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Steroids and sexual differentiation of the songbird brain Chin, Eunice Hannah 2006

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STEROIDS AND SEXUAL DIFFERENTIATION OF THE SONGBIRD BRAIN by Eunice Hannah Chin BSc, Simon Fraser University, 2004 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF SCIENCE (Zoology) THE UNIVERSITY OF BRITISH COLUMBIA December 2006 © Eunice Hannah Chin, 2006 A B S T R A C T Sexual differentiation of the songbird brain has traditionally thought to be driven by gonadal testosterone (T) and estradiol (E2). Recent studies have indicated that other sources of sex steroids may be responsible. Moreover, these alternative sources may release precursors, such as dehydroepiandrosterone (DHEA), which are subsequently converted downstream to sex steroids, such as E2, only at target tissues. This thesis examines plasma and tissue hormone profiles during development and possible alternative sources of sex steroids, including the adrenals, brain and liver, during the nestling phase in the European starling (Sturnus vulgaris). In the first experiment, wild starling chicks were blood sampled from either the brachial wing vein or the jugular vein from hatch (PO) to fledging (P20). In the second experiment, brain and peripheral tissues were collected from wild starling chicks at PO, P6 and P8. Hormones were extracted from samples using solid phase extraction, and then measured using radioimmunassays. Plasma DHEA was higher in males than females at P4 in the brachial vein, whereas plasma DHEA was higher in females than males at PO and PIO in the jugular vein. No sex difference was found in plasma E2. Brain DHEA was low, but detectable, in all regions at all ages, and a sex difference was only detected at PO. Brain E2 was non-detectable in all regions at all ages. DHEA was detectable in all peripheral tissues, with the gonadal tissue containing the highest levels of DHEA. Taken together, these data suggest that the gonads may be the primary source of circulating DHEA, and that the adrenals may be a secondary source during the nestling phase. T A B L E O F C O N T E N T S Abstract ii Table of Contents iii List of Tables iv List of Figures v Acknowledgements vi Co-Authorship Statement vii Chapter One: Literature Review 1 References 12 Chapter Two: Brain and Plasma Steroid Levels in a Free-Living Developing Songbird 16 Tables 37 Figures 40 References 51 Chapter Three: General Discussion and Conclusions 54 References 56 iii LIST OF TABLES Table 2.1: Sample sizes for all groups for (a) 2005 and (b) 2006 experiments 37 Table 2.2: Changes in hematocrit over the nestling phase in juvenile European starlings with sexes combined 38 Table 2.3: Number of detectable samples for E2 in (a) plasma and (b) brain tissue in juvenile European starlings 39 LIST OF FIGURES Figure 2.1: Pathways of steroid synthesis 40 Figure 2.2: Changes in body mass during development in European starlings 41 Figure 2.3: Steroid extraction from juvenile starling plasma 42 Figure 2.4: Steroid extraction from juvenile starling brain tissue 43 Figure 2.5: Estimating recovery of steroids from avian plasma using SPE 44 Figure 2.6: Estimating recovery of steroids from avian brain tissue using SPE 45 Figure 2.7: DHEA levels in juvenile starlings from hatch (0) to fledging (20) 46 Figure 2.8: 17f3-E2 levels in juvenile starlings from hatch (0) to fledging (20) 47 Figure 2.9: E2/DHEA ratios in juvenile starlings at each age sampled 48 Figure 2.10: Brain DHEA levels in juvenile starlings 49 Figure 2.11: Periphery DHEA levels in juvenile starlings 50 v ACKNOWLEDGEMENTS This thesis could not have been completed without the support and love of many people. Firstly, I would like to thank the National Sciences and Engineering Council of Canada (NSERC) for funding for this project. Secondly, I would like to thank my senior supervisors, Kiran Soma and Colin Brauner, for their encouragement and mentorship throughout the entire process of this thesis. I am also indebted to all the members of the Soma Lab, including Amy Newman, Thierry Charlier, Devaleena Pradhan, Amit Shah, Kelvin Po, Kim Schmidt and Lani Sheldon for all their help in the lab, the field and during the writing process of this thesis. I also wish to express my gratitude to my colleagues Oliver Love, Katrina Salvante and Tony Williams for guidance, teaching and assistance in field work - and essentially establishing a good beginning to my scientific career. To my friends outside the lab, particularly Jennifer and Kevin Striemer, Tiffany Lee, Danica Longair, Matthew Williams and Jonathan Trivett - thank you for your continued support and love. Lastly, I'd like to thank my parents, Michael and Wai Bing Chin, whom, without their love, support and continued encouragement, completion of this thesis would not have been possible. C O - A U T H O R S H I P S T A T E M E N T While the bulk of the field work and lab work for this thesis was completed by myself, E.H. Chin, the following co-authors are listed on the manuscript: Kimberly L. Schmidt, Lani D. Sheldon, Amit H. Shah, and Oliver P. Love. Both K.L. Schmidt and L.D. Sheldon assisted in field work by collecting samples, and performing daily nestbox checks and collecting growth data. Furthermore, both individuals assisted in lab work: K.L. Schmidt assisted in the bulk of the SPE extractions for both the plasma and brain tissue samples, while L.D. Sheldon assisted in genotyping. A.H. Shah assisted in the rest of the SPE extractions for the brain tissue and periphery tissue samples. O.P. Love assisted in fieldwork also by collecting samples, performing daily nestbox checks, and assisting in collection of growth data. Chapter One: Literature Review INTRODUCTION Sex steroids during development can have profound and long-lasting effects on brain, behaviour and physiology. During a critical or sensitive period, sex steroids can have organizational effects on the brain, and subsequently the behaviour controlled by affected brain regions. Presence or absence of sex steroids during this critical period can affect development, pushing brain and behaviour towards either a male or female direction. These organizational effects of steroids are often permanent and irreversible. In mammals, it is well established that the gonads are the source of sex steroids during the neonatal stage, and removal of this gonadal source during development leads to a profound change in behaviour at the adult stage of life. Guinea pigs exposed to testosterone propionate (TP) in utero will show more masculine copulatory behaviours and less feminine copulatory behaviours in adulthood compared to unexposed females (Phoenix, Goy, Gerall, and Young, 1959). In birds, circulating sex steroids during development have also been examined in precocial and altricial species. Evidence supports the gonads as the source of sex steroids in precocial species such as the Japanese quail (Coturnix japonica) and the chicken (Ottinger, Pitts, and Abdelnabi, 2001; Sayag, Snapir, Anion, Halawani, Grimm, and Robinzon, 1991). However, the source of sex steroids during development in altricial birds, particularly songbirds, is not clear. This review will examine developmental studies of circulating sex steroids in both mammalian and avian species, investigate the possible sources of these hormones, and how these sources differ between these species. 1 Mammals Several studies have looked at sex steroid levels during development in mammals, particularly in rats, monkeys and hyenas. In rats, both brain and circulating plasma levels of steroids have been examined, but mainly in the first few hours post-partum, rather than throughout development. One study examined progesterone (PROG), testosterone (T), 5a-dihydrotestosterone (5a-DHT) and 17P-estradiol (E2) levels in serum and the hypothalamus between embryonic day 21 (E21) to 24 hours post-partum (Rhoda, Corbier, and Roffi, 1984). There was no sex difference in serum and hypothalamic PROG. In males, serum and hypothalamic T levels surge 1-2 hours post-partum, and subsequently decline to levels seen at birth. At no point was T detectable in female serum or hypothalamus. 5a-DHT was not detectable in the serum or hypothalamus in either sex. There was no sex difference in serum E2, but males had higher hypothalamic levels of E2 than females throughout all the stages of life examined. Another study examined brain E2 content in newborn rats, focusing on regional differences. Immediately post-partum, males had higher E2 levels in the frontal cortex, hypothalamus and pre-optic area than females (Amanteau, Alt, Stamps, and McCarthy, 2004). By post-partum day 1 (PI), E2 levels had declined such that only the hypothalamus maintained a sex difference. In another study, serum levels of 17-OH Pregnenolone (17-OH PREG), dehydroepiandrosterone (DHEA), 17-OH PROG and androstenedione (AE) were examined in rats from birth to P98. Blood was taken from the abdominal aorta. There was no sex difference in any of the steroids measured, but all steroids decreased significantly with increasing age (Tagawa, Katagiri, and Kobayashi, 2005). At no time during any of these studies did females have higher steroid levels than males. 2 Two studies in monkeys have examined plasma and serum concentrations of sex steroids. The first study was in male crab-eating monkeys (Macaca fascicularis) from birth to adulthood. Levels of DHEA, AE, 5a-DHT, and T were measured in plasma from the cephalic, basilic or saphenous vein depending on the age of the subjects. DHEA levels were high after birth, decreased at 1 year of age, and remained stable until puberty, when levels declined further (Meusy-Dessolle and Dang, 1985). AE remained constant throughout life with a slight decrease at 3 years of age, but then increased again after 4-5 years. T and 5a-DHT remained low until puberty, when they increased in parallel and concommittantly with the establishment of spermatogenesis, and continued to increase to adulthood. Another study examined levels of AE, T, and 5<x-DHT in umbilical circulation of fetal Rhesus monkeys (Macaca mulatto) beginning at the time of gonadal differentiation. AE was higher in males than females on 79 - 84 days of gestation (Resko, Ellinwood, Pasztor, and Buhl, 1980). T was significantly higher in males than females in all gestational stages measured, and 5a-DHT was higher in males than females on 100 - 133 days of gestation as well as 140 - 163'days of gestation. Non-passerine birds The majority of work examining circulating sex steroids in precocial birds has been done in chickens (Gallus domesticus) and Japanese quail (Coturnix japonica). Work in these two species has been done in both pre- and post-hatch chicks, and has demonstrated that sex steroids are present in plasma in detectable levels in embryos as well as post-hatch chicks. 3 In chickens, plasma T levels were detectable in both male and female chicks. Male chicks had relatively high pre-hatch T levels in plasma obtained via cardiac puncture, and these levels began to increase P21 to a peak at P35 (Tanabe, Nakamura, Fujioka, and Doi, 1979). While one study reported a sex difference in plasma T from days 7 to 17 of incubation in blood collected from extraembryonic blood vessels (Woods, Simpson, and Moore, 1975), a separate study showed no sex difference in plasma T levels at embryonic ages in blood collected via cardiac puncture or venous vessels (Tanabe, Saito, and Nakamura, 1986). Meanwhile, plasma E2 levels in females (from cardiac puncture) were 10 times lower than T levels from day 17 of embryonic development (El7) to P42, while levels in male chicks were very low or non-detectable (Tanabe et al., 1979). Evidence in chickens suggests that gonads are the source of steroids both pre-and post-hatch (Galli and Wassermann, 1973; Guichard, Cedard., Mignot, Scheib, and Haffen, 1977; Tanabe et al., 1979; Woods and Erton, 1978). Early endocrinology experiments by Berthold in 1848 demonstrated that early castration of male chickens resulted in adult male chickens that were not masculinized (Nelson, 2000). However, evidence also suggests that adrenal glands are also steroidogenic in the embryonic chick. Tanabe et al (1979) measured steroids in adrenal tissue only, and discovered adrenals were capable of secreting T and E2 as well as corticosterone (Tanabe et al., 1979). It is suggested that post-hatch, adrenal glands become more specialized, producing mainly corticosteroids rather than sex steroids. Additionally, work has shown that the post-hatch chick brain contains steroids (Migues, Johnston, and Rose, 2002), suggesting that the brain may also be steroidogenic. 4 In Japanese quail, one study examined serum concentrations of testosterone, 5a-DHT, E2 and PROG in the second half of embryonic life (E9-17 of incubation) and during the first 5 weeks after hatching. Embryos were blood sampled from a large blood vessel in the egg membranes, while hatchlings were blood sampled via decaptitation. A sex difference was detected in plasma E2 over most of the age range studied (Schumacher, Sulon, and Balthazart, 1988). T peaked on El 5 in females and was significantly higher than in males. In males, T did not peak until 1 week post-hatch, and was significantly higher than in females (Schumacher et al., 1988). Another study examining sex steroids during quail development also found sex differences in androgens and E2 in blood from the chorioallantoic vein. Males had higher androgens than females at all ages sampled except for the day of hatch, where females had higher androgen levels (Ottinger et al., 2001). In contrast, plasma E2 levels were higher in females on all days sampled. Studies have also examined circulating sex steroids in each sex specifically. In females, plasma E2 in the chrorioallantoic vein remained relatively constant in the embryo, followed by a sharp decrease post-hatch in blood collected via decapitation (trunk blood) (Abdelnabi, Bakst, Woods, and Ottinger, 2000). In males, plasma androgens were elevated on E8, declined, and rose again at E l 3 and remained elevated until P3. Levels then subsequently remained low until after PI8 (Ottinger and Bakst, 1981). It has been suggested that the source of circulating steroids in quail are the gonads (Ottinger et al., 2001), but there is also evidence that the adrenals are also steroidogenic in quail (Ottinger et al., 2001). Males had higher androgen levels in gonads and adrenals than females. Male adrenal glands had higher levels than the testes (Ottinger et al., 5 2001). In contrast, females had higher E2 content in adrenals and gonads compared to males (Ottinger et al., 2001). These results are consistent with the results obtained in the chicken. Other studies examining sex steroid levels during development in non-passerine birds are in Shao ducks, white storks, and greylag geese. In Shao ducks, blood samples were collected from ducklings at PI , PI5 and P35, and then subsequently at 20 day intervals from the wing vein and analysed for T and E2. Plasma T in males increased throughout the experimental period, while plasma E2 in females increased to a peak at PI35, and then declined afterwards (Yang, Medan, Watanabe, and Taya, 2005). However, no sex differences in circulating sex steroids were assessed. In another study of white storks (Ciconia ciconia L.), plasma T levels were measured on P5 and PI 5. T was higher on PI5 than P5, but data were not analysed for sex differences (Sasvari, Hegyi, and Peczely, 1999). In greylag geese (Anser anser), excreted metabolites of gonadal steroids were measured. Samples were collected from P10 to P80. Androgen metabolites were significantly higher during the first 20 days of life than during the last 60 days before fledging, while estrogen metabolites during the first 20 days of life were not different than the last 60 days before fledging (Frigerio, Moestl, and Kotrschal, 2001). Data were not analysed for sex differences. In summary, sex differences in steroids pre- and post-hatch have been characterized in non-passerine birds. Additionally, data suggest that the adrenals may be steroidogenic in development, and may contribute to steroids for sexual differentiation of brain and behaviour during the embryonic and post-hatch stages. 6 Passerine Birds Sex steroid levels during development in altricial birds have mainly been characterized in captive zebra finches (Taeniopygia guttata). In songbirds, there is a critical period during nestling development, usually during the first week of life (Adkins-Regan, Mansukhani, Seiwert, and Thompson, 1994). During this critical period, song nuclei are sensitive to sex steroids, and exposure to E2 leads to song nuclei development in males (Gurney and Konishi, 1980). One early study examined levels of A E , T, E 2 and 5a-DHT in jugular plasma of nestling birds, focusing specifically on the critical period in zebra finches (Hutchison, Wingfield, and Hutchison, 1984). Androgens in both sexes were high on hatch date (PO), but had declined by PIO. In contrast, plasma E2 was basal in male hatchlings, but increased substantially to a peak on P4. This peak resulted in a highly significant sex difference in plasma E2, as female E2 levels did not change considerably during development. Although no evidence was presented in this study, the authors suggested gonads or adrenals as possible sources for these sex steroids. However, since then, jugular blood has been shown to be enriched with neurally formed steroids (Schlinger and Arnold, 1993). Further work expanded on this study to include pre-hatch, the second week of life and beyond fledging. Adkins-Regan and colleagues (1990) examined circulating E2, T or 5a-DHT in developing zebra finches in blood from either trunk blood source, which is a mixture of venous (jugular) and arterial (carotid) blood, or blood from the brachial vein. There was no sex difference detected in circulating steroids during the first week of life (Adkins-Regan, Abdelnabi, Mobarak, and Ottinger, 1990). However, in the second week oflife a sex difference in E2 was detected on PI 2, with males having higher levels. 7 Another study in zebra finches examined circulating plasma steroids in pooled trunk and cardiac puncture blood during the nestling phase. Again, plasma AE, T, E2 estrone (Ei), and 5a-DHT levels were examined, as well as aromatase activity. Blood data were averaged over a range of ages (PI-3, P4-6, etc.). No sex difference was detected in any of the plasma steroids (Schlinger and Arnold, 1992). Moreover, there was no sex difference in aromatase levels in the brain (Schlinger and Arnold, 1992). Sex steroids have also been examined in free-living passerines. T levels were examined in nestling European starlings (Sturnus vulgaris) trunk blood. T increased steadily post-hatch to P8, then decreased towards the end of the nestling period (Williams, Dawson, Nicholls, and Goldsmith, 1987). Moreover, there gonads were small in the nestlings, suggesting that circulating T may be of non-gonadal origin. Another study examining sex steroids during the nestling phase of wild birds was in Great tits (Parsus major). T and E2 levels were examined in jugular plasma during the nestling phase. High levels of both steroids were present in newly hatched birds of both sexes, declining to basal levels at P3 and P2 for T and E2, respectively (Silverin and Sharp, 1996). A sex difference was only detected in plasma T on PO and PI (where PO is hatch day), with males having significantly higher plasma levels than females. Electron microscopy examination of male gonads only characterized spermatogonia at all ages examined. Exogenous gonadotropin releasing hormone (GnRH) treatment did not result in T release from the gonads. This further indirectly supports the idea that gonads are not the source of circulating T during development in songbirds. Plasma E2 levels were also measured in castrated and intact song (Melospiza melodia) and swamp sparrows (Melospiza georgiana) from hatch to P200 (Marler, 8 Peters, Ball, Dufty Jr., and Wingfield, 1988). Levels in castrated and intact birds were similar, suggesting that the source of E2 in these birds was non-testicular. A similar study in swamp sparrows only measured plasma T and E 2 from 26 days to 1 year of age, the sensitive period for song acquisition and development. T was elevated between 30 and 80 days of age, as well as 260 to 360 days of age, while plasma E2 was elevated from 18 to 170 days of age, corresponding with low T levels (Marler, Peters, and Wingfield, 1987). Androgen levels were measured in wild developing African black coucals (Centropus grillii), a species with sex-role reversal, as a possible mechanism for behavioural masculinization and subsequent aggression in females. Throughout the nestling phase, plasma androgen levels were higher in males than females (Goymann, Kempenaers, and Wingfield, 2005). Another study examined pied flycatchers (Ficedula hypoleuca) at P7. Levels of T were measured in the plasma, but the data were not analysed for a sex difference in T levels (Goodship and Buchanan, 2006). Alternative Sources for Sex Steroids During Songbird Development Taken together, these studies suggest that the gonads are not the source of circulating T during songbird nestling development. Moreover, manipulative studies in songbirds have suggested that gonadal steroids may not play a role in sexual differentiation of the songbird brain (Adkins-Regan and Ascenzi, 1990; Arnold, 1975; Wade and Arnold, 1996). The alternative sources may include: the adrenal glands, the liver, and the brain. 9 Support for steroid synthesis in the adrenals was documented in work done in adult non-breeding song sparrows (Soma and Wingfield, 2001). Detectable levels of the sex steroid precursor, dehydroepiandrosterone (DHEA) were found in plasma (~1 ng/mL), even when the gonads were regressed. DHEA was also detectable in the adrenals (-12 ng/g) and testes (~14 ng/g). DHEA could be released into the plasma and converted to T and E2 in tissues such as the brain, which possess the appropriate enzymes for these conversions (Soma, Alday, Hau, and Schlinger, 2004; Tarn and Schlinger, 2006; Vanson, Arnold, and Schlinger, 1996). Alternatively, the liver may also be a source of DHEA. Experiments in young rats has demonstrated that the liver expresses P450cl7, the enzyme that converts pregnenolone (PREG) to DHEA (Katagiri, Tatsuta, Imaoka, Funae, Honma, Matsuo, Yokoi, Ishimura, Ishibashi, and Kagawa, 1998; Vianello, Waterman, Dalla Valle, and Colombo, 1997). Circulating PREG in the bloodstream may be taken up by the liver and converted to DHEA, which is subsequently released into the bloodstream. Additionally, the brain is a potential source of steroids. Brain tissue may synthesize steroids de novo from cholesterol (Mellon and Griffin, 2002). Brain slices from P25 male zebra finches synthesized E2 in vitro with PROG added to the cell culture media (Holloway and Clayton, 2001). In addition, the developing avian brain possesses the appropriate enzymes to synthesize steroids (London, Monks, Wade, and Schlinger, 2006). These alternative sources may provide an explanation for the inconsistency in detecting sex differences in circulating sex steroids during development in previous studies. However, few studies mentioned above have directly measured steroids in 10 tissue, and no previous studies have directly measured steroids in songbird brain or peripheral tissue. OBJECTIVES The general objective of this thesis was to employ a comprehensive approach to examine possible sources of steroids (specifically D H E A and E2) during the nestling phase in songbirds. Using a nestbox breeding colony of free-living European starlings, I explored the following specific objectives: (1) To determine developmental changes in circulating steroids in songbird nestlings. (2) To determine sex differences in circulating sex steroids during the nestling phase. (3) To determine the source of circulating sex steroids during development by: a. Comparing hormones in brachial and jugular plasma and b. Measuring steroid levels in gonads, adrenals, liver and brain 11 REFERENCES Abdelnabi, M. A., Bakst, M. R., Woods, J. E., and Ottinger, M. A. (2000). Plasma 17-beta estradiol levels in ovarian interstitial cell structure in embryonic Japanese quail. Poultry Science 79, 564-567. Adkins-Regan, E., Abdelnabi, M., Mobarak, M., and Ottinger, M. A. (1990). Sex steroid levels in developing and adult male and female zebra finches (Poephila guttata). General and Comparative Endocrinology 78, 93-109. Adkins-Regan, E., and Ascenzi, M. (1990). Sexual differentiation of behavior in the zebra finch: effect of early gonadectomy or androgen treatment. Hormones and Behavior 24, 114-127. Adkins-Regan, E., Mansukhani, V., Seiwert, C , and Thompson, R. (1994). Sexual differentiation of brain and behavior in the zebra finch: critical periods for effects of early estrogen treatment. Journal of Neurobiology 25(7), 865-877. Amanteau, S. K., Alt, J. J., Stamps, C. L., and McCarthy, M. M. (2004). Brain estradiol content in newborn rats: sex differences, regional heterogeneity, and possible de novo synthesis by the female telencephalon. Endocrinology 145(6), 2906-2917. Arnold, A. P. (1975). The effects of castration on song development in zebra finches (Poephila guttata). Journal of Experimental Zoology 191, 261-278. Frigerio, D., Moestl, E., and Kotrschal, K. (2001). Excreted metabolites of gonadal steroid hormones and corticosterone in Greylag Geese (Anser anser) from hatchling to fledging. General and Comparative Endocrinology 124, 246-255. Galli, F. E., and Wassermann, G. F. (1973). Steroid biosynthesis by gonads of 7- and 10-day-old chick embryos. General and Comparative Endocrinology 21, 77-83. Goodship, N. M., and Buchanan, K. L. (2006). Nestling testosterone is associated with begging behaviour and fledging success in the pied flycatcher, Ficedula hypoleuca. Proceedings of the Royal Society of London B - Biological Sciences 113,1 \-l 6. Goymann, W., Kempenaers, B., and Wingfield, J. C. (2005). Breeding biology, sexually dimorphic development and nestling testosterone concentrations of the classically polyandrous African black coucal, Centropus grillii. Journal of Ornithology 146, 314-324. Guichard, A., Cedard., L., Mignot, T. H. M., Scheib, D., and Haffen, K. (1977). Radioimmunoassay of steroids produced by cultured chick embryonic gonads: differences according to age, sex and side. General and Comparative Endocrinology 32, 255-265. Gurney, M. E., and Konishi, M. (1980). Hormone-induced sexual differentiation of brain and behavior in zebra finches. Science 208(4450), 1380-1383. Holloway, C. C , and Clayton, D. F. (2001). Estrogen synthesis in the male brain triggers development of the avian song control pathway in vitro. Nature neuroscience 4(2), 170-175. Hutchison, J. B., Wingfield, J. C , and Hutchison, R. E. (1984). Sex differences in plasma concentrations of steroids during the sensitive period for brain differentiation in the zebra finch. Journal of Endocrinology 103, 363-369. Katagiri, M., Tatsuta, K., Imaoka, S., Funae, Y., Honma, K., Matsuo, N., Yokoi, H., Ishimura, K., Ishibashi, F., and Kagawa, N. (1998). Evidence that immature rat 12 liver is capable of participating in steroidogenesis by expressing 17alpha-hydroxylase/17,20-lyase P450cl7. Journal of Steroid Biochemistry and Molecular Biology 64(1/2), 121-128. London, S. E., Monks, D. A., Wade, J., and Schlinger, B. A. (2006). Widespread capacity for steroid synthesis in the avian brain and song system. Endocrinology 147(12), 5975-5987. Marler, P., Peters, S., Ball, G. F., Duffy Jr., A. M., and Wingfield, J. C. (1988). The role of sex steroids in the acquisition and production of birdsong. Nature 336, 770-772. • Marler, P., Peters, S., and Wingfield, J. C. (1987). Correlations between song acquisition, song production and plasma levels of testosterone and estradiol in sparrows. Journal of Neurobiology 18(6), 531-548. Mellon, S. H., and Griffin, L. D. (2002). Neurosteroids: biochemistry and clinical significance. Trends in Endocrinology and Metabolism 13(1), 35-43. Meusy-Dessolle, N., and Dang, D. C. (1985). Plasma concentrations of testosterone, dihydrotestosterone, delta 4-androstenedione, dehydroepiandrosterone and oestradiol-17beta in the crab-eating monkey [Macaca fascicularis) from birth to adulthood. Journal of Reproduction and Fertility 74, 347-359. Migues, P. V., Johnston, A. N. B., and Rose, S. P. R. (2002). Dehydroepiandosterone and its sulphate enhance memory retention in day-old chicks. Neuroscience 109(2), 243-251. Nelson, R. J. (2000). An Introduction to Behavioral Endocrinology. Sinauer Associates, Inc., Sunderland. Ottinger, M. A., and Bakst, M. R. (1981). Peripheral androgen concentrations and testicular morphology in embryonic and young male Japanese quail. General and Comparative Endocrinology 43, 170-177. Ottinger, M. A., Pitts, S., and Abdelnabi, M. A. (2001). Steroid hormones during embryonic development in Japanese quail: Plasma, gonadal, and adrenal levels. Poultry Science 80(6), 795-799. Phoenix, C. H., Goy, R. W., Gerall, A. A., and Young, W. C. (1959). Organizing action of prenatally administered testosterone propionate on the tissues mediating mating behavior in the female guinea pig. Endocrinology 65, 369-382. Resko, J. A., Ellinwood, W. E., Pasztor, L. M., and Buhl, A. E. (1980). Sex steroids in the umbilical circulation of fetal rehsus monkeys from the time of gonadal differentiation. Journal of Clinical Endocrinology and Metabolism 50(5), 900-905. Rhoda, J., Corbier, P., and Roffi, J. (1984). Gonadal steroid concentrations in serum and hypothalamus of the rat at birth: aromatization of testosterone to 17-beta estradiol. Endocrinology 114(5)," 1754-1760. Sasvari, L., Hegyi, Z., and Peczely, P. (1999). Brood reduction in white storks mediated through asymmetries in plasma testosterone concentrations in chicks. Ethology 105, 569-582. Sayag, N., Snapir, N., Arnon, E., Halawani, M. E. E., Grimm, V. E., and Robinzon, B. (1991). Sexual differentiation of copulatory behaviour in the male chick requires gonadal steroids. British Poultry Science 32, 607-614. 13 Schlinger, B., and Arnold, A. (1992). Plasma sex steroids and tissue aromatization in hatchling zebra finches: implications for the sexual differentiation of singing behavior. Endocrinology 130(1), 289-299. Schlinger, B. A., and Arnold, A. P. (1993). Estrogen synthesis in vivo in the adult zebra finch: additional evidence that circulating estrogens can originate in brain. Endocrinology 133(6), 2610-2616. Schumacher, M., Sulon, J., and Balthazart, J. (1988). Changes in serum concentrations of steroids during embryonic and post-hatching development of male and female Japanese quali (Coturnix coturnix japonicd). Journal of Endocrinology 118,127-134. Silverin, B., and Sharp, P. (1996). The development of the hypothalamic-pituitary-gonadal axis in juvenile great tits. General and Comparative Endocrinology 103, 150-166. Soma, K. K., Alday, N. A., Hau, M., and Schlinger, B. A. (2004). Dehydroepiandrosterone metabolism by 3-beta-hydroxysteroid dehydrogenase in adult zebra finch brain: sex difference and rapid effect of stress. Endocrinology 145(4), 1668-1677. Soma, K. K., and Wingfield, J. C. (2001). Dehydroepiandrosterone in songbird plasma: seasonal regulation and relationship to territorial aggression. General and Comparative Endocrinology 123, 144-155. Tagawa, N., Katagiri, M., and Kobayashi, Y. (2005). Developmental changes of serum steroids produced by cytochrome P450cl7 in rat. Steroids 71(2), 165-170. Tarn, H., and Schlinger, B. A. (2006). Activities of 3beta-HSD and aromatase in slices of developing and adult zebra finch brain. General and Comparative Endocrinology Forthcoming. Tanabe, Y., Nakamura, T., Fujioka, K., and Doi, O. (1979). Production and secretion of sex steroid hormones by the testes, the ovary and the adrenal glands of embryonic and young chickens (Gallus domesticus). General and Comparative Endocrinology 39, 26-33. Tanabe, Y., Saito, N., and Nakamura, T. (1986). Ontogenetic steroidogenesis by testes, ovary, and adrenals of embryonic and post-embryonic chickens (Gallus domesticus). General and Comparative Endocrinology 63, 456-463. Vanson, A., Arnold, A. P., and Schlinger, B. A. (1996). 3[beta]-Hydroxysteroid Dehydrogenase/Isomerase and Aromatase Activity in Primary Cultures of Developing Zebra Finch Telencephalon: Dehydroepiandrosterone as Substrate for Synthesis of Androstenedione and Estrogens. General and Comparative Endocrinology 102(3), 342-350. Vianello, S., Waterman, M. R., Dalla Valle, L., and Colombo, L. (1997). Developmentally regulated expression and activity of 17alpha-hydroxylase/C-17,20-Lyase Cytochrome P450 in rat liver. Endocrinology 138(8), 3166-3174. Wade, J., and Arnold, A. P. (1996). Functional testicular tissue does not masculinize development of the zebra finch song system. PNAS 93(11), 5264-5268. Williams, T. D., Dawson, A., Nicholls, T. J., and Goldsmith, A. R. (1987). Reproductive endocrinology of free-living nestling and juvenile starlings, Sturnus vulgaris; an altricial species. Journal of Zoology (London) 212(4), 619-628. 14 Woods, J. E., and Erton, L. H. (1978). The synthesis of estrogens in the gonads of the chick embryo. General and Comparative Endocrinology 36, 360-370. Woods, J. E., Simpson, R. M., and Moore, P. L. (1975). Plasma testosterone levels in the chick embryo. General and Comparative Endocrinology 27, 543-547. Yang, P. X., Medan, M. S., Watanabe, G., and Taya, K. (2005). Developmental changes of plasma inhibin, gonadotropins, steroid hormones, and thyroid hormones in male and female Shao ducks. General and Comparative Endocrinology 143(2), 161-167. 15 Chapter 2: Brain and Plasma Steroid Levels in a Developing Free-Living Songbird 1 I N T R O D U C T I O N Sexual differentiation of the brain has traditionally been thought to be driven by gonadal hormones, particularly testosterone (T). In rodents, gonadal steroids have an organizational effect on the nervous system during a critical period, directing neural and behavioural development in either the male or female direction (Arnold, 2004; Phoenix et al., 1959). T is thought to be released by the testes and then converted to 17p-estradiol (E2) in the brain (Figure 2.1). E 2 is important in organizing the male brain and adult male reproductive behaviour. -However, sexual differentiation of the songbird brain may be an exception to this paradigm, because in songbirds, the gonads may not be the sole source of steroids affecting brain development. The nestling phase contains a critical period during which the telencephalic song nuclei are masculinized in males but not in females. In this critical period, the song nuclei are sensitive to sex steroids, particularly E 2 (Gurney and Konishi, 1980). However, removing testes in developing zebra finch males fails to prevent masculinization of the brain (Adkins-Regan and Ascenzi, 1990; Arnold, 1975). Moreover, inducing testicular tissue in embryonic zebra finch females fails to masculinize the brain (Wade and Arnold, 1996). Taken together, these data suggest that gonadal steroids are not the sole determinants for masculinizing song nuclei in males. Previous work has examined sex steroid levels during development. One study examined androstenedione (AE), T, dihydrotestosterone (DHT) and E 2 in juvenile zebra finches during development and found males had significantly higher E 2 levels than 1 A version of this chapter wil l be submitted for publication and the reference will be as follows: Chin, E.H., K . L . Schmidt, L .D. Sheldon, A . H . Shah, O.P. Love and K . K . Soma, 2006. Brain and plasma steroid levels in a free-living songbird. 16 females on post-hatch day 3 (P3, where PO is hatch day) (Hutchison et al., 1984). Another study examining AE, T, DHT and E2 in developing juvenile zebra finches found males had higher E 2 levels than females on P12 (Adkins-Regan et al., 1990). In contrast, another study looking at AE, T, DHT, E 2 and estrone (Ei) in juvenile zebra finches did not find a sex difference in E 2 during development (Schlinger and Arnold, 1992). However, these studies sampled blood from different veins. Hutchison et al (1984) collected blood from the jugular vein, whereas Adkins-Regan et al (1990) collected blood from the brachial vein, and Schlinger and Arnold (1992) collected blood from cardiac puncture. Additionally, there are no sex differences in estrogen receptor (ER) mRNA, aromatase mRNA (Perlman and Arnold, 2003), androgen receptor (AR) mRNA (Perlman, Ramachandran, and Arnold, 2003), or aromatase activity (Schlinger and Arnold, 1992) during the nestling phase. Taken together, these data suggest (a) that there may be alternative mechanisms to supply the brain with sex steroids or (b) that sex steroids may play a smaller role in sexual differentiation. One alternative source of steroids is the adrenal gland, which synthesizes sex steroid precursors such as dehydroepiandrosterone (DHEA) (Schlinger, Lane, Grisham, and Thompson, 1999; Soma, Wissman, and Wingfield, 2002). These precursors are subsequently converted locally to E 2 at target tissues, which contain the appropriate enzymes for metabolism, such as a the brain (Tarn and Schlinger, 2006; Vanson et al., 1996) (Figure 2.1). Another possible source of sex steroids is the liver, which expresses Cypl7 (17a-Hydroxylase/17,20-lyase), an enzyme required for DHEA synthesis, during development in rats (Katagiri et al., 1998). A third alternative source of sex steroids is the brain where locally formed estrogens influence neural development 17 (Adkins-Regan et al., 1990; Holloway and Clayton, 2001; London et al., 2006; Schlinger and Arnold, 1992; Schlinger, Soma, and London, 2001). Alternatively, sex steroids may not be solely responsible for sexual differentiation of the brain. It has been hypothesized that expression of sex chromosome genes in the brain drive sexual differentiation in songbirds (Arnold, 2004). While several studies have examined hormone profiles during development in captive zebra finches, few studies have focused on free-living passerines. European starlings (Sturnus vulgaris) are an excellent model for addressing this issue. Predominantly males sing in this species; however, females also sing (Cabe, 1993). Song nuclei are also larger in male starlings (Bernard, Casto, and Ball, 1993). E 2 treatment to female starling chicks partially masculinizes song nuclei (Casto and Ball, 1996). However, Williams et al (1987) did not find sex differences in plasma testosterone (T) in European starlings during development. In the present study, I examined hormone profiles during development in starling nestlings in plasma and tissue. The goals in this study were to: (1) measure DHEA and E 2 in plasma from brachial and jugular veins, and (2) measure steroid concentrations in gonads, adrenals, liver and brain. The jugular plasma is enriched with neurally derived steroids (Schlinger and Arnold, 1993). If the jugular plasma has higher levels of sex steroids, then the brain may be the source of sex steroids during development. If steroid levels in the brachial plasma are higher than the jugular plasma, then peripheral tissues such as the gonads, adrenals or liver may be the source of sex steroids during development. Male starling chicks are predicted to have higher steroid levels than female 18 starling chicks, as males develop larger song nuclei and sing more frequently than females. MATERIALS AND METHODS Field Site and Subjects This research was carried out between April and July 2005 and 2006 at the Davistead Dairy Farm in Langley, British Columbia, Canada, under a University of British Columbia Animal Care permit (A04-277), following guidelines of the Canadian Council on Animal Care. The site consists of approximately 250 nest boxes mounted on farm buildings and on posts in large fields that are used yearly by breeding starlings. All nest boxes were checked daily to determine clutch initiation and clutch completion dates as well as the laying sequence of eggs and rates of chick growth. Starlings at our field site generally lay 4-6 eggs per clutch, incubate for 10-11 days and fledge chicks 20-24 days following hatching (O.Love, unpublished data). Nestling hatch order and subsequent age were tracked using non-toxic food colouring and chick-specific feather clipping until 10 days of age, when chicks were subsequently banded with metal bands (permit #10765). Experiment 1: Steroid Levels in Plasma This experiment was conducted in 2005. We collected blood samples from developing birds from either the jugular or the brachial vein. Day of hatch was considered P0. Juvenile birds were sampled on post-hatching day 0 (P0), PI, P2, P4, P6, P8, P10, P16 and P20 (fledging age). On the day of hatch, nests were assigned two ages at which blood samples were collected. These two ages were at least a week apart. No 19 bird was blood sampled more than two times. For each blood sample collected from a particular bird, the same vein was sampled at each age. No more than 1% of total blood volume was collected per sample. Brachial blood was accessed by puncture of the brachial vein in the wing with a 26G needle and then collected with a heparinized capillary tube. Jugular blood was collected with heparinized 0.5 mL fixed needle syringes. All samples were collected between the hours of 12:00 and 16:00 and within 5 min (2.75 ± 0.07 min) of initial disturbance to avoid effects of stress. After blood collection, we obtained body measurements (mass, and length of exposed culmen, metatarsus, and wing) from the bird and returned it to its nestbox. Wing cord measurements were taken from P8 and older birds when primary feathers began to appear. Blood was centrifuged at 10,000 rpm for 10 min. Plasma was collected with a Hamilton syringe and stored at -20°C until analysis. Experiment 2: Steroid Levels in Brain Tissue This experiment was carried out in 2006. We collected tissue from juvenile starlings at P0, P6 and P8 based on previous data from (Silverin and Sharp, 1996), Casto (unpublished data) and (Williams et al., 1987) on plasma steroid levels in wild developing songbirds. All subjects were collected between the hours of 12:00 and 16:00 and sacrificed within 3 min of initial disturbance. Before sacrifice, a blood sample was collected from the jugular vein using a heparinized 0.5 mL syringe. Birds were then rapidly decapitated, and the brain and body were chilled at -20°C for 5 min prior to dissection. Brain and peripheral tissues were collected and frozen in liquid nitrogen. For the brain, the telencephalon was divided into rostral telencephalon (rTEL), caudal 20 telencephalon (cTEL), and dorsal telencephalon (dTEL). The telencephalon was not subdivided in PO chicks. The dTEL contained the song nuclei HVC and NCM, as well as the hippocampus. In PI chickens, the dTEL contains IMHV, where there are high levels of DHEA (Migues et al., 2002). To dissect out the dTEL, cuts were made on the telencephalon in the following places: halfway between rostral and caudal edges, halfway between dorsal and ventral edges, and halfway between the midline and lateral edges. The rTEL contained the song nuclei 1MAN and Area X. To dissect out the rTEL, after the dTEL was collected, tissue from the rostral edge to halfway between the rostral and caudal edges was considered rTEL and collected. The cTEL contained the song nucleus RA. After the rTEL was collected, tissue from halfway between the rostral and caudal edges to the caudal edge of the brain was considered cTEL and collected. The body was kept at 4°C while the brain was dissected. The liver, heart, gonads and adrenals were dissected out of the body. All dissections were finished within 30 min (26.87 ± 0.48 min) of initial disturbance. Steroid Extraction from Plasma Extraction methods using organic solvents did not yield a serial dilution that was parallel to the radioimmunoassay standard curve. Therefore, steroids were extracted using solid phase extraction (SPE) with CI8 columns (6 mL capacity, 500 mg, non-endcapped; United Chemical Technologies UCTCUC18156). Either 66 pL or 132 pL of plasma were prepared for extraction by bringing samples to 10 mL with deionized water (diH20). Using a vacuum manifold, each column was primed with 3 mL HPLC-grade ethanol, followed by 10 mL diH20. We then added the 10 mL sample volume. Next, the 21 columns were washed with 10 mL diHaO to remove substances that may interfere with the radioimmunoassay. Steroids were eluted with 5 mL 90% HPLC-grade methanol into 7 mL borosilicate glass scintillation vials. Each sample was evaporated to dryness under N 2 at 37°C. Depending on initial plasma volume, samples were resuspended in either 440 pL or 880 pL of phosphate buffered saline with gelatin, PBSG (5% ethanol, final concentration). 100% ethanol (absolute grade) was added first to the bottom of the vial and briefly vortexed to bring steroids into solution. PBSG was then added to bring samples up to final volume. These volumes were determined from preliminary studies and designed to place samples in the linear portion of the standard curve. Samples were then shaken at 500 rpm for 1 hr and stored overnight at 4°C. The next morning, samples were again shaken at 500 rpm for 2 min prior to radioimmunoassay. To quantify recovery of DHEA and 17P-E2, separate samples from a pool of juvenile starling plasma were spiked with 50 pg DHEA and 0.5 pg 17P-E2, then compared to unspiked plasma. Recovery of DHEA was 104.89 ± 6.09% (n = 4 pairs), and recovery of 17P- E2 was 107.45 ± 13.90% (n = 4 pairs). Therefore, plasma concentrations of DHEA and E2 were not corrected for recovery. Steroid Extraction from Tissue Steroids were also extracted from tissue using solid phase extraction (SPE). The procedure for brain tissue was the same as that for plasma, except tissue required more preparation prior to loading the samples on the columns. Tissue was weighed and transferred to 13 x 100 mm borosilicate glass test tubes. Ice-cold d i H 2 0 was added (3x times the weight of the tissue). Tissue was homogenized on ice using a Teflon pestle. Immediately, HPLC-grade methanol was added (4x the total volume of tissue and water). 22 Samples were sonicated for 15 min (Fisher Scientific FS-20), shaken for 60 min (IKA Vibrax VXR basic), and stored overnight at 4°C. The next day, samples were shaken for 90 min, and then centrifuged at 3000g for 10 min at 2°C. The supernatant was transferred to 20 mL scintillation vials. In cases where the supernatant volume was greater than 1 mL, only 1 mL of supernatant was transferred to the scintillation vial. The supernatant was brought up to 10 mL with diH^O. This 10 mL volume was loaded onto primed and equilibrated CI8 columns. Columns were washed with 10 mL of diH20. Steroids were eluted with 5 mL 90% HPLC-grade methanol into 7 mL borosilicate scintillation vials. Each sample was evaporated to dryness under N 2 at 37°C. Samples were resuspended in 440 pL PBSG (5% ethanol, final concentration). To quantify recovery of DHEA and 170-E2, separate samples from a pool of juvenile starling tissue were spiked with 50 pg DHEA and 1 pg 17p-E2, then compared to unspiked tissue. From brain, recovery of DHEA was 143.02 ± 10.42% (n = 5 pairs), and recovery of 17p- E 2 was 61.59 ± 10.68% (n = 5 pairs). From adrenal tissue, recovery of DHEA was 50.61 ± 6.14% (n = 5 pairs), and from gonadal tissue, 60.37 ± 22.22% (n = 5 pairs). All tissue concentrations were corrected for recovery and were expressed as pg steroid per g tissue wet weight. Radioimmunoassays DHEA was measured using a double antibody RIA which uses 1 2 5I-DHEA as the tracer (Diagnostic Systems Laboratories, DSL-8900). The DHEA assay was modified as per Granger et al (1999), and has been previously used with songbirds (Goodson, Evans, and Soma, 2005). Briefly, using PBSG, supplied DHEA standards were diluted lOx, the primary antibody was diluted 4x, and the tracer was diluted 4x. The DHEA antibody has a low cross-reactivity with DHEA-S (0.02%), 16p-OH DHEA (0.041%), androstenedione 23 (0.46%), testosterone (0.028%), and E 2 (< 0.004%; E. Chin, unpublished data). Assay sensitivity was 2 pg/tube. 170-E2 was measured using a double antibody RIA which uses 125I-17(3-E2 as the tracer (Diagnostic Systems Laboratories, 3rd generation E 2 RIA, DSL-39100). The 17p-E 2 assay was modified as per Shirtcliff et al (2000). Briefly, using PBSG, supplied E 2 standards were diluted 20x, the primary antibody was diluted 4x, and the tracer was diluted 3x. The 17P-E2 antibody has a low cross-reactivity with estrone (6.9%), estriol (< 0.001%), 17a-E2 (0.33%; E. Chin, unpublished data), and DHEA-S (< 0.001%). Assay sensitivity was 0.113 pg/tube. Genotyping Once plasma was collected from the blood samples, a portion of the remaining red blood cells was transferred to a piece of filter paper, which was then stored at -20°C. Starling chicks were genotyped using a polymerase chain reaction (PCR) amplification process based on techniques used by (Griffiths, S. Daan, and Dijkstra, 1996) and (Chin, Love, Clark, and Williams, 2005). DNA was isolated from the red blood cell samples using Insta-gene matrix (Bio-Rad Laboratories, Hercules, California, Cat. No. 732-6030) following the manufacturer's protocol. DNA concentration was quantified with a spectrophotometer (Beckman Coulter DU 640). PCR amplification was run using the P2 (5' -TCTGCATCGCTAAATCCTTT) and CW (5' -AGAAATCATTCCAGAAGTTCA) primer set. Adult birds of known sex (n = 5 of each sex) were used as positive controls. Negative controls contained no DNA. PCR products were run on a 3% agarose gel with ethidium bromide at 70V for 70 min. DNA was visualized with a UV transilluminator. 24 Statistics The plasma data were subjected to a two-way analysis of variance (ANOVA) followed by planned post hoc t-tests. If necessary, data were normalized by logarithm (base 10) transformation prior to running the ANOVA. Jugular plasma data from 2005 and 2006 were pooled for analysis following t-tests which determined that there was no statistically significant year difference. Plasma samples with non-detectable steroid levels were assigned the detection limit for the assay. To analyze for sex differences rather than regional differences, the tissue data were subjected to paired t-tests, or Mann-Whitney U-tests if the data had unequal variance. All tests were performed using Sigma Stat 3.0 with nestling age and sex as factors. Statistical significance was set at p = 0.05. Sample sizes for all groups are presented in Table 2.1. Results are presented as mean ± standard error of the mean. RESULTS Subject Body Mass and Hematocrit Changes in body mass with age are depicted in Figure 2.2. Male starlings hatched at 6.38 ± 0.26g and fledged at 73.61 ± 1.09g, and female starlings hatched at 6.02 ± 0.30g and fledged at 70.81 ± 1.28g. For both sexes, mass gain in juvenile starlings increased in a linear fashion from hatch to P10, after which mass subsequently plateaued to fledging. Hematocrit increased from 22.8 ± 2.0% at hatch to 47.5 ± 1.1% at fledging for both sexes (Table 2.2). 25 Validations Initial validations of a serial dilution of starling plasma using organic solvents such as dichloromethane failed to yield a serial dilution that was parallel to the standard curve (Figure 2.3a). However, serial dilutions of starling plasma and brain tissue pools extracted using SPE yielded parallelism to both the DHEA and 170-E2 standard curves (Figures 2.3, 2.4). All water blanks run during extractions were non-detectable or close to the lowest point on the standard curve for both DHEA (2.63 ± 0.24 pg; n = 15) and 17P-E2 (< 0.113 pg; n = 10). There was no effect of 100% ethanol (absolute grade) concentration for resuspension (5% or 10% final concentration) on the RIAs, thus 5% ethanol (final concentration) was chosen to resuspend samples (Figure 2.5, 2.6). Plasma Steroid Levels Plasma DHEA. All brachial DHEA samples were detectable (Figure 2.7a). For brachial DHEA, there was a main effect of age (F = 3.711; p < 0.001), but not sex (F = 1.344; p = 0.248). There was no interaction between age and sex (F = 1.793, p = 0.084). For juvenile males, brachial DHEA levels ranged from -1000 pg/mL during the first few days oflife and peaked at -2400 pg/mL at P4. Levels declined back to -1000 pg/mL by P20. For juvenile females, DHEA levels were -1000 pg/mL at hatch and increased to -1500 pg/mL at P2. Levels then declined back to 1200 pg/mL by P20. Males had 1.5 times more DHEA than females at P4, and this sex difference was highly significant (t = 3.289; p = 0.001). All jugular DHEA samples were detectable (Figure 2.7b). For jugular DHEA, there was a main effect of age (F = 2.339, p = 0.021) and sex (F = 5.333; p = 0.022). There was also a significant interaction between sex and age (F = 2.268; p = 0.025). For 26 juvenile males, jugular DHEA levels were low at hatch, 600 pg/mL, compared to brachial levels. For juvenile females, jugular DHEA was considerably higher at hatch, ~1100 pg/mL, and peaked at ~1500 pg/mL by P10. In contrast to brachial DHEA data, females had higher levels of DHEA in jugular plasma than males on P0 (t = 3.105; p = 0.002) and P10 (t = 2.329; p = 0.021). To compare differences between veins, the average brachial DHEA levels at each age were subtracted from each individual jugular DHEA levels for that age within each sex (Figure 2.7c). There was a highly significant main effect of age (F = 8.501; p < 0.001), and sex (F = 21.648; p < 0.001). There was also a highly significant interaction between age and sex (F = 5.714;p<0.001). Post hoc t-tests revealed a sex difference at P0 (t = 3.155; p = 0.002), P4 (t = 4.866; p < 0.001), and P10 (t = 4.638; p < 0.001) with females having higher difference in DHEA between the jugular than brachial vein than males. Plasma E2. In comparison, plasma E2 levels were 100 fold lower than DHEA levels in both sexes. E2 was not detectable in all brachial samples (Table 2.3a). For brachial E2, there was a main effect of age (F = 2.245; p = 0.028) but not sex (F = 1.344; p = 0.249) (Figure 2.8a). There was no significant interaction between age and sex (F = 0.484; p = 0.865). For juvenile males, brachial E2 levels were mainly non-detectable at hatch and rose to 15 pg/mL by PI. Levels dropped back to non-detectable levels at P2, but rose again to 24 pg/mL by P4. Levels subsequently declined to non-detectable by P20. For juvenile females, brachial E2 levels were also mainly non-detectable at hatch and followed a similar pattern to male levels throughout development. Levels in females 27 reached 18 pg/mL at P4 before declining to non-detectability by P20. There was no sex difference in brachial E2 levels at any age. For jugular E2, not all samples were detectable (Table 2.3a). There was a main effect of age (F = 9.475; p < 0.001), but not sex (F = 0.0441; p = 0.834) (Figure 8b). There was no significant interaction between sex and age (F = 0.576; p = 0.797). For juvenile males, levels were non-detectable at hatch, and increased to 17 pg/mL at P4. Levels declined to 10 pg/mL by P8 and remained low to P20. For juvenile females, the pattern was similar. Levels were non-detectable at hatch, peaked at 20 pg/mL at P4, and then declined to 10 pg by P8 and remained low to P20. There was no sex difference in jugular E2 levels at any age. E2 levels were also compared between the jugular and brachial veins (Figure 2.8c). There was also a highly significant main effect of age (F = 12.567; p < 0.001) and a significant main effect of sex (F = 8.041; p = 0.005). The interaction between sex and age was also highly significant (F = 3.553; p < 0.001). Post hoc t-tests revealed females having higher difference in E2 between the jugular than brachial vein than males at P4 (t = 3.577; p < 0.001), P6 (t = 3.414; p < 0.001) and P8 (t = 3.770; p < 0.001). Plasma E 2 /DHEA Ratio. To determine how much DHEA was being metabolized to E2, the ratio of E 2 t o DHEA levels for each subject was compared, potentially indicating the amount of DHEA converted to E 2 (Figure 2.9). For brachial steroid levels, there was no effect of age (F = 1.835; p = 0.077) or sex (F = 0.0518; p = 0.820). There was also no significant interaction between age and sex (F = 1.149; p = 0.336). For jugular steroid levels, there was an effect of age (F = 3.663; p < 0.001), but 28 not sex (F = 3.211; p = 0.075). There was no significant interaction between age and sex (F = 1.430; p = 0.187). Brain Steroid Levels PO. At PO, DHEA levels in the brain were detectable in all regions of the brain (Figure 2.10a). There was no sex difference in DHEA in the telencephalon (t = -0.084; p = 0.934) or the optic lobe (t = 0.362; p = 0.723). There was a sex difference detected in the diencephalon, with males having higher DHEA levels than females (t = -2.161; p = 0.048). P6. At P6, DHEA was also detectable in all regions of the brain (Figure 2.10b). There was no sex difference in DHEA levels in any of the brain regions measured: rostral telencephalon (t = -0.043; p = 0.966), caudal telencephalon (t = 1.557; p = 0.142), dorsal telencephalon (Mann Whitney, t = 76.00; p = 0.442), diencephalon (t = 0.594; p = 0.562) and optic lobes (Mann Whitney, t = 70.00; p = 0.878). P8. At P8, DHEA was also detectable in all brain regions (Figure 2.10c). There was no sex difference in DHEA levels in any of the brain regions measured: rostral telencephalon (t = -1.157; p = 0.267), caudal telencephalon (t = -0.639; p = 0.533), dorsal telencephalon (t = -0.750; p = 0.466), diencephalon (t = -0.268; p = 0.793), and optic lobes (Mann Whitney, t = 58.00; p = 0.328). Brain E2 Levels. Surprisingly, E2 was not detectable in any brain region at any age. The detection limit for all ages is presented in Table 2.3. However, positive controls were run to ensure that the extraction technique was efficient. E2 was extracted and measured in adult zebra finch ovary, and the adult male zebra finch brain. E2 was 29 detectable in all samples of adult zebra finch ovary and adult male zebra finch brain, as well as spiked brain tissue samples. Peripheral Tissue DHEA Levels PO. At PO, DHEA was detectable in all tissues collected (Figure 2.1 la). There was no sex differences detected in any of the tissues measured: gonads (Mann Whitney; t = 45.00; p = 1.00), adrenals (t = -1.079; p = 0.299), and liver (Mann Whitney; t = 63.00; p = 0.645). The heart (negative control) had lower levels of DHEA than the jugular plasma. E 2 was not measured because plasma levels were low at this age. P6. At P6, DHEA was detectable in all tissues collected (Figure 2.1 lb). There was no sex difference in DHEA levels in tissues collected: gonads (Mann Whitney; t = 53.00; p = 0.130), adrenals (t = -0.193; p = 0.849) or liver (Mann Whitney; t = 62.00; p = 0.574). The heart (negative control) had lower levels of DHEA than the jugular plasma. E 2 was not measured because plasma levels were low. P8. At P8, DHEA was detectable in all tissues (Figure 2.1 lc). There was a sex difference detected in the gonads, with the testes containing higher DHEA levels than the ovaries (Mann Whitney; t = 46.00; p = 0.021). There was no sex difference detected in the adrenals (t = -0.643; p = 0.531) or the liver (t - 0.506; p = 0.621). The heart (negative control) had lower levels of DHEA than the jugular plasma. E 2 was not measured because plasma levels were low. DISCUSSION This study investigated the changes in steroid levels during development in a free-living songbird. Assessing plasma DHEA and E 2 levels revealed higher DHEA levels 30 than E2 levels. Males had higher DHEA levels than females at P4 in the brachial vein, while in contrast, females had higher DHEA levels than males at PO and PIO in the jugular vein. No sex difference was detected in either vein at any age for E2. In the brain, DHEA levels were low, and a sex difference was only detected at PO in the diencephalon. E2 was not detectable in the brain at any age or either sex. In the periphery, DHEA was detectable in all tissues. This is the first study to directly measure steroids in plasma, brain and peripheral tissues from songbirds. Plasma D H E A and E 2 In the brachial plasma, males exhibited higher levels of DHEA than females on P4. The timing of elevated levels of DHEA in males corresponds to timing of elevated E2 levels in previous work done with zebra finches (Hutchison et al., 1984). In addition, this peak in DHEA occurs during the first week of life, which has been suggested to be the critical period for sexual differentiation of the song nuclei, as previously determined in zebra finches (Adkins-Regan et al., 1994). Moreover, this suggests there is a peripheral source of DHEA. In jugular plasma, the opposite sex difference was observed at PO and PIO, with the females having higher levels than the males. This may suggest that males may take up and metabolize DHEA in the brain more than females. This may be linked to differential enzyme activity in the songbird brain. Previous work has demonstrated that there is a sex difference in 3p-Hydroxysteroid dehydrogenase (3p-HSD) in the adult songbird brain, which converts DHEA to AE (Soma et al., 2004) (Figure 2.1). This enzyme is also present in the developing songbird brain (Schlinger and London, 2006). 31 Should there also be a sex difference during development, this may result in differential metabolism of DHEA, accounting for the higher levels of DHEA in the females. To further elucidate the idea that the brain takes up DHEA synthesized in the periphery, the average brachial DHEA for each sex and age group was subtracted from each jugular DHEA value for that sex and age group. The difference between the two veins was representative of the steroid levels the brain removed from the plasma. At PO and PIO, females had higher difference in DHEA between the jugular than brachial vein than males, suggesting that the female brain at these ages were not taking up DHEA synthesized in the periphery. Moreover, at P4, the males had less DHEA in the jugular plasma compared to the brachial, suggesting the P4 male brain was taking up DHEA. Moreover, females had higher jugular than brachial levels of E 2 from P2 to P6, suggesting the male brain was also taking up E 2 during this period. These ages fall within the critical period during which the brain is sensitive to steroids (Adkins-Regan et al., 1994; Gurney and Konishi, 1980). The low ratio of E 2 to DHEA suggests that the DHEA produced and circulated in the blood either (1) was not being converted to E 2 or (2) that the E 2 was being further converted downstream to other estrogens. This is surprising as E 2 has been suggested to be the primary sex steroid responsible for brain sexual differentiation in birds based on elevating levels of E 2 during the critical period (Adkins-Regan et al., 1994; Casto and Ball, 1996; Gurney and Konishi, 1980). This may suggest that further metabolism of E 2 is required during sexual differentiation. Circulating high levels of DHEA during development may be a physiological strategy employed to avoid the negative impacts of high circulating levels of T. DHEA 32 can only affect tissues which possess the enzymes to convert DHEA further downstream to T and E2. In contrast, high circulating T has been connected to decreased immune function and increased metabolic rate (Owen-Ashley, Hasselquist, and Wingfield, 2004; Wikelski, Lynn, Breuner, Wingfield, and Kenagy, 1999), both of which are important physiological factors for juveniles. By circulating DHEA, only target tissues will be affected by T or E2, and such important physiological factors will not be impacted during development. It has been shown that DHEA does not highly impact immune function (Owen-Ashley et al., 2004). Brain DHEA and E 2 Levels DHEA was detectable in all brain regions at all ages. However, there was no sex difference detected in any region at any age. Moreover, DHEA levels were lower in the brain than in plasma. There was also only one sex difference in DHEA levels detected: at PO, in the diencephalon. The results of the current study suggest that the brain may be metabolizing DHEA. DHEA may be converted to E2. However, E2 measurements in the brain in this study do not indicate that this is the case. Previous work has demonstrated that the brain contains enzymes to metabolize DHEA (London et al., 2006; Soma et al., 2004). Another possibility may be that DHEA is converted into another estrogen. Previous work looking at human pregnancy has suggested that the fetal adrenal produces the sulfated ester, DHEA-S. This DHEA-S is hydroxylated at the 16a position by the fetal liver, and then aromatized to estriol (E3) by the placenta. It is conceivable that perhaps a similar process is occurring in the juvenile songbird brain, and that the sex difference may lie in E 3 , not DHEA or E2 (Diczfalusy, 1984). 33 Surprisingly, E 2 was not detectable in any brain regions at any age, despite detectable levels in circulating plasma. This does not support the hypothesis that E 2 is primarily responsible for brain sexual differentiation, and further supports the findings of low E 2 in circulation (Casto, 2001). There may be several explanations for the low levels of E 2 in the brain. Firstly, E 2 may be further converted to other estrogens, such as estrone (Ei) or estriol (E3). Ei is detectable in plasma in developing songbirds, and levels are higher than circulating E 2 (Schlinger and Arnold, 1992). Ei may be the active estrogen in the brain after conversion from E 2 , which may explain why levels in the plasma are also quite low. Secondly, the method of dissections of the brain may have diluted out high concentrations of E 2 . Small punches of brain tissue may reveal more localized concentrations of E 2 . Thirdly, 17(3-E2 may not be the active estrogen in the brain. A recent study has suggested that the isomer, 17a-E2 is a neuroactive estrogen (Toran-Allerand, Tinnikov, Singh, and Nethrapalli, 2005). This is the first study to measure steroid levels in songbird brain tissue, and further work is needed to determine if E 2 levels in the brain are more localized. Peripheral DHEA DHEA was detectable in all tissues at all ages. Particularly, DHEA levels were high in the gonads, 50 to 200 times higher than the plasma, depending on age. This suggests that the gonads are the primary source of peripheral DHEA. Previous studies have suggested that the gonads are not sources of sex steroids required for brain sexual differentiation (Wade and Arnold, 1996), and that gonadal tissue did not actively synthesize steroids during the nestling phase (Schlinger and Arnold, 1992; Silverin and Sharp, 1996). However, these studies only considered T and E 2 levels, and did not 34 consider the capacity of the gonads to synthesize DHEA from cholesterol. The data in the current study suggest that these tissues have Cypl7 activity during the nestling phase, the enzyme required to convert PREG to DHEA (Figure 2.1). These data support the findings from previous studies which have found that the gonads express Cypl7 mRNA (Freking, Nazairians, and Schlinger, 2000). It is possible that while the gonads do not possess all the enzymes for synthesizing T and E2 during the critical period, they may still be capable of synthesizing and releasing DHEA into circulation, which can be subsequently converted at target tissues. Moreover, the induced testes in juvenile female zebra finches may have secreted DHEA, but the brain did not have the appropriate enzymes to convert the DHEA further downstream to affect brain anatomy. DHEA was also detectable in the adrenals, suggesting that the adrenals may contribute to peripheral levels of DHEA. It has been previously demonstrated that the avian adrenal is capable of synthesizing DHEA (Soma and Wingfield, 2001). The adrenals contribute to peripheral levels of DHEA in wintering song sparrows, when the gonads are regressed and are not synthesizing T. Juveniles are physiologically similar to wintering songbirds, as their gonads are regressed. It is possible that the adrenals may be a secondary source of DHEA. In the event that gonads are regressed or removed all together, the adrenals may take over as the primary source of DHEA. This may explain why castration had no effect on song behaviour in male zebra finches (Arnold, 1975). Taken together, the data support the idea that the gonads and adrenals secrete DHEA, which may be converted to T and E2 at target tissues. DHEA was also detectable in the liver, but not at levels higher than the plasma. Previous studies in developing rodents have suggested that the liver is capable of 35 synthesizing DHEA as there is Cypl7 activity in the liver during development (Katagiri et al., 1998; Tagawa et al., 2005). While it is possible that the liver is a source of DHEA during development, further studies should examine possible Cypl7 activity in the avian liver. The liver is a large organ in developing songbirds, and may express large amounts of Cypl7. The whole liver was not collected here, and DHEA levels in the liver were low. Further studies should examine Cypl7 activity as well as DHEA content of the entire liver. The results of this study both support new hypotheses as well as challenge older ones. Firstly, the high levels of DHEA in the gonads and adrenals support the hypothesis that the adrenals secrete DHEA into the bloodstream to be subsequently converted at target tissues. Secondly, high levels of DHEA detected in the gonads suggest that the gonads do play a role in sex steroid synthesis during development and are not completely inactive. Thirdly, the non-detectable levels of E 2 in the brain suggest that E 2 may not be the active estrogen in the brain during development. However, these data do not rule out the hypotheses that the brain is capable of synthesizing steroids, or that there may be a direct genetic effect on sexual differentiation of the brain. Further studies should focus on other potential active estrogens in the brain, more localized measurements of E 2 in the brain, as well as enzyme activity, such as Cypl7 and 3p-HSD, in tissues analyzed in this study. 36 Tables Table 2.1: Sample sizes for all groups for (a) 2005 and (b) 2006 experiments. (a) Age Males Females Brachial Jugular Brachial Jugular PO 6 7 4 7 PI 12 10 5 11 P2 8 9 4 7 P4 9 8 11 8 P6 8 7 13 7 P8 8 6 4 8 PIO 7 7 8 8 P16 10 13 9 7 P20 9 10 9 7 (b) Age Males Females P0 8 8 P6 8 8 P8 8 8 37 Table 2.2: Changes in hematocrit over the nestling phase in juvenile European starlings with sexes combined. Values are presented as means ± SEM. Age Hematocrit (%) Sample Size PO 22.7 ±2.0 5 PI 23.7 ± 1.5 4 P2 21.9±2.0 14 P4 24.4 ± 1.6 8 P6 34.1 ± 1.5 17 P8 36.6 ±2.1 16 PIO 36.8 ± 1.8 20 P16 40.8 ±0.8 18 P20 47.4 ± 1.0 22 38 Table 2.3: Number of detectable samples for E 2 in (a) plasma and (b) brain tissue in juvenile European starlings. Values are presented as means ± SEM and number of detectable samples in brackets. Detection limit for plasma was 9 pg/mL. Detection limit for brain tissue is presented in the table. Age Males Females Brachial Jugular Brachial Jugular PO 0/6 1/7 1/4 1/7 PI 6/12 4/10 4/5 3/11 P2 6/8 6/9 3/3 5/7 P4 9/9 7/8 11/11 8/8 P6 8/8 111 7/13 5/7 P8 6/8 2/6 3/4 3/8 PIO 3/7 3/7 4/8 7/8 P16 6/10 7/13 3/9 5/7 P20 5/9 4/10 8/9 3/7 Age Sex TEL rTEL cTEL dTEL DIEN OL PO Female 32.2 ± 1.6 i n S i 65.7 ± 6 . 9 (0/8) 36.4 ±5 .6 (0/8) Male 31."- 1.2 (0/N) 77.1 ± 10.6 (0/8) 39.4 ±5 .4 (0/8) P6 Female 23.3 I 2.4 HI N i 26.3 ± 3.5 ( I S ) 23.4 ± 2.7 (0/8) 30.1 ± 3.8 (1/8) 21.6 ± 2.1 (0/8) Male 28.8 :• 5.0 H I S , 17.9- 0.5 H I S , 11 1 .. "> 0>X) 26.8 ± 5.8 H I S ) 32.1 ±4 .7 (0/8) 46.1 ±25.5 (0/8) P8 Female 18.9 0.4 (0/8) 17.0-0.4 H i S i 22.4 ± 4.7 (2/8) 20.8 ± 1.4 (0/8) Male 21.5 : 2.9 i l l X . 21.2 ± 1.4 ... s. 19.0-1 0.9 HI S . 19.5 ±1 .1 (0/8) 19.1 ±0 .6 (0/8) 39 F i g u r e 2.1: Pathways of steroid synthesis. Adapted from Mellon and Griffin, 2002. Cholesterol see Pregnenolone 3&-HSD -• Progesterone CYP1T CYP17 Dehydroeplandrosterone • Androstsnedlone 4 • Testosterone AROMATASE AROMATASE Estrone 4-17P-HSO -*• Estradiol 40 Figure 2.2: Changes in body mass during development in European starling 1 Figure 2.3: Steroid extraction from juvenile starling plasma using (a) dichloromethane to extract DHEA, (b) solid phase extraction (SPE) to extract DHEA and (c) SPE to extract 17P-E2. Circles represent the standard curve for each assay, and triangles are the serial dilution of juvenile starling plasma. Results are plotted on a log scale x-axis. (a) Dichloromethane Extraction 100 T Standard Curve Starling Plasma 10 100 DHEA (pg) or plasma volume (pl_) Solid Phase Extraction • Standard Curve * Starting Plasma 10 100 D H E A (pg) or plasma volume (uL) Solid Phase Extraction • Standard Curve * Starling Plasma 0-1 1 10 E 2 (pg) or plasma volume (p±) 42 Figure 2.4: Steroid extraction from juvenile starling brain tissue using SPE. Circles represent the standard curve for each assay, and triangles are the serial dilution of juvenile starling brain tissue. 100 -> 1 10 100 1000 D H E A (pg) or brain t issue (mg) 43 Figure 2.5: Estimating recovery of steroids from avian plasma using SPE with (a) labeled steroids and (b) unlabelled steroids. (a) 100 [ H] DHEA [3H] 17p-E 2 80 60 40 20 (b) X I 0% 5% 10% % Ethanol in Resuspension < UJ X Q O J Q . 100 -i 80 60 H Unspiked F7F?H Spiked 40 20 H 50 pg DHEA added Recovery = 113% X 50 pg DHEA added Recovery = 120% i 5% 10% % Ethanol in Resuspension 44 Figure 2.6: Estimating recovery of steroids from avian brain tissue using SPE with (a) labeled steroids and (b) unlabelled steroids. 5% ethanol, final concentration, was used in resuspension. (a) 80 60 > § 40 a: 0s • [3H] DHEA • [3H]17p-E, 20 (b) X x 0% 5% % Ethanol in Resuspension 10% 80 60 H S5 § 40 H cn o. 20 Unspiked 25 pg DHEA added Recovery = 80% I 50 pg DHEA added Recovery = 72% 0.5 Brain tissue (mg) 45 Figure 2.7: DHEA levels in (a) brachial plasma (b) jugular plasma and (c) jugular brachial levels in juvenile starlings from hatch (0) to fledging (20). Females are represented by black circles, and males are represented by gray circles. Age (days) Figure 2.8: 17p-E2 levels in (a) brachial plasma (b) jugular plasma and (c) jugular brachial levels in juvenile starlings from hatch (0) to fledging (20). Females are represented by black circles, and males are represented by gray circles, (a) Brachial E 2 10 12 14 16 18 20 22 (c) Jugular E, 8 10 12 14 16 18 20 22 47 Figure 2.9: E 2/DHEA ratios in (a) brachial plasma and (b) jugular plasma in juvenile starlings at each age sampled. (a) < LU X Q LU ro o ro 0.026 0.024 0.022 0.020 0.018 0.016 0.014 0.012 0.010 0.008 0.006 0.004 0.002 0.000 - •— Females Males (b) ~ i i i i i i 1 1 1 1 1 0 2 4 6 8 10 12 14 16 18 20 22 Age (days) 0.026 - i 0.024 -0.022 -0.020 -< 0.018 -LU X 0.016 -Q C M 0.014 -LU i_ 0.012 -ra 0.010 -D) —) 0.008 -0.006 -0.004 -0.002 -0.000 -8 10 12 14 16 18 20 22 Age (days) 48 Figure 2.10: Brain DHEA levels in (a) PO chicks (b) P6 chicks and (c) P8 chicks. Samples sizes are n = 8 for both sexes at all ages. Regions measured included telencephalon (TEL) (PO chicks only), rostral telecephalon (rTEL), caudal telencephalon (cTEL), dorsal telencephalon (dTEL) (P6 and P8 chicks only), diencephalon (DIEN) and optic lobes (OL) (all chicks). (a) | 600 o PO Females Males III J L (b) £ 600 A Q P6 cTEL dTEL DIEN (c) 1400 1200 • 1000 -O) 800 -CL Q 600 -D 400 -200 -0 -1 P8 I i l iJU rTEL cTEL 49 Figure 2.11: Periphery DHEA levels in (a) PO chicks (b) P6 chicks and (c) P8 chicks. Sample sizes are n = 8 for both sexes at all ages. (a) 350000 300000 250000 H a> a> 200000 Q. < X 150000 Q 100000 PO • • i Females ^ s m m Males 1 i (b) 80000 i pQ i Q 1 (c) . g 30000 a § 20000 P8 r" Adrenal Tissue 50 R E F E R E N C E S Adkins-Regan, E., Abdelnabi, M., Mobarak, M., and Ottinger, M. A. (1990). Sex steroid levels in developing and adult male and female zebra finches (Poephila guttata). General and Comparative Endocrinology 78, 93-109. Adkins-Regan, E., and Ascenzi, M. (1990). Sexual differentiation of behavior in the zebra finch: effect of early gonadectomy or androgen treatment. Hormones and Behavior 24, 114-127. Adkins-Regan, E., Mansukhani, V., Seiwert, C , and Thompson, R. (1994). Sexual differentiation of brain and behavior in the zebra finch: critical periods for effects of early estrogen treatment. Journal of Neurobiology 25(7), 865-877. Arnold, A. P. (1975). The effects of castration on song development in zebra finches (Poephila guttata). Journal of Experimental Zoology 191, 261-278. Arnold, A. P. (2004). Sex chromosomes and brain gender. Nature Reviews Neuroscience 5(9), 701-708. Bernard, D. J., Casto, J. M., and Ball, G. F. (1993). Sexual dimorphism in the volume of song control nuclei in European starlings: assessment by a Nissl stain and autoradiography for muscarinic cholinergic receptors. Journal of Comparative Neurology 334, 559-570. Cabe, P. R. (1993). European Starling. The Birds of North America 48,1-24. Casto, J. M. (2001). Development and hormonal regulation of sex differences in the song system of European starlings (Sturnus vulgaris), Johns Hopkins University. Casto, J. M., and Ball, G. F. (1996). Early administration of 17beta-estradiol partially masculinizes song control regions and alpha-2 adrenergic receptor distribution in European Starlings (Sturnus vulgaris). Hormones and Behavior 30, 387-406. Chin, E. H., Love, O. P., Clark, A. M., and Williams, T. D. (2005). Brood size and environmental conditions sex-specifically affect nestling immune response in the European starling (Sturnus vulgaris). Journal of Avian Biology 36, 549-554. Diczfalusy, E. (1984). The early history of estriol. Journal of Steroid Biochemistry 20(4B), 945-953. Freking, F., Nazairians, T., and Schlinger, B. A. (2000). The expression of the sex steroid-synthesizing enzymes CYP11A1, 3beta-HSD, CYP17, and CYP19 in gonads and adrenals of adult and developing zebra finches. General and Comparative Endocrinology 119, 140-151. Goodson, J. L., Evans, A., and Soma, K. K. (2005). Neural responses to aggressive challenge correlate with behavior in nonbreeding sparrows. Neuroreport 16(5), 1719-1723. Griffiths, R., S. Daan, and Dijkstra, C. (1996). Sex identification in birds using two CHD genes. Proceedings of the Royal Society of London B 263, 1251-1256. Gurney, M. E., and Konishi, M. (1980). Hormone-induced sexual differentiation of brain and behavior in zebra finches. Science 208(4450), 1380-1383. Holloway, C. C , and Clayton, D. F. (2001). Estrogen synthesis in the male brain triggers development of the avian song control pathway in vitro. Nature neuroscience 4(2), 170-175. 51 Hutchison, J. B., Wingfield, J. C , and Hutchison, R. E. (1984). Sex differences in plasma concentrations of steroids during the sensitive period for brain differentiation in the zebra finch. Journal of Endocrinology 103, 363-369. Katagiri, M., Tatsuta, K., Imaoka, S., Funae, Y., Honma, K., Matsuo, N., Yokoi, H., Ishimura, K., Ishibashi, F., and Kagawa, N. (1998). Evidence that immature rat liver is capable of participating in steroidogenesis by expressing 17alpha-hydroxylase/17,20-lyase P450cl7. Journal of Steroid Biochemistry and Molecular Biology 64(1/2), 121-128. London, S. E., Monks, D. A., Wade, J., and Schlinger, B. A. (2006). Widespread capacity for steroid synthesis in the avian brain and song system. Endocrinology 147(12), 5975-5987. Mellon, S. H., and Griffin, L. D. (2002). Neurosteroids: biochemistry and clinical significance. Trends in Endocrinology and Metabolism 13(1), 35-43. Migues, P. V., Johnston, A. N. B., and Rose, S. P. R. (2002). Dehydroepiandosterone and its sulphate enhance memory retention in day-old chicks. Neuroscience 109(2), 243-251. Owen-Ashley, N. T., Hasselquist, D., and Wingfield, J. C. (2004). Androgens and the immunocompetence handicap hypothesis: unravelling direct and indirect pathways of immunosuppression in song sparrows. The American Naturalist 164(4), 490-505. Perlman, W. R., and Arnold, A. P. (2003). Expression of estrogen receptor and aromatase mRNAs in embroynic and posthatch zebra finch brain. Journal of Neurobiology 55(2), 204-219. Perlman, W. R., Ramachandran, B., and Arnold, A. P. (2003). Expression of androgen receptor mRNA in the late embryonic and early posthatch zebra finch brain. Journal of Comparative Neurology 455, 513-530. Phoenix, C. H., Goy, R. W., Gerall, A. A., and Young, W. C. (1959). Organizing action of prenatally administered testosterone propionate on the tissues mediating mating behavior in the female guinea pig. Endocrinology 65, 369-382. Schlinger, B., and Arnold, A. (1992). Plasma sex steroids and tissue aromatization in hatchling zebra finches: implications for the sexual differentiation of singing behavior. Endocrinology 130(1), 289-299. Schlinger, B. A., and Arnold, A. P. (1993). Estrogen synthesis in vivo in the adult zebra finch: additional evidence that circulating estrogens can originate in brain. Endocrinology 133(6), 2610-2616. Schlinger, B. A., Lane, N. I., Grisham, W., and Thompson, L. (1999). Androgen Synthesis in a Songbird: A Study of Cypl7 (17[alpha]-Hydroxylase/C17,20-Lyase) Activity in the Zebra Finch. General and Comparative Endocrinology 113(1), 46-58. Schlinger, B. A., and London, S. E. (2006). Neurosteroids and the songbird model system. Journal of Experimental Zoology 305A, 743-748. Schlinger, B. A., Soma, K. K., and London, S. E. (2001). Neurosteroids and brain sexual differentiation. TRENDS in Neurosciences 24(8), 429-431. Silverin, B., and Sharp, P. (1996). The development of the hypothalamic-pituitary-gonadal axis in juvenile great tits. General and Comparative Endocrinology 103, 150-166. 52 Soma, K. K., Alday, N. A., Hau, M., and Schlinger, B. A. (2004). Dehydroepiandrosterone metabolism by 3-beta-hydroxysteroid dehydrogenase in adult zebra finch brain: sex difference and rapid effect of stress. Endocrinology 145(4), 1668-1677. Soma, K. K., and Wingfield, J. C. (2001). Dehydroepiandrosterone in songbird plasma: seasonal regulation and relationship to territorial aggression. General and Comparative Endocrinology 123, 144-155. Soma, K. K., Wissman, A. M. B., E.A., and Wingfield, J. C. (2002). Dehydroepiandrosterone (DHEA) increases territorial song and the size of an associated brain region in a male songbird. Hormones and Behavior 41(2), 203-212. Tagawa, N., Katagiri, M., and Kobayashi, Y. (2005). Developmental changes of serum steroids produced by cytochrome P450cl7 in rat. Steroids 71(2), 165-170. Tarn, H., and Schlinger, B. A. (2006). Activities of 3beta-HSD and aromatase in slices of developing and adult zebra finch brain. General and Comparative Endocrinology Forthcoming. Toran-Allerand, C. D., Tinnikov, A., Singh, R. J., and Nethrapalli, I. S. (2005). 17{alpha}-estradiol: a brain active estrogen? Endocrinology, en.2004-1616. Vanson, A., Arnold, A. P., and Schlinger, B. A. (1996). 3[beta]-Hydroxysteroid Dehydrogenase/Isomerase and Aromatase Activity in Primary Cultures of Developing Zebra Finch Telencephalon: Dehydroepiandrosterone as Substrate for Synthesis of Androstenedione and Estrogens. General and Comparative Endocrinology 102(3), 342-350. Wade, J., and Arnold, A. P. (1996). Functional testicular tissue does not masculinize development of the zebra finch song system. PNAS 93(11), 5264-5268. Wikelski, M., Lynn, S., Breuner, C. W., Wingfield, J. C , and Kenagy, G. J. (1999). Energy metabolism, testosterone and corticosterone in White-Crowned Sparrows. Journal of Comparative Physiology A 185, 463-470. Williams, T. D., Dawson, A., Nicholls, T. J., and Goldsmith, A. R. (1987). Reproductive endocrinology of free-living nestling and juvenile starlings, Sturnus vulgaris; an altricial species. Journal of Zoology (London) 212(4), 619-628. 53 Chapter 3: General Discussion and Conclusions The goals of this study were to characterize plasma DHEA and E 2 levels in male and female starling chicks during development, as well as measure steroids in the songbird brain and peripheral tissues in selected ages during development. The findings of this study both support the hypothesis regarding alternative sources of steroids, as well as challenge the idea that the gonads do not play a role in steroid synthesis during development. Previous work has focused on the gonads secreting T, which is converted in the brain to E 2 . However, the data from this thesis indicate that DHEA is released by the gonads into the blood, suggesting that the gonads may play a role in steroid synthesis during development. Previous work in this field has focused primarily on 17p*-E2 as the primary steroid responsible during sexual differentiation of the brain - particularly because administering exogenous E 2 to juvenile female songbirds during the critical period yields a partial masculinization in adulthood (Adkins-Regan et al., 1994; Casto and Ball, 1996; Gurney and Konishi, 1980). However, this study revealed that plasma levels of E 2 are low during development. It is possible that it is not E 2 levels that are important, but the metabolism of E 2 downstream that plays a role in sexual differentiation of the brain. While this is the first study to examine steroid levels in the songbird brain and peripheral tissue, there are still more studies to pursue. Low levels of DHEA and non-detectable levels of E 2 in the brain were surprising. However, it is possible that levels of DHEA and E 2 are more localized in the brain, and this effect was diluted out by the dissection method employed. Another possibility is that DHEA is being converted to another estrogen, such as 17a-E2, Ei or E 3 in the brain. Future studies should focus on 54 taking more specific areas of the brain, such as HVC, NCM or RA, as well as measuring other estrogens in tissue and plasma that were not previously considered as 'active'. Moreover, only part of the liver was analyzed for DHEA during this experiment. As the avian liver is quite large, future studies should examine the whole liver for DHEA synthesis. In addition, tissue samples should be collected from more ages during development. This study focused on a limited number of ages, and several key important ages were missed, such as P4 and PIO. The ages chosen in this study were made a priori based on from previous studies (Casto, 2001; Hutchison et al., 1984; Silverin and Sharp, 1996; Williams et al., 1987). However, the results of this study suggest that a more intensive sampling during development may be useful. The findings of this study support the hypothesis that there are alternative sources of sex steroids, but challenge the idea that the gonads are inactive during the nestling phase. However, this study does not rule out the hypotheses that the brain is steroidogenic (neurosteroids) or that there may be effects of sex chromosome genes on the brain during sexual differentiation. Future studies should examine these two possibilities and their roles during sexual differentiation of the songbird brain. 55 REFERENCES Adkins-Regan, E., Mansukhani, V., Seiwert, C , and Thompson, R. (1994). Sexual differentiation of brain and behavior in the zebra finch: critical periods for effects of early estrogen treatment. Journal of Neurobiology 25(7), 865-877. Casto, J. M. (2001). Development and hormonal regulation of sex differences in the song system of European starlings (Sturnus vulgaris), Johns Hopkins University. Casto, J. M., and Ball, G. F. (1996). Early administration of 17-beta estradiol partially masculinizes song control regions and alpha-2 adrenergic receptor distribution in European Starlings (Sturnus vulgaris). Hormones and Behavior 30, 387-406. Gurney, M. E., and Konishi, M. (1980). Hormone-induced sexual differentiation of brain and behavior in zebra finches. Science 208(4450), 1380-1383. Hutchison, J. B., Wingfield, J. C , and Hutchison, R. E. (1984). Sex differences in plasma concentrations of steroids during the sensitive period for brain differentiation in the zebra finch. Journal of Endocrinology 103, 363-369. Silverin, B., and Sharp, P. (1996). The development of the hypothalamic-pituitary-gonadal axis in juvenile great tits. General and Comparative Endocrinology 103, 150-166. Williams, T. D., A. Dawson, Nicholls, T. J., and Goldsmith, A. R. (1987). Reproductive endocrinology of free-living nestling and juvenile starlings, Sturnus vulgaris; an altricial species. Journal of Zoology (London) 212(4), 619-628. 56 

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