig. 2D; effect of treatment, F ⫽ 0.428, P ⫽ 0.5198; effect of time, F ⫽ 0.761, P ⫽ 0.4735; interaction, F ⫽ 1.668, P ⫽ 0.2003).
Effects of Restraint Stress In the white-crowned sparrows, juncos, and western scrub-jays, there was no effect of time on PRL levels in saline-injected birds (white-crowned sparrow, F ⫽ 0.545, P ⫽ 0.704; junco, t ⫽ 1.821, P ⫽ 0.128; west-
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FIG. 2. Plasma PRL response to intravenous VIP or saline in several passerine species. Error bars represent SEM. Data are normalized as described under Materials and Methods. See text for statistics. chPRL-IR, chicken prolactin immunoreactivity; RSP-IR, recombinant starling prolactin immunoreactivity. (A) Captive white-crowned sparrows exposed to 30–50 days of photostimulation. (B) Free-living dark-eyed juncos injected during April and May. (C) Free-living Florida scrub-jays injected during March and April. (D) Captive western scrub-jays, on natural photoperiod, injected during August.
ern scrub-jay, F ⫽ 1.137, P ⫽ 0.349). In the Florida scrub-jay, saline injection and restraint significantly inhibited plasma PRL (ANOVA, F ⫽ 6.226, P ⫽ 0.016), which was apparent within 15 min of treatment (P ⬍ 0.02).
DISCUSSION In this study, intravenous VIP induced a rapid rise in plasma PRL in the white-crowned sparrow, the darkeyed junco, and the Florida scrub-jay (Figs 2A–2C). These results resemble those of Vleck and Patrick
(1999), who found that VIP increases plasma PRL significantly in blue jays, zebra finches, and Mexican jays. Our results are also consistent with those of Dawson and Sharp (1998), who reported a suppression of plasma PRL after immunization against VIP in starlings. The absolute levels we found appear to be physiological; in the white-crowned sparrow posttreatment PRL peaked at a level similar to that which is seen at the onset of photorefractoriness (see Maney et al., 1999). Our results suggest that VIP control of PRL secretion may be widespread among birds. Our most detailed investigation was performed using white-crowned sparrows (Fig. 2A). In this species, the lowest dose of VIP (50 ng) did not significantly
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affect PRL levels. However, the 100 and 500 ng doses increased plasma PRL within 2 min of treatment. This rapid effect is consistent with the results of Macnamee et al. (1986), who report stimulation of PRL secretion within 2 min of VIP injection in the bantam hen. Figure 2A suggests that 500 ng VIP may have increased plasma PRL more than 100 ng VIP, but this apparent dose dependence was not significant. We used relatively low doses of VIP compared to those reported to stimulate PRL secretion in other species (Lea and Vowles, 1986; Vleck and Patrick, 1999), perhaps administering more VIP would elicit a dose-dependent response. One VIP dose was given and fewer blood samples were collected in the studies of other species. One thousand nanograms of VIP increased plasma PRL in juncos (Fig. 2B) and Florida scrub-jays (Fig. 2C) at the earliest time point analyzed (15 min for the juncos and 5 min for the jays). Earlier sampling from the juncos may have revealed a stronger PRL response. Figure 2D suggests that in the western scrub-jay, VIP injection may have affected PRL secretion slightly at 5 min after treatment. However, this effect was not significant. The lack of an effect in this species may be explained by the time of year and a seasonal insensitivity to VIP at the level of the pituitary. This hypothesis is supported by data from turkeys and juncos. In turkeys, the concentration of VIP in hypothalamopituitary portal blood is the same in laying and nonreproductive hens, despite nonreproductive birds having lower plasma levels of PRL (Youngren et al., 1996). Exogenous VIP does stimulate PRL release in nonreproductive hens, although significantly less so than during laying or incubation (Pitts et al., 1996). In juncos, despite evidence of seasonal differences in circulating plasma levels of PRL (Ketterson et al., 1990), Saldanha et al. (1994) found no differences in hypothalamic content of VIP between long- and short-day birds. The evidence that VIP is a PRL-releasing factor in juncos (Fig. 2B) and bantam hens (Macnamee et al., 1986), combined with the lack of seasonal differences in VIP despite seasonal differences in PRL suggests that pituitary lactotrophs may be insensitive to VIP in the nonbreeding season. The mechanism of this insensitivity could involve either a downregulation of pituitary VIP receptors or an inhibition of PRL gene transcrip-
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Maney et al.
tion, synthesis, or storage. For example, PRL mRNA half-life decreases significantly in unphotostimulated turkeys compared with laying or incubating turkeys (Tong et al., 1997). Seasonal modulation of lactotroph sensitivity to VIP could be mediated by reproductive hormones. Photostimulated turkeys, starlings, and great tits (Parus major) that have low circulating sex steroids as a result of gonadectomy respond to sex steroid replacement with an increase in circulating PRL (El Halawani et al., 1983; Dawson and Goldsmith, 1984; Silverin and Goldsmith, 1997). Additionally, turkey pituitary cells in vitro release more PRL in response to VIP when the culture medium contains gonadal steroid hormones (Knapp et al., 1988). Thus, it is possible that the lack of PRL response in western scrub-jays is attributable to the phase in the reproductive cycle during which the experiment was conducted. However, since ovariectomized turkeys respond to VIP injection with a significant rise in PRL (Opel and Proudman, 1988), we might expect a blunted rather than absent response in nonbreeding jays. Experiments measuring responses to VIP injection at different times of year in the same species are needed. Our protocol for taking serial blood samples before and after VIP injection involves restraining the birds in a cloth bag between sampling. This treatment increases glucocorticoid secretion in many species of passerine (e.g., Wingfield 1994, Wingfield et al., 1998), and has often been used to study the endocrine response to stress. In the sparrows, juncos and western scrub-jays, there was no effect of capture and handling on PRL secretion in animals receiving saline. The Florida scrub-jays, however, decreased their plasma PRL, opposite from what would be expected were PRL involved in the stress response in this species. This is consistent with Schoech et al. (1996), who report a negative correlation between plasma PRL and time after capture in Florida scrub-jays. PRL levels increase rapidly during restraint stress in mammals, and PRL is often considered part of the classical stress response (see de Vlaming, 1979, for review). A role for PRL in the stress response of birds, however, is less clear; for example, the stress of receiving an injection and subsequent blood sampling may decrease PRL in bantam hens (see Sharp et al., 1989). The effects of stress on PRL secretion may
VIP and PRL in Passerines
depend on reproductive state. Opel and Proudman (1986) report that PRL increases in most turkeys subjected to restraint stress; however, incubating hens show a reduction in PRL levels. One might conclude that these results are analogous to ours from Florida scrub-jays, which were also sampled mostly during incubation (see Fig. 2C). However, unlike in the turkey studies, the jay sample was composed of individuals of both sexes and status (i.e., breeders and nonbreeders) in a species in which only female breeders incubate. Adding further to the uncertainty of PRL’s role in the avian stress response, Delehanty et al. (1997) compared interyear differences in the effects of environmental stressors on PRL titers. In a year of drought and extreme high temperatures, incubating male Wilson’s phalaropes (Phalaropus tricolor) had lower PRL levels than in other years. Based on rates of weight loss and nest abandonment, the researchers concluded that the phalaropes were stressed. Together with our results, these studies suggest that the stress response in birds may differ from mammals with regard to PRL.
ACKNOWLEDGMENTS We thank A. F. Parlow (Pituitary Hormones and Antisera Center, UCLA-Harbor Medical Center, Torrance, CA) for his generous donation of chicken PRL antiserum and standard. We are grateful to Lynn Erckmann for expert animal care, to Aaron Kim, Chris Goode, and Pete Wilson for technical assistance, and to Nancy Temkin for help with statistical analysis. Thanks as well to Marty Morton and Maria Pereyra for field assistance and accommodations. This work was supported by NSF Grants DCB-9005081, IBN-9408013, and IBN-9631350 to J.C.W. and IBN-9224397 to S.J.S.
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330 turkey by vasoactive intestinal polypeptide. Proc. Soc. Exp. Biol. Med. 187, 455–460. Opel, W., and Proudman, J. (1986). Plasma prolactin responses to serial bleeding in turkeys. Dom. Anim. Endocrinol. 3, 19–207. Pitts, G. R., Youngren, O. M., Phillips, R. E., and El Halawani, M. E. (1996). Photoperiod mediates the ability of serotonin to release prolactin in the turkey. Gen. Comp. Endocrinol. 104, 265–272. Rozenboim, I., Tabibzadeh, C., Silsby, J. L., and El Halawani, M. E. (1993). Effect of ovine prolactin administration on hypothalamic vasoactive intestinal peptide (VIP), gonadotropin releasing hormone I and II content, and anterior pituitary VIP receptors in laying turkey hens. Biol. Reprod. 48, 1246–1250. Saldanha, C. J., Deviche, P. J., and Silver, R. (1994). Increased VIP and decreased GnRH expression in photorefractory dark-eyed juncos (Junco hyemalis). Gen. Comp. Endocrinol. 93, 128–136. Sharp, P. J., Sterling, R. J., Talbot, R. T., and Huskisson, N. S. (1989). The role of hypothalamic vasoactive intestinal polypeptide in the maintenance of prolactin secretion in incubating bantam hens: Observations using passive immunization, radioimmunoassay, and immunocytochemistry. J. Endocrinol. 122, 5–13. Silver, R. (1984). Prolactin and parenting in the pigeon family. J. Exp. Zool. 232, 617–625. Silverin, B., and Goldsmith, A. (1997). Natural and photoperiodically induced changes in plasma prolactin levels in male great tits. Gen. Comp. Endocrinol. 105, 145–154. Schoech, S. J., Ketterson, E. D., and Nolan, V., Jr. (1999). Exogenous testosterone and the adrenocortical response in the dark-eyed junco, Junco hyemalis. Auk, in press. Schoech, S. J., Ketterson, E. D., Nolan, V., Sharp, P. J., and Buntin, J. D. (1998). The effect of exogenous testosterone on parental behavior,
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Maney et al. plasma prolactin, and prolactin binding sites in dark-eyed juncos. Horm. Behav. 34, 1–10. Schoech, S. J. (1996). The effect of supplemental food on body condition and the timing of reproduction in a cooperative breeder, the Florida scrub-jay. Condor 98, 234–244. Schoech, S. J., Mumme, R. L., and Wingfield, J. C. (1996). Prolactin and helping behavior in the cooperatively breeding Florida scrub-jay (Aphelocoma coerulescens). Anim. Behav. 52, 445–456. Tong, Z., Pitts, G. R., Foster, D. N., and El Halawani, M. E. (1997). Transcriptional and posttranscriptional regulation of prolactin during the turkey reproductive cycle. J. Mol. Endocrinol. 18, 223–231. Vleck, C. M., and Patrick, D. J. (1999). Effects of vasoactive intestinal peptide on prolactin secretion in three species of passerine birds. Gen. Comp. Endocrinol., in press. Wingfield, J. C. (1994). Modulation of the adrenocortical response to stress in birds. In ‘‘Perspectives in Comparative Endocrinology’’ (K. G. Davey, R. E. Peter, and S. S. Tobe, Eds.), pp. 520–528. National Research Council Canada, Ottawa. Wingfield, J. C., and Farner, D. S. (1993). Endocrinology of reproduction in wild species. In ‘‘Avian Biology’’ (D. S. Farner and J. R. King, Eds.), Vol. 9, pp. 163–327. Academic Press, London. Wingfield, J. C., Maney, D. L., Breuner, C. W., Honey, P., Jacobs, J. D., Lynn, S., Ramenofsky, M., and Richardson, R. D. (1998). Ecological basis of hormone-behavior interactions: The ‘‘emergency life history stage.’’ Am. Zool. 38, 191–206. Youngren, O., Chaiseha, Y., Phillips, R., and El Halawani, M. (1996). Vasoactive intestinal peptide concentrations in turkey hypophysial portal blood differ across the reproductive cycle. Gen. Comp. Endocrinol. 103, 323–330.
Effects of Vasoactive Intestinal Peptide on Plasma Prolactin in Passerines Donna L. Maney,1 Stephan J. Schoech,* Peter J. Sharp,† and John C. Wingfield Department of Zoology, Box 351800, University of Washington, Seattle, Washington 98105; *Department of Biology and Center for the Integrative Study of Animal Behavior, Indiana University, Bloomington, Indiana 47405; and †Roslin Institute, Roslin, Midlothian EH25 9PS, United Kingdom Accepted November 2, 1998
Vasoactive intestinal peptide (VIP) is a potent releaser of prolactin (PRL) in domestic fowl, turkey, and ring doves. However, few comparative studies have investigated this in wild species. We tested the effects of intravenously administered chicken VIP on plasma PRL concentrations in four passerine species: the white-crowned sparrow (Zonotrichia leucophrys gambelii), the dark-eyed junco (Junco hyemalis), the Florida scrub-jay (Aphelocoma coerulescens), and the western scrub-jay (A. californica). In the white-crowned sparrow, junco, and Florida scrub-jay, which were tested during the breeding season, VIP induced a rapid increase in plasma PRL. Serial plasma samples taken after VIP injection in the white-crowned sparrow show a 10-fold increase in PRL within 2 min of treatment, followed by a gradual decline. Effects of VIP, as compared to saline, remained significant for at least 20 min after treatment. Western scrub-jays did not respond to intravenous VIP with a significant rise in PRL secretion, possibly because they were tested after termination of the breeding season. This study indicates that VIP control of PRL release may be widespread among avian species, and that seasonal changes in plasma PRL may be mediated in part at the level of the pituitary. In addition, analysis of the control data revealed no increase in plasma PRL as a result of injection or restraint, suggesting that unlike in mammals, PRL is not released during acute stress in passerines. r 1999 Academic Press
Key Words: prolactin; vasoactive intestinal peptide; avian; releasing factor. In birds, the pituitary hormone prolactin (PRL) is involved in many aspects of reproductive physiology and behavior. It is widely thought to play a role in parental behavior by mediating increases in incubation, crop milk secretion, feeding of young, and nest defense (Silver, 1984; Janik and Buntin, 1985; Lea et al., 1986; Buntin et al., 1991). After eggs are laid, PRL may inhibit GnRH and thus facilitate gonadal regression (Rozenboim et al., 1993). PRL is also thought to play a role in postnuptial molt at the termination of breeding in passerines (Dawson and Sharp, 1998). Secretion of PRL follows a highly seasonal pattern in most avian species studied (e.g., Hiatt et al., 1987). Increasing day length in spring triggers a slow rise in plasma PRL, which then increases sharply as parental behavior commences. In passerines a surge of PRL accompanies the onset of photorefractoriness, which is characterized by the termination of breeding and hypothalamic insensitivity to long day length (see Nicholls et al., 1988, for review). In domestic fowl (Gallus domesticus), turkey (Meleagris gallopavo), and ring doves (Streptopelia risoria), PRL release is controlled by vasoactive intestinal peptide (VIP), a hypothalamic releasing factor (Macnamee et al., 1986; Lea and Vowles, 1986; Opel and Proudman, 1988; El Halawani et al., 1990). Until recently, the effects of VIP on PRL secretion
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Current address: Department of Psychology, Johns Hopkins University, Baltimore, MD 21218. 0016-6480/99 $30.00 Copyright r 1999 by Academic Press All rights of reproduction in any form reserved.
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had not been tested in a passerine or a wild avian species. Dawson and Sharp (1998) found an inhibition of PRL secretion after immunization against VIP in European starlings (Sturnus vulgaris). Here we report that VIP acts as a PRL releasing factor in three other wild passerines: white-crowned sparrows (Zonotrichia leucophrys gambelii), Florida scrub-jays (Aphelocoma coerulescens), and dark-eyed juncos (Junco hyemalis). Our results are consistent with those of Vleck and Patrick (1999), who report that intravenous VIP elevates plasma PRL significantly within minutes of treatment in blue jays (Cyanocita cristata), zebra finches (Poephilla guttata), and Mexican jays (A. ultramarina). Because the protocol to test the effects of VIP on PRL release requires that animals be restrained, we were also able to test the effects of capture and handling stress on PRL secretion.
MATERIALS AND METHODS White-Crowned Sparrows (Zonotrichia leucophrys) Male Z. l. gambelii were captured during their autumnal migration in Central Washington State in 1994 and 1995. They were maintained in outdoor aviaries for 2 months before being transferred to individual cages in an environmental chamber with controlled light and temperature (20°C). Sex was determined by laparotomy prior to transfer. Day length in the chamber approximated a natural winter photoperiod (8 L:16 D) while the birds acclimated to captive conditions. After 1 week, the light cycle was changed to long days (20 L:4 D) to simulate conditions during breeding in the Arctic. Injections began 30 days after transfer to long days, at which time gonadal development reaches its peak (see Wingfield and Farner, 1993). Immediately prior to control or VIP injection, a blood sample was taken by alar venipuncture to determine baseline PRL. Birds then received an intravenous (jugular) injection of 0, 50, 100, or 500 ng chicken VIP (chVIP; Peninsula Laboratories, Belmont, CA) in 20 µl saline. Additional blood samples were taken at 2, 5, 10, and 20 min after injection. These doses correspond approximately to 2, 4, and 20 µg/kg. There were eight birds in each dosage group, and each bird was injected only once. Birds
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were held in a cloth bag during the intervals between samples. Once the first bird was removed from the chamber for sampling, the chamber was entered approximately every 6 to 15 min until sampling was completed. The time of sampling with respect to when the chamber door was first opened was carefully recorded. Regression analysis revealed no relationship between initial preinjection PRL levels and the time since the chamber door was first opened (r2 ⫽ 0.006, P ⫽ 0.72). Samples were kept cool on ice until they were centrifuged and the plasma harvested. Plasma samples were stored at ⫺20°C until PRL assay in the laboratory of J.C.W. (see below).
Dark-Eyed Juncos (Junco hyemalis) Juncos were captured during April and May of 1997 in the immediate vicinity of Mountain Lake Biological Station in western Virginia (for a more complete description of the study site see Ketterson et al., 1990; Schoech et al., 1999). All birds were free-living at the time of treatment; they were captured in the field and held temporarily before being released at the capture site. Treatment occurred 1–3 weeks prior to nest initiation. All birds were captured singly in mist nets and removed as soon as possible. Nine juncos were injected intravenously with 1000 ng chVIP (approximately 50 µg/kg) in 25 µl saline, and six were injected with saline only. Blood samples were collected immediately prior to injection and at 15 min after. In all cases the initial preinjection sample was collected between 1 and 2 min of the time the bird first entered the net. Plasma samples were shipped to the United Kingdom for PRL assay in the laboratory of P.J.S. (see below).
Florida Scrub-Jays (Aphelocoma coerulescens) During March and April of 1997, Florida scrub-jays were trapped at Archbold Biological Station in southcentral Florida (see Schoech, 1996, for details). At this time, the jays were at the incubation stage of their reproductive cycle. Individual jays were captured in continuously monitored Potter traps. Eight jays were injected with 1000 ng chVIP (approximately 13 µg/kg) in 50 µl saline, and six jays received saline only. Blood samples were collected immediately prior to and at 5 and 15 min following injection. In all cases, birds were removed from the traps and the initial sample col-
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lected within 1 min of time of capture. All birds were free-living at the time of treatment and were released shortly after the last blood sample was collected. Plasma samples were shipped on dry ice to the United Kingdom for PRL assay in the laboratory of P.J.S. (see below).
Western Scrub-Jays (Aphelocoma californica) Western scrub-jays were captured in Sunland, California, in July of 1994 and transported to Seattle, Washington. They were housed in an outdoor aviary under ambient conditions and given ad libitum food (peanuts and dog food) and water until treatment in August of 1994. Twelve jays received an intravenous injection of 500 ng chVIP (approximately 6 µg/kg) in 20 µl saline, and 12 jays received saline only. Blood samples were collected immediately prior to and at 5, 10, and 20 min after injection. When tested, each individual was removed from the aviary and an initial sample collected within 2 min of entering the aviary. Upon completion of treatment (i.e., injection with saline or VIP and collection of serial samples), the jay was returned to the aviary and another individual was captured. The nearly 30 min that passed between visits to the aviary to release one jay and capture the subsequent one was most likely sufficient for any hormonal effects of our previous visit to pass. Samples were kept cool on ice until they were centrifuged and the plasma harvested. Plasma samples were stored at ⫺20°C until PRL assay in the lab of J.C.W. (see below).
PRL Assay Z. leucophrys and A. californica plasma PRL levels were determined by a postprecipitation, double antibody radioimmunoassay (RIA) using purified chicken PRL as a standard and an anti-serum against PRL raised in rabbits. Both standard and antiserum were generously donated by Dr. A. F. Parlow (Pituitary Hormones and Antisera Center, UCLA-Harbor Medical Center, Torrance, CA). We have shown previously that this assay can be used to determine plasma PRL levels in Aphelocoma jays (Schoech et al., 1996). Because we had not measured PRL levels in sparrows, we validated the assay for Z. leucophrys by comparing inhibition curves for chicken standard and pooled white-crowned sparrow plasma (see below, Fig. 1).
A. coerulescens and J. hyemalis PRL levels were assayed in the laboratory of P.J.S. using antisera against recombinant-derived starling PRL (Bentley et al., 1997). Inhibition curves for plasma of both species are parallel to the standard curve in this assay, which has been used previously to measure PRL levels in J. hyemalis (Schoech et al., 1998). Although the absolute levels of PRL determined by this assay usually exceed those determined by the Parlow PRL assay, relative differences in PRL levels are well conserved (see Maney et al., 1999). The two assays have similar coefficients of variation; interassay variation is approximately 15 and 16% for the Parlow and starling assays, respectively, and intraassay variation is approximately 10 and 8%, respectively.
Statistical Analysis PRL levels were normalized by dividing each posttreatment level by the pretreatment level. Data were then log-transformed to reduce heteroscedasticity. Most analyses consisted of a two-way repeated measures ANOVA with time as the repeated measure and VIP dose as the independent variable. If this test showed significant effects (P ⬍ 0.05), post hoc tests (F tests followed by Scheffe tests or t tests) were used to examine within- and between-treatment differences. A difference was considered significant if the P value remained below 0.05 after correction for the number of comparisons. Since PRL was measured at only one posttreatment time in the junco study, these data were analyzed by unpaired t test only. To test the effects of restraint stress on PRL secretion, we also looked for an effect of time on PRL levels following saline injection. Analyses were performed using a one-way repeated measured ANOVA followed by Scheffe tests where appropriate, with the exception of the junco data, which were analyzed using paired t tests.
RESULTS Radioimmunoassay Validation White-crowned sparrow plasma inhibited the binding of radiolabeled chicken PRL to the chicken PRL
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antibody (Fig. 1). The near-parallel dilution curves (F1,11 ⫽ 1.064, P ⫽ 0.324, test for homogeneity of slopes) of chicken standard and white-crowned sparrow plasma indicate that this assay provides accurate measurement of relative PRL levels in Z. leucophrys. In addition, we have used this assay to quantify photoinduced PRL secretion in three subspecies of Z. leucophrys (Maney et al., 1999). PRL levels during photostimulation follow a typical avian pattern when measured by this assay. In this study, PRL for both whitecrowned sparrows and western scrub-jays fell within the physiological range reported for white-crowned sparrows and Aphelocoma jays when using this assay (Maney et al., 1999; Schoech, 1996; Schoech et al., 1996). The junco and Florida scrub-jay samples were measured with the recombinant starling PRL assay, which measures PRL levels in these species as approximately one order of magnitude higher than levels in the species measured with the Parlow PRL assay. Both assays reliably detect similar relationships among samples measured by both methods (Maney et al., 1999). The levels reported here are similar to those previously obtained using the recombinant starling
FIG. 1. Standard curve for chicken PRL (chPRL) and the inhibition curve from pooled white-crowned sparrow plasma. The standard curve shows displacement of 125I-labeled chPRL by the reference standard. The near-parallel dilution curve from white-crowned sparrow plasma indicates that this RIA can be used to assess relative levels of plasma PRL in this species (F1,11 ⫽ 1.064, P ⫽ 0.324, test for homogeneity of slopes).
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Maney et al.
PRL assay for European starlings (Bentley et al., 1997), juncos (Schoech et al., 1998), and white-crowned sparrows (Maney et al., 1999).
Intravenous VIP Injections In photostimulated white-crowned sparrows, intravenous VIP injection significantly affected plasma PRL (Fig. 2A; effect of dose, F ⫽ 9.505, P ⫽ 0.0002; effect of time, F ⫽ 6.164, P ⫽ 0.0008; interaction, F ⫽ 1.953, P ⫽ 0.0558). The higher two doses of VIP significantly elevated plasma PRL, compared to saline treatment, within 2 min of injection (for 100 ng, F ⫽ 5.068, P ⫽ 0.026; for 500 ng, F ⫽ 5.68, P ⫽ 0.0152). This effect was still significant at 5 min following treatment (for 100 ng, F ⫽ 4.776, P ⫽ 0.034; for 500 ng, F ⫽ 5.403, P ⫽ 0.0192). At 10 min after injection, 100 ng VIP continued to elevate plasma PRL compared with saline (F ⫽ 5.748, P ⫽ 0.0144), and with 50 ng VIP (F ⫽ 4.882, P ⫽ 0.0308), whereas the effect of 500 ng VIP was no longer significant. One hundred nanograms of VIP continued to elevate plasma PRL compared with saline at 20 min after treatment (F ⫽ 5.203, P ⫽ 0.0232). In dark-eyed juncos (J. hyemalis), plasma PRL was significantly higher 15 min after injection with 1000 ng VIP than after saline injection (Fig. 2B; unpaired t test, t ⫽ ⫺2.997, P ⫽ 0.0103). In Florida scrub-jays (A. coerulescens), injection of 1000 ng VIP significantly increased plasma PRL (Fig. 2C; effect of treatment, F ⫽ 13.545, P ⫽ 0.0031; effect of time, F ⫽ 15.931, P ⫽ 0.0018; interaction, F ⫽ 0.274, P ⫽ 0.6104). Plasma PRL increased significantly within 5 min of VIP injection compared with saline injection (t ⫽ ⫺3.663, P ⫽ 0.0066). This effect remained significant at 15 min following treatment (t ⫽ ⫺3.45, P ⫽ .0096). In Western scrub-jays (A. californica) there was no effect of VIP injection on plasma PRL (Fig. 2D; effect of treatment, F ⫽ 0.428, P ⫽ 0.5198; effect of time, F ⫽ 0.761, P ⫽ 0.4735; interaction, F ⫽ 1.668, P ⫽ 0.2003).
Effects of Restraint Stress In the white-crowned sparrows, juncos, and western scrub-jays, there was no effect of time on PRL levels in saline-injected birds (white-crowned sparrow, F ⫽ 0.545, P ⫽ 0.704; junco, t ⫽ 1.821, P ⫽ 0.128; west-
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FIG. 2. Plasma PRL response to intravenous VIP or saline in several passerine species. Error bars represent SEM. Data are normalized as described under Materials and Methods. See text for statistics. chPRL-IR, chicken prolactin immunoreactivity; RSP-IR, recombinant starling prolactin immunoreactivity. (A) Captive white-crowned sparrows exposed to 30–50 days of photostimulation. (B) Free-living dark-eyed juncos injected during April and May. (C) Free-living Florida scrub-jays injected during March and April. (D) Captive western scrub-jays, on natural photoperiod, injected during August.
ern scrub-jay, F ⫽ 1.137, P ⫽ 0.349). In the Florida scrub-jay, saline injection and restraint significantly inhibited plasma PRL (ANOVA, F ⫽ 6.226, P ⫽ 0.016), which was apparent within 15 min of treatment (P ⬍ 0.02).
DISCUSSION In this study, intravenous VIP induced a rapid rise in plasma PRL in the white-crowned sparrow, the darkeyed junco, and the Florida scrub-jay (Figs 2A–2C). These results resemble those of Vleck and Patrick
(1999), who found that VIP increases plasma PRL significantly in blue jays, zebra finches, and Mexican jays. Our results are also consistent with those of Dawson and Sharp (1998), who reported a suppression of plasma PRL after immunization against VIP in starlings. The absolute levels we found appear to be physiological; in the white-crowned sparrow posttreatment PRL peaked at a level similar to that which is seen at the onset of photorefractoriness (see Maney et al., 1999). Our results suggest that VIP control of PRL secretion may be widespread among birds. Our most detailed investigation was performed using white-crowned sparrows (Fig. 2A). In this species, the lowest dose of VIP (50 ng) did not significantly
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affect PRL levels. However, the 100 and 500 ng doses increased plasma PRL within 2 min of treatment. This rapid effect is consistent with the results of Macnamee et al. (1986), who report stimulation of PRL secretion within 2 min of VIP injection in the bantam hen. Figure 2A suggests that 500 ng VIP may have increased plasma PRL more than 100 ng VIP, but this apparent dose dependence was not significant. We used relatively low doses of VIP compared to those reported to stimulate PRL secretion in other species (Lea and Vowles, 1986; Vleck and Patrick, 1999), perhaps administering more VIP would elicit a dose-dependent response. One VIP dose was given and fewer blood samples were collected in the studies of other species. One thousand nanograms of VIP increased plasma PRL in juncos (Fig. 2B) and Florida scrub-jays (Fig. 2C) at the earliest time point analyzed (15 min for the juncos and 5 min for the jays). Earlier sampling from the juncos may have revealed a stronger PRL response. Figure 2D suggests that in the western scrub-jay, VIP injection may have affected PRL secretion slightly at 5 min after treatment. However, this effect was not significant. The lack of an effect in this species may be explained by the time of year and a seasonal insensitivity to VIP at the level of the pituitary. This hypothesis is supported by data from turkeys and juncos. In turkeys, the concentration of VIP in hypothalamopituitary portal blood is the same in laying and nonreproductive hens, despite nonreproductive birds having lower plasma levels of PRL (Youngren et al., 1996). Exogenous VIP does stimulate PRL release in nonreproductive hens, although significantly less so than during laying or incubation (Pitts et al., 1996). In juncos, despite evidence of seasonal differences in circulating plasma levels of PRL (Ketterson et al., 1990), Saldanha et al. (1994) found no differences in hypothalamic content of VIP between long- and short-day birds. The evidence that VIP is a PRL-releasing factor in juncos (Fig. 2B) and bantam hens (Macnamee et al., 1986), combined with the lack of seasonal differences in VIP despite seasonal differences in PRL suggests that pituitary lactotrophs may be insensitive to VIP in the nonbreeding season. The mechanism of this insensitivity could involve either a downregulation of pituitary VIP receptors or an inhibition of PRL gene transcrip-
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Maney et al.
tion, synthesis, or storage. For example, PRL mRNA half-life decreases significantly in unphotostimulated turkeys compared with laying or incubating turkeys (Tong et al., 1997). Seasonal modulation of lactotroph sensitivity to VIP could be mediated by reproductive hormones. Photostimulated turkeys, starlings, and great tits (Parus major) that have low circulating sex steroids as a result of gonadectomy respond to sex steroid replacement with an increase in circulating PRL (El Halawani et al., 1983; Dawson and Goldsmith, 1984; Silverin and Goldsmith, 1997). Additionally, turkey pituitary cells in vitro release more PRL in response to VIP when the culture medium contains gonadal steroid hormones (Knapp et al., 1988). Thus, it is possible that the lack of PRL response in western scrub-jays is attributable to the phase in the reproductive cycle during which the experiment was conducted. However, since ovariectomized turkeys respond to VIP injection with a significant rise in PRL (Opel and Proudman, 1988), we might expect a blunted rather than absent response in nonbreeding jays. Experiments measuring responses to VIP injection at different times of year in the same species are needed. Our protocol for taking serial blood samples before and after VIP injection involves restraining the birds in a cloth bag between sampling. This treatment increases glucocorticoid secretion in many species of passerine (e.g., Wingfield 1994, Wingfield et al., 1998), and has often been used to study the endocrine response to stress. In the sparrows, juncos and western scrub-jays, there was no effect of capture and handling on PRL secretion in animals receiving saline. The Florida scrub-jays, however, decreased their plasma PRL, opposite from what would be expected were PRL involved in the stress response in this species. This is consistent with Schoech et al. (1996), who report a negative correlation between plasma PRL and time after capture in Florida scrub-jays. PRL levels increase rapidly during restraint stress in mammals, and PRL is often considered part of the classical stress response (see de Vlaming, 1979, for review). A role for PRL in the stress response of birds, however, is less clear; for example, the stress of receiving an injection and subsequent blood sampling may decrease PRL in bantam hens (see Sharp et al., 1989). The effects of stress on PRL secretion may
VIP and PRL in Passerines
depend on reproductive state. Opel and Proudman (1986) report that PRL increases in most turkeys subjected to restraint stress; however, incubating hens show a reduction in PRL levels. One might conclude that these results are analogous to ours from Florida scrub-jays, which were also sampled mostly during incubation (see Fig. 2C). However, unlike in the turkey studies, the jay sample was composed of individuals of both sexes and status (i.e., breeders and nonbreeders) in a species in which only female breeders incubate. Adding further to the uncertainty of PRL’s role in the avian stress response, Delehanty et al. (1997) compared interyear differences in the effects of environmental stressors on PRL titers. In a year of drought and extreme high temperatures, incubating male Wilson’s phalaropes (Phalaropus tricolor) had lower PRL levels than in other years. Based on rates of weight loss and nest abandonment, the researchers concluded that the phalaropes were stressed. Together with our results, these studies suggest that the stress response in birds may differ from mammals with regard to PRL.
ACKNOWLEDGMENTS We thank A. F. Parlow (Pituitary Hormones and Antisera Center, UCLA-Harbor Medical Center, Torrance, CA) for his generous donation of chicken PRL antiserum and standard. We are grateful to Lynn Erckmann for expert animal care, to Aaron Kim, Chris Goode, and Pete Wilson for technical assistance, and to Nancy Temkin for help with statistical analysis. Thanks as well to Marty Morton and Maria Pereyra for field assistance and accommodations. This work was supported by NSF Grants DCB-9005081, IBN-9408013, and IBN-9631350 to J.C.W. and IBN-9224397 to S.J.S.
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