Chromosomal Based Sex Differences in the Brain

1. Chromosomal Based Sex Differences in the Brain Jennifer Yoon

2. Classical view of brain sexual differentiation is that gonadal steroid hormones act directly to promote sexual dimorphisms in neural and behavioral development.

3. Dimorphic Brain Areas male female • The sexually dimorphic nucleus of the preoptic area (SDN-POA) is larger in male rats (a) than in females (b) • In contrast, the volume of the rat posterodorsal medial amygdala (MePD), which is about 1.5 times larger in males (c) than in females (d) • In zebra finches, the robustus archistriatum (RA) nucleus is crucial for song production and has a greater volume in males (e) than in females (f)

4. Discovery of the SRY • four men seeking treatment for infertility - found that they were genetic females, with an XX female sex-chromosome pattern. Yet all four were anatomically male and had testes. • Further studies showed that they had a small fragment of the Y chromosome tacked onto one of their X chromosomes. • enough to reverse their sexual anatomy but not endow them with other male genes to enable their testes to make sperm. • identified an area of 5kb specific to the males side – SRY (sex region on the Y chromosome)

5. Evidence to support SRY as the sex determining gene • The Sry gene is expressed strongly in Sertoli cells at 11.5 dpc, which is the correct moment in testis development • XY females with a mutant SRY gene (fathers did not have the same mutation) • Sxr (sex reversed) strain of mice where the Sxr gene contains a copy of the SRY and converts females to males

6. Are there such things as ovary determining genes?? • One would predict that a loss of function mutation in such gene would have no effect in males but that a gain of function mutation would lead to XY sex reversal. These predictions would be the other way round in females.

7. Dax1 • Bardoni et al. (1994) discovered a syndrome known as Dosage Sensitive Sex Reversal • Duplication of the chromosome band Xp21 gives rise to XY females. • Dax1, in the mouse, is expressed in developing gonads. It seems to be repressed by Sry. • Deletion of this gene caused adrenal hypoplasia. • But the deletion didn’t cause any sex reversal in males. • Over expression of Dax1 transgenes does not cause sex reversal in mice except in the case of the 'weak' Y chromosome of Mus poschiavinus.

8. Recent research, however, indicates that some sexual dimorphisms in the brain and other tissues cannot be easily explained as the result of only gonadal steroid action.

9. (days in vitro) • Used mice in which the genetic sex of the brain was independent of gonadal phenotype • Dissociated cell cultures made from E14.5 mesencephalon & labeled with THir and MAP2ir • XY- culture had more THir neurons than XX cultures regardless of gonadal phenotypes • cell culture with Sry had a higher number of dopaminergic neuron than cell cultures without (XY-Sry > XY-) Black bars: males Gray bars: females Carruth et al 2002

10. Differences in sex chromosome effects were attributed to • expression of Y genes that are normally present only in male cells • differences in expression of X genes • differences in paternal imprinting of X genes

11. Sexually dimorphic gene • Differential expression of genes verified by RT-PCR & microarray at 10.5 dpc (before any gonadal differentiation) • Detection of 51 genes with differential expression between males and females • Majority of genes are involved in: • Cellular differentiation & proliferation • Transcriptional regulation • Signaling molecules • ¼ have unknown functions Dewing et al. 2003

12. HVc of the Zebra Finch • females treated with fadrozole, aromatase inhibitor, it blocked ovarian development and allowed testicular development • but despite the presence of functional testicular tissue and secretion of androgens, females still had feminine pattern of neural development

13. found that XY and XX brain cells differed in phenotype even before the effects of gonadal hormones • found that genes were dimorphicly expressed even before the effects of gonadal hormones • found that females developed a female brain even in the presence of male hormones

15. Animal Models

16. Avian Sex Chromosome • males ZZ, females ZW • Is it the Z or W chromosome that regulates sex in birds?

17. Avian Sex Chromosomes Two Possibilities • The W chromosome contains a gene whose expression in the early embryonic stage triggers gene expression towards female sex determination • double dosage of a gene on the Z chromosome causes male determination whereas a single dosage causes female sex determination

18. Dominant W Hypothesis • A dominant W chromosome gene is expressed in the early embryonic stage triggering gene expression towards female sex determination

19. PKCIW • PKCI (PKC inhibitor) = ASW (Avian, Sex-specific, W-linked) • strongly expressed in the female genital ridge from day 4-5 in chicken embryos prior to sexual differentiation • PKCIZ is expressed in small amounts in both sexes • hypothesized that PKCIW may interfere with PKCIZ function via heterodimerization

20. Gynandromorphic Finches male female

21. Gynandromorphic Finches male female male female • Fig.1 Photomicrographs of histological sections of the gonads. R dimorphic testis with germ cells, L ovarian tissue with follicles • Fig.2 Photomicrographs of in situ hybridization A)mRNA for PKCIW located in left but absent in right B)mRNA for PKCIZ was ubiquitous but higher on the right side of the brain

22. PKCIW • The uniformly high expression of PKCIW mRNA can be seen in left-side Purkinje cells and granule cells, extending to the midline (arrows). (cerebellum)

23. CHD1 • CHD proteins appear to function as facilitators or inhibitors of gene expression • Chromodomain-Helicase DNA binding protein • chromodomain similar to heterochromatin protein 1 & polycomb which function to repress gene expression • helicase domain is homologous to SNF2/SWI1 and brahma which are critical for gene activation • DNA-binding domain contains motifs characteristic of protein H1 and HMG I/Y

24. CHD1 r l r l right left • (A) PCR products representing fragments of CHD1Z and CHD1W amplified from genomic DNA from the right and left sides of the gynandromorph brain. CHD1W (upper band) was predominantly restricted to the left side of the brain. CHD1Z (lower band) was found on both sides of the brain. (B) Southern blot of genomic DNA from leg muscle and feather pulp of the gynandromorph shows greater amount of PKCIW in genomic DNA from the left side than from the right CHD1Z

25. Developmental Expression of CHD1 various tissues of the zebra finch • CHD1Z (upper band) and CHD1W (lower band) • CHD1Z expression higher in males than females • CHD1W expressed primarily in females

26. Z dosage hypothesis • higher dose of a Z-linked gene in ZZ embryos triggers male development while a lower dose allows female development • and so the Z-linked gene must escape dosage compensation

27. DMRT1 • DMRT1 (drosophila doublesex and C elegans Mab3 related transcription factor #1) • best candidate because it is present on the Z chromosome and has no homologue on the W and appears to escape dosage compensation • expression found in genital ridge & wolffian ducts of chickens in embryonic stage 25 prior to sexual differentiation • suggests DMRT1 becomes testis specific & in adults it is expressed exclusively in the testis

28. DMRT1 • ZW females that have been sex-reversed with an aromatase inhibitor which upregulates DMRT1 in parallel with testis differentiation despite being present in a single copy • DMRT1 maps to chromosome 9p24.3 and deletions in distal 9p have been implicated in gonadal dysgenesis and sex reversal in 46 XY individuals • XY Dmrt1-/- knockouts fail to produce differentiated testes and experience germ cell death

29. MHM Region • MHM (male hypermethylated region) • this region becomes hypermethylated and transcriptionally inactive in males • in females, this region is hypomethylated and is transcribed but the RNA is not tranlated

30. So what’s the purpose of transcribing a gene that isn’t translated for a functional use?

31. MHM region • The MHM region is not translated but the non-coding RNA that is transcribed coats the Z chromosome of the female adjacent to the DMRT1 locus and possibly causes repression • Appears that the W chromosome contains a factor that mediates the methylation status of the Z chromosome • But if repression occurs, it must only be partial because DMRT1 is expressed in ZW gonads, though in lower levels than in males

32. Research in the dimorphic expression and function of chromosomal genes is still fairly recent.

33. Methods

34. So where is the research on the chromosomal aspect of sexual differentiation?