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Neonatal Mice Possessing an Sry Transgene Show a Masculinized Pattern of Progesterone Receptor Expression in the Brain Independent of Sex Chromosome Status

Department of Psychology and Center for Neuroscience Research (C.K.W., J.L.P., P.S.Q.), University at Albany-State University of New York, Albany, New York 12222; Department of Physiological Science and Laboratory of Neuroendocrinology of the Brain Research Institute (J.X., A.P.A.), University of California, Los Angeles, California 90095; and Center for Neuroendocrine Studies (P.S.Q., G.J.D.), University of Massachusetts, Amherst, Massachusetts 01003

Address all correspondence and requests for reprints to: Christine K. Wagner, Department of Psychology, Social Science 369, 1400 Washington Ave, University at Albany, Albany, New York 12222. E-mail: cwagner{at}albany.edu.

Abstract

To assess the relative roles of sex chromosome genes and gonadal steroid hormones in producing sex differences in progesterone receptor (PR) expression in the forebrain of neonatal mice, we used mice in which the Sry gene had been deleted from the Y-chromosome and inserted as a transgene on an autosome in both XX and XY genotypes. Levels of PR immunoreactivity (PRir) in the anteroventral periventricular nucleus, the medial preoptic nucleus, and the ventromedial nucleus were significantly higher in mice that possessed an Sry transgene compared with mice that lacked an Sry transgene, regardless of their complement of sex chromosomes (XX vs. XY). This result suggests that sexual differentiation of PR expression in these regions is likely controlled mainly by gonadal hormones, not by the genetic sex of the brain cells. No differences in PRir were detected between wild-type XY mice with the Sry gene on the Y-chromosome and XY mice with the Sry transgene, suggesting that testicular hormones produced in these two genotypes have comparable effects on neural tissue.

THE TENET THAT gonadal steroid hormones are solely responsible for sexual differentiation of nongonadal tissues, including the brain, has endured for several decades. However, possible exceptions to the rule have been reported for neural and nonneural tissues of birds and mammals (1, 2, 3, 4). Recent evidence suggests that the complement of sex chromosomes (XX vs. XY) in brain cells may also contribute to sex differences in the brain. Sex differences in the expression of X and Y genes have been reported in the neonatal mouse brain (5). The relative contributions of gonadal hormones and genetic sex of brain cells can be assessed in mice in which gonadal sexual phenotype is independent of genetic sex of the brain, as for example in mice in which the testis-determining gene, Sry, is deleted from the Y-chromosome and inserted as a transgene into an autosome in both XX and XY genotypes (6, 7, 8). These mice produce offspring that include XX and XY males (i.e. mice that have the Sry transgene and therefore develop testes) and XX and XY females (i.e. mice that lack the Sry transgene and therefore develop ovaries). Comparing these mice in adulthood has shown a sex chromosomal effect on vasopressin innervation independent of gonadal sex (9). However, it is difficult in the case of sex differences observed in adulthood to determine whether sex chromosomes influenced the development of neural tissue directly. Sex chromosome complement may alter circulating levels of testicular hormones during development or may render the brain differentially sensitive to gonadal hormones during periods of sexual differentiation. A sex difference present during neural development that is sensitive to the effects of gonadal hormones could shed light on this issue. The expression of the progesterone receptor (PR) gene in developing brain lends itself well to this question

During perinatal life, there is a dramatic and transient sex difference in the expression of PR within several regions of the rat and mouse forebrain (10, 11, 12, 13). PR expression is significantly higher in fetal and neonatal male mice in the anteroventral periventricular nucleus (AVPv), the medial preoptic nucleus (MPN), and the ventromedial nucleus (VMN) (11). PR expression is induced in the developing male brain by estradiol, a metabolite of testosterone, acting via estrogen receptor (11, 12, 13). Although the higher levels of testosterone in neonatal males appear to be the main factor in the sex difference in PR expression, whether sex chromosome genes contribute directly to this sex difference has not been explored.

The present study examined PR immunoreactivity in the brains of neonatal XX and XY mice that either possessed or lacked an Sry transgene. In this regard, PR expression provides a unique marker of sexual differentiation because the sex difference lies in the expression of a single gene that is directly regulated by estradiol and its nuclear receptor. In addition, the sex differences in PR are not permanent but are transitory during development (13), offering a model of developmental plasticity not provided by other sexually differentiated systems of the central nervous system.

Materials and Methods

Animals
All mice, gift of Dr. Paul Burgoyne, were of the random-bred MF1 strain, except for the Y-chromosome, which derived from strain 129 (129/SvEv-Gpil^c Y) (14). In some mice, the Y-chromosome was deleted for the testis-determining gene Sry [Tdy^m1 mutation of Lovell-Badge and Robertson (6) and Gubbay et al. (7)], forming the Y^--chromosome, so that XY^- mice have ovaries and thus are called females. In some mice, an Sry transgene was inserted into an autosome, creating XXSry and XY^- Sry mice that develop testes (8). XY^- Sry males were bred with normal MF1 XX females to produce four genotypes in which gonadal status (ovaries vs. testes) was independent of sex chromosome status (XX vs. XY^-) (i.e. XX and XY^- have ovaries, XXSry and XY^- Sry have testes). In addition, wild-type MF1 XY¹²⁹ males were compared with XY^- Sry males to test for any differences attributable to the transgenic vs. the endogenous Sry gene.

Mice were housed and bred at the University of California, Los Angeles (UCLA) and all experimental procedures were approved by the UCLA Institutional Animal Care and Use Committee. Genotype was determined by PCR using analysis of the presence of Y long arm gene family Ssty and of the Sry transgene (14). Pregnant females were allowed to deliver normally. On the day of birth (postnatal d 0; P0) neonates were killed by decapitation. Brains were removed and immediately immersion fixed in 5% acrolein in 0.1 M phosphate buffer for 6 h at 4 C, then immersed in 30% sucrose in 0.1 M PBS overnight at 4 C. Brains were shipped to the University of Massachusetts, where they were sectioned coronally at 50 µm thickness on a sliding microtome and stored in cryoprotectant (30% sucrose, 0.1% polyvinyl-pyrrolidone-40 in ethylene glycol and 0.1 M phosphate buffer) at -20 C until immunocytochemical processing.

Immunocytochemistry
Immunocytochemistry was performed on free-floating sections according to previously published methods (11, 13). The primary antibody was a rabbit polyclonal antiserum (Dako Inc., Glostrup, Denmark) directed against the DNA binding domain of the human PR. This antiserum detects both the A and B isoforms of PR (15). PR antiserum was diluted to 1:1000 in TBS (pH 7.6) containing 2% normal goat serum, 0.3% Triton X-100, and 0.02% sodium azide for 72 h at 4 C. After 60 min in biotinylated goat antirabbit IgG (5 µg/ml; Vector Laboratories, Burlingame, CA) and incubation in ABC reagent (Vectastain Elite Kit, Vector Laboratories) for 60 min, tissue was incubated in TBS containing 0.05% diaminobenzidine, 0.75 mM nickel ammonium sulfate, 0.15%-ß-D-glucose, 0.04% ammonium chloride and 0.001% glucose oxidase for approximately 20 min. The sections were rinsed and mounted onto gelatin-coated slides and coverslipped. Preabsorption of the PR antiserum overnight with human PR (A and B isoforms) at 10 times higher molar concentration than that of the primary antiserum, or omission of the PR antiserum abolished all immunoreactivity (11, 12).

Analysis
Representative sections in the AVPv, the MPN, and the VMN for each animal were selected for image analysis by an experimenter blind to the genotype of the animals. Sections for analysis were chosen within the rostral MPN, the mid-AVPv and the caudal aspect of the ventrolateral VMN, which most closely corresponds with plate nos. 8/9 for the AVPv, plate no. 9 for the MPN and plate no. 17 for the VMN of the E22 rat brain atlas of Altman and Bayer (16). Microscopic images of the PRir in the AVPv, MPN and VMN were captured with an Olympus BH-2 microscope fitted with a CCD72 (Dage MTI, Michigan City, MI) camera that was connected to a QuickCapture frame grabber board (Data Translation Inc., Marlboro, MA) in a MacIntosh IIfx computer. National Institutes of Health (NIH) Image software (W. Rasband, NIH, Bethesda, MD) was used to analyze captured images. The total amount of PRir was determined for each region by measuring the area (µm²) covered by thresholded pixels [i.e. those pixels with a gray level higher than a defined threshold density (specific immunoreactive staining)]. Threshold was defined as the mean OD five times the SD higher than the mean background density. The mean background density was measured in a region devoid of PRir immediately lateral to the analyzed region containing PRir. Statistical analysis was performed using separate two-way ANOVAs (gonadal status x sex chromosome status) for each region. Preplanned comparisons were made using Student-Newman-Keuls (P < 0.05). Direct comparisons between wild-type males and transgenic XY^- Sry mice were made using Student’s t test (P < 0.05).

Results

There was no difference in PRir levels in the AVPv, MPN, and VMN of wild-type XY males and XY^- Sry mice (P = 0.53 for AVPv; P = 0.47 for MPN; P = 0.70 for VMN; Fig. 1). Levels of PRir were significantly higher in all three brain regions examined in mice that possessed an Sry transgene compared with mice that lacked an Sry transgene in both XX and XY^- sex chromosomal genotypes (Fig. 2). Two-way ANOVA revealed a significant main effect of Sry transgene in the AVPv (F_1,31 = 36.091, P < 0.0001), the MPN (F_1,34 = 116.466, P < 0.0001) and the VMN (F_1,34 = 201.301, P < 0.0001). There was no significant main effect of sex chromosome complement (XX vs. XY^-), nor a significant interaction between Sry transgene (i.e. gonadal status) and sex chromosome status in any of the regions. Post hoc analysis revealed that male XXSry and XY^- Sry male mice had significantly higher levels of PRir compared with XX and XY^- female mice respectively in all three regions (P < 0.05). In no region was there a significant difference between XXSry and XY^- Sry or XX and XY^-.

View larger version (15K): [in this window] [in a new window]   FIG. 1. Relative amount of PRir in the AVPv, the MPN, and the VMN in wild-type XY and XY- Sry male mice.

View larger version (15K): [in this window] [in a new window]   FIG. 2. Relative amount of PRir in the AVPv, the MPN, and the VMN in XX and XY neonatal mice that either lack or possess an Sry transgene on an autosome. *, Significantly different from Sry (P < 0.05).

The present study tested the influence of gonadal secretions as well as genetic sex on the expression of PR in perinatal brains. Mice with testes (XY, XY^- Sry, and XXSry) differed dramatically from the two groups without testes (XX and XY^-) in all three regions examined. In no case was there a group difference attributable to the complement of sex chromosomes. Thus, XY^- Sry males did not differ from XX males and XY^- females did not differ from XX females. In addition, there was no interaction between sex chromosome complement and the presence or absence of testes. These results indicate that XX and XY^- cells in these brain regions can respond fully, at least in terms of PR expression, to the differentiating influences of gonadal steroid hormones irrespective of their complement of sex chromosome genes.

The present results suggest that males with XXSry and XY^- SRY genotype have functionally equivalent levels of gonadal hormones prenatally as do females with XX and XY^- genotype. Sex differences in the expression of PR on the day of birth depend on the presence of gonadal steroids prenatally. Treatment of females prenatally with either testosterone or estrogen increased PR expression and abolished the sex difference in PR seen in fetal rats (12). Likewise, the aromatase inhibitor, 1,4,6-androstatriene-3,17-dione, decreased PR in fetal male rats to levels normally seen in females (12). Moreover, the sex difference in PR is abolished in estrogen receptor knockout mice. PR expression in knockout males is not different from that seen in wild-type females (11). In this regard, PR expression on the day of birth is a very sensitive barometer of the prenatal actions of gonadal steroids, particularly the testosterone metabolite estradiol.

The present study reveals that the expression of PR is not different between any of the groups with testes, whether they possessed the endogenous Sry gene, or the Sry transgene on either an XY- or an XX-chromosome background. Similarly, the two female groups were indistinguishable from one another in PR expression. These results suggest that sex chromosome complement does not significantly influence prenatal gonadal hormone levels or the ability of the fetal brain to respond to estradiol. These findings are consistent with the idea that the effects of sex chromosome complement on sex differences in vasopressin innervation in adult mice (9) cannot simply be explained by differences in prenatal levels of testosterone or its metabolite, estradiol. Similarly, the present results demonstrating that males with the endogenous Sry gene and males with the Sry transgene do not differ in the expression of PR on the day of birth suggest that differences in sexual behavior and neural morphology found previously between these groups (9) are more likely attributable to differential genetic effects of the transgene vs. the wild-type gene rather than to differences in prenatal levels or actions of gonadal steroid hormones.

Acknowledgments

We thank Dr. Paul Burgoyne for the gift of the mice.

Footnotes

This work was supported by NIH Grants MH59268 and NS43196 (to A.P.A.), NIH Grant HD37244 (to C.K.W.), and National Research Service Award MH65874 (to P.S.Q.).

Abbreviations: AVPv, Anteroventral periventricular nucleus; MPN, the medial preoptic nucleus; PR, progesterone activity; PRir, PR immunoreactivity; VMN, ventromedial nucleus.

Received September 15, 2003.

Accepted for publication November 18, 2003.

References

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