Human Molecular Genetics, 2002, Vol. 11, No. 12 1409-1419
© 2002 Oxford University Press
Sex differences in sex chromosome gene expression in mouse brain
1Department of Physiological Science and Laboratory of Neuroendocrinology of the Brain Research Institute, University of California, Los Angeles, CA 90095-1606, USA and 2Division of Developmental Genetics, MRC National Institute for Medical Research, Mill Hill, London NW7 1AA, UK
Received January 25, 2002; Accepted March 29, 2002
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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A major question is whether genes encoded on the sex chromosomes act directly in non-gonadal tissues to cause sex differences in development or function, or whether all sex differences in somatic tissues are induced by gonadal secretions. As part of this question we asked whether mouse X–Y homologous gene pairs are expressed in brain in a sex-specific fashion. Using RT–PCR and northern blot analysis, we assessed mRNA expression in brain of eight Y-linked genes as well as their X-linked homologues, at three ages: 13.5 days post coitum, the day of birth (P1) and adult. Transcripts of six Y genes were expressed at one or more ages: Usp9y, Ube1y, Smcy, Eif2s3y, Uty and Dby. Their expression also occurred in XY female brain, and therefore does not require testicular secretions. Six X-linked homologues (Usp9x, Ube1x, Smcx, Eif2s3x, Utx and Dbx) were also expressed in brain, and in adulthood all of these transcripts were expressed at significantly higher levels in brains of females than in brains of males, irrespective of their X-inactivation status. For five of these gene pairs, the expression of the Y-linked homologue in males was not sufficient to compensate for the female bias in X gene expression. Three X–Y gene pairs, Usp9x/y, Ube1x/y and Eif2s3x/y, appeared to be differentially regulated (expressed in brain in a different age- or tissue-dependent pattern), and hence may not be functionally equivalent. These sex differences in X–Y gene expression suggest several mechanisms by which these genes may participate in sex differences in brain development and function.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Gonadal steroid hormones are thought to be the primary molecular signals that initiate sexual differentiation of non-gonadal tissues. This concept has been proven for numerous sexually dimorphic tissues such as the sex ducts, genitalia and brain (1–3). In mammals, male fetuses and neonates experience higher levels of testosterone, secreted from the testes, than females. Testosterone (or its metabolite estradiol) initiates a program of male-specific development in a number of brain regions, resulting in anatomical sexual dimorphisms that control sexually dimorphic behaviors and functions (4,5). Recent evidence, however, indicates that some sexual dimorphisms in brain and other tissues cannot be easily explained as the result of gonadal steroid action (6). For example, primary cell cultures of rat and mouse midbrain or hypothalamus undergo sexual differentiation in vitro even though the tissue is harvested prior to the onset of sexually dimorphic gonadal secretions (7,8). In wallabies, the scrotum and pouch begin to differentiate before the gonads – not as the result of gonadal secretions but determined by the number of X chromosomes (9,10). In mice, XY males differ from XX males, and XY females differ from XX females, in the density of vasopressin fibers in the lateral septum, a sexually dimorphic character (G. De Vries, E. Rissman, R. Simerly, L.-Y. Yang, E. Scordalakes, C. Auger, A. Swain, R. Lovell-Badge, P. Burgoyne and A. Arnold, manuscript in preparation). Other evidence implicates the Y chromosome in the control of aggression and other behavioral and neural phenotypes (11,12).
These studies raise the question whether sex chromosome genes, which are present in different quantities in the genomes of males and females, might be expressed in the brain and cause sex-specific patterns of development and/or function. Male mammals possess genes on the non-recombining region of the Y chromosome (NRY) that are not present in females, and which might act in the brain to cause masculine patterns of neural development. Alternatively, genes on the non-pseudoautosomal portion of the X chromosome (NPX), which are present in two doses in females but only a single dose in males, could cause sex-specific neural development because of such dosage differences. For most NPX genes, dosage in the two sexes is thought to be balanced because of X inactivation, which silences expression of one of the two X copies. Nevertheless, a significant percentage of NPX genes escape inactivation (at least in humans), so that dosage in the two sexes may not be equivalent (13).
For most mouse Y genes, relatively little is known about their expression in brain. Some Y genes have been reported to be expressed in the brain at one or more ages and in different brain regions (see e.g. 14–18), but there has been little systematic study with an eye to discovering possible induction of sex-specific patterns of neural development. We therefore set out to analyze expression of a panel of NRY genes in the brain of fetal, neonatal and adult mice, as a first step towards understanding whether any of these genes is in a position to contribute to sex differences in the brain. NRY genes have previously been divided into two classes for humans – those expressed ‘ubiquitously’ and those expressed in a ‘testis-specific’ manner (19) – although exceptions to this classification are already apparent (20,21). In mice, a similar classification has been employed for the eight NRY genes examined here – those expressed in numerous tissues, including Smcy, Uty, Eif2s3y and Dby (16–18,22), and those expressed (at least in adults) predominantly or exclusively in testis, such as Rbmy, Ube1y, Usp9y and Zfy1/2 (23–27). We have not included Sry in the current study, because its expression in brain has been studied more extensively than that of other NRY genes (14,28–31).
We also studied the androgen dependence of Y-gene expression indirectly by comparing expression in XY male and XY female mice. The XY female mice carry a 11 kb deletion in the Y chromosome (hereinafter designated Y-) which removes the testis-determining gene Sry (32). Thus, XY- mice possess ovaries and hence are called females. We also studied XY-Sry male mice in which the Sry deletion has been complemented by an autosomally located Sry transgene (33). XY-Sry mice are fully functional males that experience sufficient androgen secretion to masculinize the genitalia and specific regions of the central nervous systems (34). If the expression of NRY genes in brain requires testosterone, one would predict that these two types of mice show different patterns of NRY gene expression.
Determining if these genes have a male-specific effect is complicated by the fact that most NRY genes have a closely related NPX homologue. These X–Y homologues are thought to be ancient alleles, but because the NPX and NRY segments of the sex chromosomes no longer recombine in males, these genes have evolved as distinct forms (35). If NPX and NRY homologues are functionally equivalent, any potential sex difference in brain function due to expression of an NRY gene could be eliminated by the expression of its NPX partner, particularly if it is exempt from X inactivation in females. We have therefore examined the expression of six NPX genes that are homologues of NRY genes. In each case, the NPX gene is expressed at higher level in female than in male brain, and in most cases the Y homologue is expressed at a level short of that required to compensate for this sex difference.
Our results confirm previous studies indicating that ‘ubiquitously expressed’ NRY genes are expressed in brain. Moreover, we have found that Usp9y, previously thought to be testis-specific, is expressed in brain. For the NRY genes reported here to be expressed in brain, the homologous X partners are detected in the brains of both sexes at all three ages examined. We also find that, in some cases, the developmental profile and tissue distribution of Y-gene expression appear not to parallel those of its NPX partner gene, suggesting that the two genes may be differentially regulated and thus may not be equivalent in function.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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RT–PCR analysis of neural expression of a panel of eight Y genes
We first evaluated the tissue specificity of gene expression in adulthood for eight NRY genes using cDNA from testis, brain, muscle, liver, heart and kidney. The results obtained were in agreement with previously published RT–PCR data. The four ‘ubiquitously expressed’ Y genes were detected in every tissue examined, whereas Ube1y was detected in testis and in brain, and Usp9y, Rbmy and Zfy1 only in the testis (not shown).
We then used RT–PCR to test further for brain expression at 13.5 days post coitum (dpc) and the day of birth (P1), using adult brain for comparison. As expected, the transcripts of the ubiquitously expressed NRY genes (Smcy, Uty, Eif2s3y and Dby) were also detected in the developing brain (Fig. 1A). Transcripts of Rbmy and Zfy1 were not detected in brain at any age (not shown). Transcripts of the two other genes – Ube1y (previously detected at a very low level in adult brain) and Usp9y (previously described as testis-specific) – were detected in the developing brain at 13.5 dpc and P1 (Fig. 1A). The expression of Usp9y in adult brain in this experiment was extremely weak (Fig. 1A) – on the borderline of detectability.
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Two Uty PCR products were generated when the region between bp 1279 and bp 1529 [GenBank accession no. AF057367, (18)] was amplified (Figs 1–3). Sequence comparison between Uty cDNA (GenBank accession no. AF05767) and the genomic sequence (which is within the BAC clone GSMB-187H15, GenBank accession no. AC006508) revealed that there are 25 exons in the Uty gene. The forward and reverse primers (F and R1) are located within exons 12 and 14, respectively. The sequences of the two Uty PCR products indicated that exon 13 was absent in the shorter isoform. To further confirm this result, a second reverse primer R2 was designed to span the junction of exons 13 and 14, so that only the longer isoform containing exon 13 was amplified (data not shown). Because exon 13 contains 135 bases, its deletion does not cause a frameshift.
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Y-gene expression does not require the presence of a testis
We next examined the expression of NRY genes in XY- female and XY-Sry male mice and, as controls, in their sibling XX females and XXSry males. At P1, the XY- females and XY-Sry males showed a similar expression pattern of NRY transcripts (Fig. 2A), indicating that the expression of NRY genes was not dependent on testicular secretions, including male levels of androgens. Because we have not used RT–PCR quantitatively, these results do not rule out more subtle regulation of Y-gene expression by gonadal secretions.
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Because Usp9y expression was lower in adult male brain than in P1 male brain (Fig. 1A), and the RT–PCR product of Usp9y appeared to be less abundant in XY-Sry males than in XY- females at P1 (Fig. 2A), we speculated that androgens might downregulate the expression of Usp9y in adulthood. As a partial test of this idea, we performed PCR amplification of Usp9y on cDNA derived from brains of adult XY- female mice and XY-Sry male mice (Fig. 2B). Usp9y transcripts were again very low or undetectable in these adults, consistent with the idea that the absence of Usp9y in adult male brain is not caused by testicular secretions such as androgens. Usp9y was readily detected in cDNA derived from the positive control, adult C57BL/6 XY testis (Fig. 2B). Usp9y was not detected from adult MF1Y129 XY- females and XY-Sry males (Fig. 2B), in minor contrast to the very low level detected in C57BL/6 males (Fig. 1A).
For three Y genes (Eif2s3y, Dby and Smcy ) for which our probes detect specific expression of the Y homologue in northern blots, we quantified group differences in Y-homologue expression in adult XY- and XY-Sry mice by probing a northern blot of mRNA derived from three independent samples from each group (data not shown). In each case, no group difference was detected (ANOVA, P>0.05).
Are Y-linked and X-linked homologues expressed in parallel in males?
Because the NPX and NRY homologues are thought to be functionally quite similar, we used RT–PCR to determine if the neural expressions of X and Y homologues covary as a function of age. Of the six NRY genes expressed in the developing brain, all have NPX homologues. Five of these NPX genes were expressed at all three ages and thus have a pattern of expression similar to their NRY partners, although the level of NPX expression appears higher than that of NRY genes (Fig. 1A,B). In contrast, Usp9x was expressed at all three ages, whereas Usp9y was clearly detected at 13.5 dpc and P1, but only weakly in the adult.
To compare NRY and NPX expression across tissues, we assayed P1 male brain, lung and liver (Fig. 3A). The four ubiquitously expressed Y genes and their X homologues were detected in all three tissues tested. In contrast, Ube1y and Usp9y showed an expression pattern apparently different to that of their NPX homologues. Ube1y was only detected in the brain, whereas Ubelx was detected in all tissues. Usp9y was found in both brain and lung, with the level of PCR amplification being much higher in the brain than in the lung, whereas Usp9x was expressed in all tissues tested (Fig. 3B).
We used northern blot analysis to evaluate differential expression of the X–Y homologous gene transcripts at various times of development or in various tissues. This analysis was restricted to three gene pairs (Smcx/y, Eif2s3x/y and Dbx/y), for which our probes allow some discrimination between X and Y transcripts (Fig. 4). We found that X and Y transcripts had similar patterns of expression for Smcx/y and Dbx/y, but not for Eif2s3x/y. Three mRNA isoforms of Eif2s3y were detected, but only one for Eif2s3x. In brain, isoform A of Eif2s3y was predominant at 13.5 dpc, but was considerably reduced in adult brain and in the other tissues tested. Eif2s3y and Eif2s3x were not expressed in a completely parallel fashion, in that the ratio of embryonic to adult expression of Eif2s3y was larger than that of Eif2s3x, and the ratio of brain to non-brain expression in adults was lower for Eif2s3y than for Eif2s3x. We detected three isoforms for Smcx and two for Smcy. Smcx and Smcy were expressed at a higher level at 13.5 dpc than in adulthood. In adult males, expression of each of these was higher in brain than in heart, kidney and liver. Dbx and Dby also had a similar expression pattern. Neural expression was higher than that of peripheral tissues, and relatively little change was observed between embryonic and adult brains.
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We further analyzed the coordinated expression of these three X–Y pairs by quantifying the levels of expression found in northern blots and calculating the correlation coefficient relating X and Y mRNA levels (of the most abundant isoform) in the five male tissues shown in Figure 4. Smcx/y were significantly correlated (r=0.99, P<0.001) and Dbx/y were nearly significantly correlated (r=0.79, P=0.13), but Eif2s3x/y were not (r=0.08, P=0.91).
Six NPX genes are expressed at a higher level in females than in males; the sex difference is not compensated by expression of Y homologues
Both male and female brains express all six NPX genes examined, among which Smcx, Eif2s3x, and Utx have been reported to escape X inactivation (18,19,22,36,37), whereas Ube1x is subjected to X inactivation (38). The X-inactivation status of Dbx is unclear and Usp9x has been suggested to be X-inactivated based on expression profiles (26). We quantified sex differences in the expression of these six NPX genes in whole adult brain by probing a northern blot of mRNA derived from four independent samples each of male and female adult brains. Surprisingly, expressions of Usp9x, Ube1x, Smcx, Eif2s3x, Utx and Dbx were all significantly higher in female brain than in male brain (Fig. 5A,B), independent of the reported inactivation status of these genes.
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An important question is whether expression of the Y homologues in males is sufficient to compensate for the lower level of expression of the X genes examined in Figure 5. To answer this, we measured the summed expression of the X and Y homologues in northern blots using probes designed to recognize equally the X and Y forms. Probes were synthesized single-stranded cDNA fragments complementary to stretches of virtually identical homology between the X–Y partners of Eif2s3x/y and Smcx/y (Fig. 6). In both cases, the summed expression of the X and Y mRNAs in males was lower than the expression of the X form in females (P<0.05), indicating that although the expression of the Y gene partially compensated for the lower expression of the X form in males, the compensation was not complete. A similar conclusion is possible for the other three X–Y gene pairs studied here: Usp9x/y, Ube1x/y and Utx/y. Expression of the Y homologue in these cases was not detected in our poly(A)+ northern blots despite our repeated attempts using sensitive antisense riboprobes that were as long as those used to detect X-homologue expression. Thus, the level of expression of the Y partner in these cases was much lower than that of the X partner, and could not sum with X-homologue expression to eliminate the sex difference in expression. In the case of Dbx/y, we were not able to synthesize probes that equally recognized the X and Y forms, so we were not able to measure the summed expression levels.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The present study bears on the issue of whether sex chromosome genes are expressed in the brain and act there to induce sex differences in neural function. Transcripts of six of the eight NRY genes that we surveyed can be detected by RT–PCR analysis at one or more of the three ages tested: 13.5 dpc (around or before the onset of sexually dimorphic gonadal secretions), immediately after birth and in adulthood. The expression of these NRY genes at P1 does not require the presence of testes, since the same mRNAs are detected in XY- mice that possess ovaries. Therefore, if any of these NRY genes contributes to sex differences in brain function, it would represent an exception to the classic model of sexual differentiation of non-gonadal tissues (2,3), which attributes all sex differences in non-gonadal phenotypes to the male-specific action of testicular hormones.
The demonstration of NRY expression in brain could be irrelevant to functional sex differences, however, if these transcripts play no functional role (which is unlikely for all of the NRY genes tested), or if their role in males is balanced by the action of similar genes in females. The genes most likely to balance the effects of NRY genes are their X homologues. Because of the conservation of sequence of X–Y gene pairs throughout evolution, it has been widely suspected that the X–Y pairs are functionally quite similar, and thus do not have distinct functional roles (39,40). For instance, human RPS4X and RPS4Y are both able to rescue the deficiency in cell division caused by a mutation in the RPS4 gene in a hamster cell line (41). If males have two doses of equivalent genes – one from Y and one from X – and females have two doses from the X chromosomes, then the inequality of the X dose in the two sexes is balanced by the Y dose. The present results provide two lines of evidence that raise questions about this scenario. Firstly, functional equivalence of X–Y genes would require that the two genes be expressed in parallel, at the same times of development and in the same tissues. This requirement is not met in the current data for Usp9x/y, Ube1x/y and Eif2s3x/y, for which we detected apparent X–Y differences in the developmental course of their expression (Figs 1 and 4) and/or in their distribution across tissues (Fig. 3). The non-parallel expression of these three X–Y gene pairs suggests that they may be differentially regulated, and therefore may have different functional effects. Secondly, the analysis of levels of expression of X–Y homologous genes suggests that the mRNAs encoding six NPX genes are expressed at higher levels in adult female brain than in male brain – a sex difference that is not compensated by expression of the NRY homologue in most cases. For most NPX genes expressed in adult brain, expression of the NRY homologue fell short of that needed to compensate for the sex difference in NPX expression. For instance, the difference in summed expression of Eif2s3x/y (Fig. 6) was only 33% less than the sex difference in Eif2s3x expression (Fig. 5); the magnitude of the sex difference in expression of Smcx (Fig. 5) was reduced only by 3% when the summed expression of Smcx and Smcy was calculated (Fig. 6). The resulting sex difference in mRNA expression could contribute to sex differences in brain function.
The consistent sex differences in NPX expression in adult brain are surprising because the magnitudes of the differences are similar and are not related to the published X-inactivation status of these genes. Smcx, Eif2s3x, and Utx are reported to escape X inactivation (22,36,37), whereas Ube1x is subjected to X inactivation (38). The X-inactivation status of Dbx and Usp9x is unclear. It may be difficult to predict expressed dose from the previously established X-inactivation status, partly because X inactivation may be specific to developmental stage or tissue type (42–44). The published inactivation status of NPX genes studied here may not be accurate for neural tissue at the ages examined in this study.
The retention of ‘ubiquitously expressed’ Y genes in the face of strong degenerative pressure on the Y chromosome has been explained as a mechanism of dosage compensation for their functionally equivalent X homologues, which usually escape X inactivation (20,35,39,40). In this light, the sex difference in summed expression of X–Y homologues is unexpected, especially because the level of expression of several Y homologues was less than that of X homologues. One possibility is that the sex difference in mRNA expression is unrelated to levels of translation, and that the dosages of the homologous X–Y proteins are not sexually dimorphic. A second possibility is that compensation of sex differences in X dosage is critically important, but in a tissue other than brain, or at a developmental stage other than those that we have studied. Alternatively, retention of Y genes may be favored if these genes contribute to a masculine function in brain or other tissues, not as compensation for unequal X-gene dose. Similarly, the higher dosage of X genes may be advantageous for female brain functions.
The sex differences in NPX gene expression found here could theoretically be the result of sex differences in numbers of specific cell types in the brain. This explanation is unlikely, because the known sex differences in numbers of specific cells in brain are generally either large (2–8-fold) but limited to very small brain regions (5), or small differences (10–20%) in cell number distributed over larger areas (45). Neither of these differences in cell number would explain 2-fold differences in level of expression of specific genes in the entire brain.
These NRY genes detected in brain exert distinct biological activities. Usp9y and Ube1y encode enzymes involved in the ubiquitin–proteasome pathway, which regulates protein degradation (15,25,26). Dby and Eif2s3y are likely involved in initiation of translation. Dby encodes a putative RNA-binding protein with a conserved DEAD box motif. Eif2s3y encodes the subunit of eukaryotic initiation factor 2, a component of the 40S ribosomal complex (22). Uty encodes a protein containing a tetratricopeptide repeat motif, which is believed to mediate protein–protein interaction and is found in a variety of functionally distinct proteins (17).
Usp9y expression in brain is particularly interesting. It was limited to the earliest ages sampled (13.5 dpc and P1), when the brain is undergoing rapid growth and differentiation, and it was expressed at a higher level in brain than other tissues. The proteins encoded by Usp9x/y belong to a family of ubiquitin-specific proteases that removes ubiquitin from protein–ubiquitin conjugates and in turn regulates protein degradation (46), a process involved in regulating cell proliferation and/or differentiation. In Drosophila, mutation of the Usp9x/y homologue causes a defect in neuronal differentiation (47), and overexpression in neurons leads to a significant increase in the number of synaptic boutons, an elaboration of the synaptic branching pattern and a disruption of synaptic function (48). Usp9x mRNA also has high expression in brain (49). Usp9x prevents the ubiquitination of (and therefore stabilizes) AF-6 and ß-catenin, both of which accumulate at cell–cell contact sites and participate in the transduction of intercellular signals to specify cell fate in the brain (50,51). Together, this evidence suggests that Usp9x/y plays a role in neural development.
An important caveat is that our studies, like many previous studies of expression of X–Y homologous genes, rely on measures of mRNA expression. The issues raised by the study of transcripts can be resolved only by further study of the expression of these genes at the protein level, and of their functional effects.
NRY genes have been implicated in the control of aggressive behavior in mice (12,52), hippocampal size asymmetry (53), hippocampal mossy fiber distribution (54), open-field activity (55,56), apomorphine-induced-stereotyped behaviors (57), entrainment of circadian rhythms (58), copulatory behaviors (59) and discrimination learning (53). The six NRY genes studied here, together with Sry (14,30), are candidates for these putative Y effects on brain and behavior.
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MATERIALS AND METHODS |
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Animals
C57BL/6 mice, bred at UCLA from stocks obtained from the Jackson Laboratory (Bar Harbor, ME) and MF1 mice, bred at UCLA from stocks of the Division of Developmental Genetics of the National Institute for Medical Research, Mill Hill, London, UK, were maintained on a 14 : 10 light : dark cycle with food and water available ad libitum. Timed pregnant females were generated by pairing adults and performing daily vaginal plug examination each morning at 9 a.m. The day that a plug was found was designated as 0.5 dpc.
To harvest tissues, embryos and neonates were decapitated, and adult animals were anesthetized with metofane (methoxyflurane; Pitman-Moore, Mundelein, IL), after which the tissues of interest were rapidly dissected out and frozen immediately on dry ice and kept at -80°C until processing.
To examine whether the expression of Y genes in males depends on androgens, MF1 XY- and XY-Sry mice were generated by breeding XY-Sry mice with XX mice. XY-Sry mice have the 129/SvEv–Gpi1c strain Y chromosome that carries the Tdyml mutation and is designated the Y- chromosome (32). The mutation is a 11 kb deletion that removes the testis-determining gene Sry. These mice also carry a fully penetrant Sry transgene in an autosomal location, which counteracts the Sry deletion, so that XY-Sry mice possess testes. Breeding XY-Sry males with MF1 XX females generates progeny of four genotypes included in this study: XX females, XY- females, XY-Sry males and XXSry males. Brains were dissected out at P1 and in adulthood, and expression of Y genes was examined by RT–PCR.
Determining genotype
To determine the genotypic sex of 13.5 dpc embryos and P1 pups as well as the genotypes of the XY-Sry offspring, genomic DNA was extracted from tail biopsies by proteinase K digestion at 50°C overnight, followed by a phenol/chloroform extraction. The presence of the Sry gene was detected by PCR with Sry-specific primers (Table 1), and the presence of a Y chromosome was determined by PCR amplification of the Ssty gene family. Myogenin served as an amplification control.
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RT–PCR
The expression of Y-chromosome genes and their X homologues in the brain was examined in C57BL/6 mice at 13.5 dpc, at P1 and in adulthood. At 13.5 dpc and P1, tissue was pooled from 6–8 animals. Total RNA was isolated with Trizol (Gibco BRL, Rockville, ML), and 6 µg was reverse-transcribed in a 40 µl reaction. A 1.0 µl aliquot was then added to a 25 µl PCR reaction containing 1 µM of each primer. The primer sequences are shown in Table 1. PCR conditions were similar to those given in the references, with slight modifications. Typically, 30–35 cycles of amplification were used. The products were separated on a 2% agarose gel stained with ethidium bromide. All PCR reactions were replicated at least once.
Validation of the RT–PCR products
The authenticity of the RT–PCR products of NPY and NRX genes was confirmed by DNA sequencing using an ABI Prism 7700 Sequence Detection System. The sequence of the Ube1y PCR product, amplified from 13.5 dpc, P1 and adult male brain cDNA, was identical to the Ube1y sequences found in GenBank (accession nos AF150963 and NM_011667). The sequence of the Usp9y PCR product was identical to AJ307017, the mouse Usp9y sequence found in GenBank. Sequences of the PCR products of the four ubiquitously expressed Y genes also matched the corresponding sequences in GenBank.
Because primers for Usp9y and Ube1y were derived from different exons, PCR products from reverse-transcribed cDNAs could be distinguished from the longer products amplified from the genomic DNA (for Usp9y, 272 bp versus 400 bp; for Ube1y, 283 bp versus 490 bp). Furthermore, no amplification of Rbmy was observed in any cDNA sample tested, except adult testis. Rbmy PCR is a sensitive assay for genomic contamination, because this gene is present in multiple copies on the Y chromosome.
Northern analysis
Poly(A)+ RNA was isolated with the PolyATract mRNA Isolation System (Promega), and 4–5 µg from each tissue was electrophoresed through a 1.25% agarose gel containing 2 M formaldehyde. RNA was transferred to a MagnaGraph membrane (MSI) using 10xSSC. The membranes were hybridized overnight using probes randomly labeled with 32P (1–5x106 cpm/ml), then washed sequentially at low stringency (2xSSC, 0.1% SDS at 42°C) and high stringency (0.1xSSC, 0.1% SDS at 55°C), followed by autoradiography on Kodak X-OMAT film. The volume intensities of hybridization were quantified with a PhosphorImager, and normalized relative to the intensity of hybridization of a Gapdh probe.
The figures of PCR and Northern blots were scanned digitally and then adjusted to increase contrast using Adobe Photoshop. In no cases were the images altered except to increase contrast.
Probes
Templates for synthesis of probes encoding single genes were purified PCR products, except for Utx and Usp9x, which were cDNA clones provided by Drs A. Greenfield and S. Wood, respectively. The Utx cDNA fragment is 1.1 kb (37), and the Usp9x cDNA is a 766 bp fragment encompassing bp 98 through bp 864 [GenBank accession no. U67874, (49)]. DNA probes were synthesized by randomly labeling with [32P]dCTP, except that the Usp9x probe was an antisense riboprobe generated with a StripEZ kit (Ambion).
Three probes were synthesized to recognize equally the two homologues of Dbx/y, Eif2s3x/y and Smcx/y. These were synthesized single-stranded cDNA oligonucleotides end-labeled with [
-32P]ATP using T4 Polynucleotide Kinase (PNK; Promega, Madison, WI). The Eif2s3x/y probe was CCT CTT CTT ATC TGR CCC CAA CCA ATT AAA CGC CAG TGT TTC TCA ACT C, derived from the sequence for Eif2s3x (GenBank accession no. NM012010, bp 1345–1393) and Eif2s3y (GenBank accession no. NM12011, bp 1342–1390). The Smcx/y probe was TTC TGT RGG TAC CAT ATG CAC AGG CAT GTT GAA GTA GTC AGC, derived from the sequence for Smcx (GenBank accession no. AF127245, bp 1505–1546) and for Smcy (GenBank accession no. AF127244, bp 1229–1270). These probes were tested as probes on Southern blots of restriction-digested genomic male and female DNA, to determine if they equally recognized the X and Y forms. The probes for Eif2s3x/y and Smcx/y hybridized approximately equally to the X and Y restriction fragments in the male DNA, or approximately equally to male and female DNA. Because this analysis did not completely validate the equality of hybridization of the Dbx/y probe, we present only results based on the analysis with the Eif2s3x/y and Smcx/y probes.
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ACKNOWLEDGEMENTS |
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We thank Drs Nabeel Affara, Andy Greenfield and Stephen Wood for gifts of cDNAs and information, Dr Baskaran Ramachandran for advice and assistance, and Dr Eric Vilain for comments on an earlier version of this manuscript. This work was supported by NIH MH59268.
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FOOTNOTES |
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* To whom correspondence should be addressed at: Department of Physiological Science, UCLA, 621 Charles E. Young Drive South, Los Angeles, CA 90095-1606, USA. Tel:+1 310 825 2169; Fax:+1 310 825 8081; Email: arnold{at}ucla.edu
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REFERENCES |
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