The effects of estradiol on 17β-hydroxysteroid dehydrogenase type IV and androgen receptor expression in the developing zebra finch song system

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  The effects of estradiol on 17β-hydroxysteroid dehydrogenase type IV and androgen receptor expression in the developing zebra finch song system
  THE EFECTS OF ESTRADIOL ON 17 β -HYDROXYSTEROIDDEHYDROGENASE TYPE IV AND ANDROGEN RECEPTOREXPRESSION IN THE DEVELOPING ZEBRA FINCH SONGSYSTEM J. Bayley Thompson a , Eldin Dzubur  a , Juli Wade b , and Michelle Tomaszycki a,CA a  Wayne State University, Department of Psychology, Detroit MI 48202, USA b  Michigan State University, Department of Psychology and Neuroscience Program, EastLansing, MI 48824, USA  Abstract Recent work in zebra finches suggests that genes and hormones may act together to masculinizethe brain. This study tested the effects of exogenous estradiol (E2) on 17 β -Hydroxysteroid Dehydrogenase type IV (HSD17B4) and the co-localization of HSD17B4 and androgen receptor (AR) mRNA. We asked three primary questions: First, how does post-hatching E2 treatment affectHSD17B4 mRNA expression in males and females? Second, is this gene expressed in the samecells as AR, and, third, if so does E2 modulate co-expression? Female finches implanted with50 μ g of E2 on the third day post-hatching showed a significant increase in the density of cellsexpressing HSD17B4 and AR in HVC at day 25. Co-localization of AR cells that also expressed HSD17B4 was high across groups (>81%). We found significant sex differences in co-localizationin both the HVC and Area X of control animals, with males showing a higher percentage of cellsexpressing AR mRNA that also expressed HSD17B4 in comparison to females. However,although E2 treatments significantly increased the number of cells expressing HSD17B4 mRNAand AR mRNA in the HVC of females, the percentage of HSD17B4 cells co-expressing AR wasreduced in HVC and Area X in E2-treated animals. These results lend support to the hypothesisthat genes and hormones may act in concert to modulate the sexually differentiation of the zebrafinch song system. Further, the data suggest that a single hormonal mechanism cannot mimic thecomplex development of male singing behavior and associated song nuclei. Keywords Zebra finch; masculinization; estradiol; song nuclei; genes; hormones; 17 β  hydroxysteroid dehydrogenase type IV; in situ  hybridization © 2010 Elsevier B.V. All rights reserved.Corresponding author: Michelle L. Tomaszycki, PhD, Department of Psychology, Wayne State University, 5057 Woodward, DetroitMI, 48202, Phone: (313)577-0341, Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may bediscovered which could affect the content, and all legal disclaimers that apply to the journal pertain.  NIH Public Access Author Manuscript  Brain Res . Author manuscript; available in PMC 2012 July 15. Published in final edited form as: Brain Res  . 2011 July 15; 1401: 66–73. doi:10.1016/j.brainres.2011.05.031. NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t    1. Introduction Sex differences in behavior are widespread across species and can be linked tocorresponding sex differences in the brain. For example, zebra finches exhibit strikingsexual dimorphisms in singing behavior that parallel differences in brain morphology. Malefinches sing and females do not, and the brain areas controlling song are substantially larger in males. For example, Area X, a region important for song learning, is present in adultmales, but does not develop in females (Nottebohm & Arnold, 1976). The lateralmagnocellular nucleus of the anterior nidopallium (LMAN) is also important for songlearning. Although LMAN is sexually monomorphic in volume, the LMAN of males haslarger somas and nucleoli than that of females (Nixdorf-Bergweiler, 2001). HVC (proper name) and the robust nucleus of the arcopallium (RA) are responsible for the motor aspectsof song and these areas are larger in volume, have more cells, and larger somas in malesthan in females (Wade, 2001).Research has focused primarily on hormonal control of song system masculinization. Infemales, E2 administered early in development can partially masculinize song nuclei and can even result in the production of male-typical song, although results vary greatly betweenindividual females (Holloway & Clayton, 2001; Simpson & Vicario, 1991a, 1991b).However, no hormonal treatments have reliably masculinized females, and few studies havedemasculinized males by limiting hormone exposure. For example, treatments with anti-estrogens (Mathews & Arnold, 1990; Mathews & Arnold, 1991; Mathews et al., 1988) or estrogen-synthesis inhibitors (Balthazart et al., 1995; Wade & Arnold, 1994) do not inhibitmasculinization (Mathews & Arnold, 1990). Castration concurrent with flutamide treatment(an androgen antagonist) does not completely prevent masculinization (Bottjer & Hewer,1992). However, treating slices from male song nuclei with estrogen antagonists inhibitsmale-typical connections between HVC and RA (Holloway & Clayton, 2001), suggestingsome role for hormones, albeit in vitro. More recent research has revealed that male-typicalcell sizes in HVC and RA can be demasculinized by direct intracranial injections of anestrogen receptor antagonist (Bender & Veney, 2008). Thus, the role for hormones in thesexual differentiation of the song system remains unclear.One might expect that E2 masculinizes females by acting at estrogen receptors (ERs) insong nuclei. However, adult zebra finches have relatively few ERs in the song system; theseare detectable only in HVC and at low levels (Metzdorf et al., 1999). In contrast, androgenreceptors (ARs) are abundant in Area X, LMAN, RA and HVC (Gahr & Metzdorf, 1997;Kim et al., 2004). AR expression is sexually dimorphic in HVC as early as P9 (Gahr &Metzdorf, 1999a) and parallels the increase in volume of HVC in male zebra finches (Kim,et al., 2004). Despite the abundance of ARs in song nuclei, androgen treatments in femalezebra finches have little effect on masculinization, and treatments with an androgen receptor antagonist, flutamide only partially demasculinize song regions in males (Grisham et al.,2007). However, flutamide prevents the masculinizing effects of E2 in females, suggestingthat ARs are an important component of the process through which masculinization occurs(Grisham et al., 2002). Additionally, E2 up-regulates AR mRNA expression in Area X and HVC (Kim, et al., 2004; Nordeen et al., 1986), and treatments of fadrozole (an aromataseinhibitor) decrease AR mRNA expression in these same regions (Kim, et al., 2004).In addition to effects of steroid hormones, it is likely that the masculinization of the songsystem involves direct genetic effects. This idea was first illustrated by a gynadromorphiczebra finch. The right side of this spontaneously occurring bird had a male gonad, masculine plumage, and male-biased expression of sex chromosome genes (males birds are ZZ;females ZW), while the right was genotypically and phenotypically female (Agate et al.,2003). Morphology of the forebrain song control system indicated roles for both direct Thompson et al.Page 2  Brain Res . Author manuscript; available in PMC 2012 July 15. NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t    genetic effects and one or more diffusible factors on sexual differentiation. Studies haveidentified specific genes that are both sex-linked and expressed at increased levels in themale than the female song system (Chen et al., 2005; Duncan & Carruth, 2007; Tang et al.,2007; Tang & Wade, 2006; Tomaszycki et al., 2009). Recent research suggests that some of these genes may interact with AR to facilitate masculinization (Tang & Wade, 2010; Wu, etal., 2010).The present study investigated the Z-linked gene encoding HSD17B4, which converts E2into a less active estrogen, estrone (de Launoit & Adamski, 1999). Genomic analysis of HSD17B4 confirms a role of this gene in steroid synthesis and, indeed, ERa responseelements have been predicted from this analysis (London & Clayton, 2010). HSD17B4mRNA is sexually dimorphic in the song system across a range of ages. At day 25 post-hatching, this gene is expressed in 3 song regions: LMAN, Area X and HVC (Tomaszycki,et al., 2009). Expression is higher in the male zebra finch LMAN as early as day 5 (Londonet al., 2010) and is significantly increased in males than in females in HVC at post hatch day25 (Tomaszycki, et al., 2009). This study asks three primary questions: First, how does E2treatment affect HSD17B4 mRNA expression in day 25 males and females? This age is particularly relevant to development of male brain morphology and behavior. The forebrainsong circuit is rapidly differentiating, and it is early in the period when males learn songfrom their fathers. We hypothesize that E2 will increase HSD17B4 expression in the HVCof females, since E2 can partially masculinize females and one plausible mechanism is anincrease in male-biased gene expression. Second, is this gene located in the same cells asAR? If cells express both AR and HSD17B4, it will suggest that they may act in concert to produce masculinization. Third, does E2 treatment also affect co-localization of AR and HSD17B4? If so, E2 may influence masculinization by modulating the interaction betweenthese two genes. 2. Results 2.1 HSD17B4 Two-way (sex by treatment) ANOVAs were conducted for each brain region. We examined three song nuclei: HVC, LMAN and Area X. RA was not analyzed because it did not showspecific staining for HSD17B4 in this or a previous study (Tomaszycki, et al., 2009). InHVC, there was no significant main effect of sex ( F  =2.550,  p =0.127) or treatment ( F  <1) onHSD17B4 mRNA expression. However, a significant sex by treatment interaction for HSD17B4 was detected ( F   =7.213,  p =0.015, fig 1). Control males had a greater number of cells expressing HSD17B4 than did control females ( t   =4.188,  p =0.002) and treatment withE2 increased the expression of HSD17B4 mRNA expression in females but decreased it inmales, such that E2-treated animals did not significantly differ from each other ( t  <1).In Area X, no significant main effects were detected for [sex: ( F  <1), treatment: ( F  =1.961,  p =0.178)], nor was there a significant interaction ( F  <1, fig 2). The same was true for HSD17B4 mRNA expression in LMAN [sex: ( F  <1), treatment: ( F  =1.964,  p =0.177),interaction: ( F  <1), fig 3]. 2.2 Androgen Receptor  For HVC, no main effect of either sex ( F  <1) or treatment ( F  <1) existed. However, asignificant sex  by treatment interaction was found for AR mRNA expression in this brainregion ( F  =17.780,  p =0.001, fig 1). E2 increased the number of cells expressing AR  in theHVC of females and decreased expression in the same region in males, such that E2 maleshad lower levels of expression than did E2 females ( t   =3.318,  p =0.008). Also, control males Thompson et al.Page 3  Brain Res . Author manuscript; available in PMC 2012 July 15. NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t    had a significantly higher number of cells expressing AR than did control females ( t   =2.990,  p =0.014).In Area X, a main effect of treatment was observed for cells expressing AR mRNA ( F  =11.348,  p =0.003, fig 2). Estrogen-treated animals had significantly fewer cells expressingAR mRNA than did controls. However, there was no significant effect of sex, ( F  <1), nor was there a significant sex by treatment interaction, ( F  <1).In LMAN, AR mRNA expression did not differ overall between the sexes, ( F  <1), or acrosstreatments, ( F  =2.756,  p =0.113), nor was there a significant interaction between the two,( F  =0.428,  p =0.521, fig 3). 2.3 Co-localization of HSD17B4 and AR mRNA ANOVAs were run on arcsine transformed percentages to test the effects of sex and E2treatment on the co-localization of HSD17B4 and AR mRNA. Co-localization wasexamined in two ways: the percentage of AR mRNA cells expressing HSD17B4 and the percentage of HSD17B4 mRNA cells expressing AR, to obtain a more complete picture of co-localization patterns.The percentage of AR mRNA cells expressing HSD17B4 was high across groups and brainregions (>81%). However, males (regardless of treatment) had a greater percentage of AR mRNA expressing cells that also expressed HSD17B4 than did females in HVC ( F  =6.482,  p =0.020), as well as Area X ( F  =4.653,  p =0.044, see Table 1). This effect did not occur inLMAN ( F  <1). Significant effects of treatment were not detected in any of the regionsexamined [HVC: ( F  <1); Area X: ( F   =2.76,  p =0.113); LMAN: ( F  <1)]. Also, there were nosignificant sex by treatment interactions in HVC ( F  =1.83,  p =0.192), Area X ( F   =2.22,  p= 0.153), or LMAN ( F  <1).The percentage of HSB17B4 expressing cells that co-expressed AR mRNA was also high inHVC across groups, but lower and more variable within Area X and LMAN. While an effectof sex was not detected [HVC: ( F  <1); Area X: ( F= 1.313,  p =0.266); LMAN: ( F  =1.008,  p =0.328), see Table 1], E2 significantly decreased co-localization in Area X (E2: F  =6.602,  p= 0.019) and LMAN ( F  =5.094,  p =0.036), but not in HVC ( F  <1). Finally, no significant sex by treatment interactions were seen in any of the song regions [HVC: ( F  =2.240,  p =0.151);Area X: ( F  <1); LMAN: ( F  <1)]. 3. Discussion Overall, we confirmed findings that sexual dimorphisms in HSD17B4 mRNA expression at post hatch day 25 are confined to HVC (Tomaszycki et al., 2009). We further demonstrated that E2 treatments beginning at post hatch day 3 increased expression of HSD17B4 mRNAin the HVC of females and confirmed E2-induced increases in AR mRNA expression in thefemale HVC as well (Kim, et al., 2004). These same treatments decreased HSD17B4 and AR mRNA expression in the HVC of males. Co-localization of HSD17B4 and AR was highacross sexes and treatment groups, although males had a significantly greater degree of co-localization than did females in HVC and Area X, but not in LMAN. Taken together, theseresults suggest that E2 may partially masculinize females by separately increasingHSD17B4 and AR in the song system, but the inability of E2 to completely masculinizefemales may relate to the failure to increase the co-localization of these two factors. 3.1 Effects of E2 on HSD17B4 and AR mRNA expression3.1.1 HVC— The higher number of cells expressing HSD17B4 mRNA in the HVC of malescompared to females is consistent with the hypothesis that this gene is involved in Thompson et al.Page 4  Brain Res . Author manuscript; available in PMC 2012 July 15. NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t    masculinization. Since HSD17B4 converts E2 into estrone, for which estrogen receptorshave a lower affinity (de Launoit & Adamski, 1999), HSD17B4 may effectively decreaseactivity at ERs in HVC. In parallel, ER-immunoreactivity decreases in males in this regionat post hatch day 30 (Schlinger, 1997). Further research should evaluate this relationship.Sex differences in HSD17B4 mRNA expression, however, may not lead to sexuallydifferentiated downstream responses. Recently, London et al. (2010) showed that adult sexdifferences in HSD17B4 mRNA expression do not translate into a sex difference in enzymeactivity. Further research should determine whether or not this is the case duringdevelopment.Given that E2 can partially masculinize females, we predicted that E2 might accomplish thisat least in part by increasing expression of male-biased genes, such as HSD17B4. Our hypothesis was supported, suggesting that E2 may partially masculinize females byincreasing the activity of Z-linked genes. Surprisingly, E2 treatments decreased HSD17B4expression in males. At least two explanations are plausible, the first of which fits with thehypothesis above: (1) too much E2 may demasculinize males, or (2) E2 may not masculinizemales at all. One should also consider, however, that group differences in the number of cells within a specified area (as measured here) may not reflect relationships in the totalnumber of cells expressing HSD17B4 or the level of its activity. These patterns should beinvestigated before firm conclusions can be drawn.We also replicated previous findings on sex differences in AR mRNA expression in theHVC of males compared to females (Gahr & Metzdorf, 1999a) and an increase in AR mRNA expression in females as the result of E2 treatments (Kim, et al., 2004), using alower dosage of E2 (50 μ g vs. 83 μ g).We predicted that HSD17B4 would be located in the same cells as androgen receptors. Thisco-localization might play a part in the masculinization process, since estrogen receptors arelow in the song system (Gahr & Metzdorf, 1999b). Males had a significantly higher degreeof co-localization (AR cells expressing HSD17B4) in HVC in comparison to females. Theseresults mirror those found with sorting nexin 2 and ribosomal proteins L17 and L37 (Tang &Wade, 2010; Wu et al., 2010), and collectively point to the possibility that the co-localization of a variety of Z-chromosome genes and AR may be important for themasculinization of the song system. However, E2 treatments had no effect on the extent of the co-localization in the present study. This failure of E2 to increase co-localization alongwith the up-regulation of AR and HSD17B4 may explain why E2 only partiallymasculinizes the female zebra finch. Simply mimicking male levels of expression is notenough; male-typical patterns of co-localization may be necessary for completemasculinization. Previous research has shown that E2 treatments do not reliably masculinizesinging behavior in females (Arnold, 1997; Simpson & Vicario, 1991a), and we may nowhave at least one underlying mechanism for this result. It is possible that there are other mechanisms by which male-typical levels of co-localization are achieved. Perhaps femaleshave a complementary system by which genes on the W chromosome may act to inhibitmasculine patterns of co-localization (Arnold, 1996).Thus, in HVC at least, we have some novel information that might elucidate the mechanisms by which E2 masculinizes females, via HSD17B4 and AR. However, the processes do notaccount for normal masculinization, which suggests that separate mechanisms are likelyinvolved. 3.1.2 Area X— Lesions of Area X during development lead to deficits in song productionin males (Doupe & Solis, 1997), suggesting that sex differences in this region at day 25 may Thompson et al.Page 5  Brain Res . Author manuscript; available in PMC 2012 July 15. NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t  
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